Periaqueductal Gray Neurons Research Articles

Effects of inflammatory pain on CB1 receptor in the midbrain periaqueductal gray.

The periaqueductal gray (PAG) mediates the antinociceptive properties of analgesics, including opioids and cannabinoids. Administration of either opioids or cannabinoids into the PAG induces antinociception. However, most studies characterizing the antinociceptive properties of cannabinoids in the PAG have been conducted in naive animals. Few studies have reported on the role of CB1 receptors in the PAG during conditions which would prompt the administration of analgesics, namely, during pain states. To examine inflammatory pain-induced changes in CB1 receptor expression and function in the midbrain periaqueductal gray. In this study, we used the Complete Freund Adjuvant model to characterize CB1 receptor expression and G-protein coupling during persistent inflammatory pain. Inflammatory pain induced an upregulation in the expression of synaptic CB1 receptors in the PAG. Despite this pain-induced change in CB1 expression, there was no corresponding upregulation of CB1 mRNA after the induction of inflammatory pain, suggesting a pain-induced recruitment of CB1 receptors to the synaptic sites within PAG neurons or increased coupling efficiency between the receptor and effector systems. Inflammatory pain also enhanced ventrolateral PAG CB1 receptor activity, as there was an increase in CP55,940-stimulated G-protein activation compared with pain-naïve control animals. These findings complement a growing body of evidence which demonstrate pain-induced changes in brain regions that are responsible for both the analgesic and rewarding properties of analgesic pharmacotherapies. Because much of our understanding of the pharmacology of cannabinoids is based on studies which use largely pain-naïve male animals, this work fills in important gaps in the knowledge base by incorporating pain-induced adaptations and cannabinoid pharmacology in females.

Persistent Activity of Metabotropic Glutamate Receptor 5 in the Periaqueductal Gray Constrains Emergence of Chronic Neuropathic Pain

Pain sensation is powerfully modulated by signal processing in the brain, and pain becomes chronic with the dysfunction of the pain modulatory system; however, the underlying mechanisms are unclear. We found that the metabotropic glutamate receptor 5 (mGluR5) in the periaqueductal gray (PAG), the key area of endogenous pain modulation, is persistently active in normal conditions to maintain an appropriate sensory perception. In the neuropathic pain condition, Homer1a, an activity-dependent immediate early gene product, disrupted the persistent mGluR5 activity resulting in chronic pain. Remarkably a single-time blockage of the mGluR5 resulted in chronic neuropathic pain-like symptoms even in the absence of nerve injury. The decline of mGluR5 activity induced the pain modulatory dysfunction with a profound reduction of excitability of PAG neurons. These findings uncover the role of the persistent mGluR5 activity invivo and provide new insight into how pain becomes chronic with the maladaptive coping of the PAG to pain sensation.

Dopamine neurons in the ventral periaqueductal gray modulate isoflurane anesthesia in rats.

AimsGeneral anesthesia has been applied in surgery for more than 170 years, and there is little doubt that GABAA receptors have an important role as anesthetic molecular targets, but its neural mechanisms remain unclear. Increasing researchers have shown that dopaminergic pathways in the brain are crucial for sleep and wake. General anesthesia‐induced unconsciousness and natural sleep share some neural correlates. However, the role of GABAA receptors in ventral periaqueductal gray (vPAG) dopamine (DA) neurons in the isoflurane‐induced unconsciousness has yet to be identified.MethodsIn the present study, we used calcium fiber photometry recording to explore that the activity of ventral periaqueductal gray (vPAG) neurons. Then, rats were unilaterally microinjected with 6‐hydroxydopamine into the vPAG area to determine the role of vPAG‐DA neurons in isoflurane‐induced‐anesthesia. Furthermore, thirty SD rats were divided into three groups: a GABAAR agonist‐muscimol group, a GABAAR antagonist‐gabazine group, and a control group. Finally, whole‐cell patch clamp was used to examine the effects of isoflurane and GABAA receptor agonist/antagonist on vPAG‐DA neurons.ResultsThe vPAG neurons were markedly inhibited during isoflurane anesthesia induction and that these neurons were activated during emergence from isoflurane anesthesia. Lesion to the vPAG‐DA neurons shortened the induction time and prolonged the emergence time while increasing δ power in isoflurane anesthesia. Intracerebral injection of the GABAA receptor agonist (muscimol) into the vPAG accelerated the induction of anesthesia and delayed recovery from isoflurane anesthesia, with a decrease of δ power and an augment of β power. Injection of GABAA receptor antagonist gabazine generated the opposite effects. Isoflurane enhanced GABAergic transmission, and GABAA receptor agonist partly increased isoflurane‐induced inhibition of vPAG‐DA neurons, while GABAA receptor antagonist evidently attenuated GABAergic transmission.ConclusionOur results suggest that vPAG‐DA neurons are involved in isoflurane anesthesia through activation of the GABAA receptor.

GPR30 receptor promotes preoperative anxiety-induced postoperative hyperalgesia by up-regulating GABAA-\u03b14\u03b21\u03b4 subunits in periaqueductal gray in female rats

BackgroundG-protein coupled estrogen receptor 30 (GPR30) was proved the specific estrogen receptor relating to mechanical hyperalgesia. Studies have shown that the GABAA receptor subunits α4, β1, and δ in the periaqueductal gray (PAG) neurons promote the descending facilitation system. This study inquired into whether and how GPR30 and GABAA-α4β1δ in the PAG promote preoperative anxiety-induced postoperative hyperalgesia in female rats.MethodsAll the female rats were subjected to the single prolonged stress (SPS) to stimulate preoperative anxiety. Subsequently, mechanical allodynia was evaluated before and after the incision, based on the paw withdrawal mechanical threshold (PWMT). The selective GPR30 agonist G1 and antagonist G15 were locally microinjected into the PAG. The expression of GPR30, protein kinase A (PKA), and GABAA receptor subunits α4, β1, and δ in the PAG neurons were detected using western blotting and immunofluorescence.ResultsBehavioral testing revealed that Group S and Group I decreased the nociceptive threshold levels of PWMT in female rats. PWMT in Group S + I decreased more than that of Group S and Group I. Further, results of western blotting showed the expression of GPR30, PKA, and GABAA α4, β1, and δ subunits significantly up-regulated in Group S + I, and immunofluorescence indicated that the neurons of PAG in Group S + I appeared simultaneously immunopositive for GPR30 and GABAA α4, β1, and δ receptors. After microinjection of G1 into the PAG, female rats with plantar incision continued to exhibit significant hyperalgesia until postoperative 48 h. On the other hand, microinjection of G15 with SPS and plantar incision procedure relieved postoperative hyperalgesia in female rats. Western blotting demonstrated that intra-PAG injection of G15 markedly decreased the GPR30, PKA, and GABAA α4, β1, and δ levels in Group G15 + I.ConclusionsOur results indicate that the GPR30-PKA-GABAAα4β1δ pathway in the PAG promotes preoperative anxiety-induced postoperative hyperalgesia in female rats. This mechanism might be a potential novel therapeutic target for hyperalgesia in females.

Control of Non-REM Sleep by Midbrain Neurotensinergic Neurons

The periaqueductal gray (PAG) in the midbrain is known to coordinate behavioral and autonomic responses to threat and injury through its descending projections to the brainstem. Here, we show that neurotensin (NTS)-expressing glutamatergic neurons in the ventrolateral PAG (vlPAG) powerfully promote non-rapid eye movement (NREM) sleep partly through their projection to the caudal medulla. Optogenetic and chemogenetic activation of vlPAG NTS neurons strongly enhanced NREM sleep, whereas their inactivation increased wakefulness. Calcium imaging and optrode recording showed that they are preferentially active during NREM sleep.The NREM-promoting effect of vlPAG NTS neurons ispartly mediated by their projection to the caudal ventromedial medulla, where they excite GABAergic neurons. Bidirectional optogenetic and chemogenetic manipulations showed that the medullary GABAergic neurons also promote NREM sleep, and they innervate multiple monoaminergic populations. Together, these findings reveal a novel pathway for NREM sleep generation, in which glutamatergic neurons drive broad GABAergic inhibition of wake-promoting neuronal populations.

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 Specialized Neural Circuit Gates Social Vocalizations in the Mouse

Vocalizations are fundamental to mammalian communication, but the underlying neural circuits await detailed characterization. Here, we used an intersectional genetic method to label and manipulate neurons in the midbrain periaqueductal gray (PAG) that are transiently active in male mice when they produce ultrasonic courtship vocalizations (USVs). Genetic silencing of PAG-USV neurons rendered males unable to produce USVs and impaired their ability to attract females. Conversely, activating PAG-USV neurons selectively triggered USV production, even in the absence of any female cues. Optogenetic stimulation combined with axonal tracing indicates that PAG-USV neurons gate downstream vocal-patterning circuits. Indeed, activating PAG neurons that innervate the nucleus retroambiguus, but not those innervating the parabrachial nucleus, elicited USVs in both male and female mice. These experiments establish that a dedicated population of PAG neurons gives rise to a descending circuit necessary and sufficient for USV production while also demonstrating the communicative salience of male USVs. VIDEO ABSTRACT.

The Role of Glutamatergic and Dopaminergic Neurons in the Periaqueductal Gray/Dorsal Raphe: Separating Analgesia and Anxiety.

The periaqueductal gray (PAG) is a significant modulator of both analgesic and fear behaviors in both humans and rodents, but the underlying circuitry responsible for these two phenotypes is incompletely understood. Importantly, it is not known if there is a way to produce analgesia without anxiety by targeting the PAG, as modulation of glutamate or GABA neurons in this area initiates both antinociceptive and anxiogenic behavior. While dopamine (DA) neurons in the ventrolateral PAG (vlPAG)/dorsal raphe display a supraspinal antinociceptive effect, their influence on anxiety and fear are unknown. Using DAT-cre and Vglut2-cre male mice, we introduced designer receptors exclusively activated by designer drugs (DREADD) to DA and glutamate neurons within the vlPAG using viral-mediated delivery and found that levels of analgesia were significant and quantitatively similar when DA and glutamate neurons were selectively stimulated. Activation of glutamatergic neurons, however, reliably produced higher indices of anxiety, with increased freezing time and more time spent in the safety of a dark enclosure. In contrast, animals in which PAG/dorsal raphe DA neurons were stimulated failed to show fear behaviors. DA-mediated antinociception was inhibitable by haloperidol and was sufficient to prevent persistent inflammatory pain induced by carrageenan. In summary, only activation of DA neurons in the PAG/dorsal raphe produced profound analgesia without signs of anxiety, indicating that PAG/dorsal raphe DA neurons are an important target involved in analgesia that may lead to new treatments for pain.

A Novel Role for the Periaqueductal Gray in Consummatory Behavior.

The periaqueductal gray (PAG) has a well-established role in pain processing, autonomic function and behavioral responses to fear. Anatomical work suggests the PAG may mediate food intake and reward processing as it has extensive reciprocal connections within brain circuits that mediate appetitive processes and consummatory behaviors such as prefrontal cortex, hypothalamus, amygdala, parabrachial nucleus (PBN) and ventral tegmental area (Kelley et al., 2005). Therefore, we investigated if the PAG of hungry rats has a functional role in appetitive and consummatory behaviors. To address this, PAG was pharmacologically inactivated during a spatial working memory task with muscimol (0.1–0.3 μg), a GABAA agonist via intracranial infusion. Inactivation of PAG led to reduced intake of food rewards and increased errors on this task. To focus on the specific effects PAG inactivation had on food consumption, PAG was inactivated during two separate food intake tasks in a separate group of rats. Again, PAG inactivation resulted in a significant decrease in food consumption, as well as an increased latency to consume food. We next investigated PAG neural responses to reward encounters. A different group of rats performed the same task used in Experiment 1 while the in vivo activity of PAG neurons was recorded. In a subset of PAG neurons, reward encounters elicited phasic excitation. A separate subset of PAG neurons were inhibited during reward encounters. These responses scaled with the size of the reward, with sustained excitation or inhibition in response to large rewards compared to small. Our data also show that separate groups of PAG neurons in awake behaving animals display either increased and decreased neural responses to reward encounters. Additionally, a proportion of neurons were modulated by the animals’ velocity. This study is the first to show that PAG neurons process reward-related information, perhaps to mediate consummatory behaviors related to food consumption.

Involvement of the periaqueductal gray in the descending antinociceptive effect induced by the central nucleus of amygdala.

Here we studied whether descending control of mechanical nociception by glutamate in the central nucleus of the amygdala (CeA) of healthy control animals is induced by amygdaloid NMDA receptors and relayed through the midbrain periaqueductal gray (PAG). Mechanical nociception in the hind paws was assessed in rats with chronic guide cannulae for glutamate administration in the right CeA and for inducing local anesthesia in the PAG. In a separate electrophysiological study, ON-like PAG neurons giving an excitatory response to noxious pinch of the tail were recorded in anesthetized rats following glutamate administration into the CeA. A high dose of glutamate (100 microg) in the CeA induced mechanical antinociception in the contra- but not ipsilateral hind limb. Antinociception was prevented by an NMDA receptor antagonist in the CeA or local anesthesia of the PAG. Discharge rate of ON-like PAG neurons was increased by a high dose of glutamate (100 microg) in the CeA and this increase was prevented by an NMDA receptor antagonist in the CeA. The results indicate that amygdaloid NMDA receptors in the CeA may induce contralaterally mechanical antinociception through a circuitry relaying in the PAG. Activation of ON-like PAG neurons is associated with the descending antinociceptive effect. Mechanisms and causality of this association still remain to be studied.

Central Amygdala Circuits Mediate Hyperalgesia in Alcohol-Dependent Rats.

Alcohol withdrawal symptoms contribute to excessive alcohol drinking and relapse in alcohol-dependent individuals. Among these symptoms, alcohol withdrawal promotes hyperalgesia, but the neurological underpinnings of this phenomenon are not known. Chronic alcohol exposure alters cell signaling in the central nucleus of the amygdala (CeA), and the CeA is implicated in mediating alcohol dependence-related behaviors. The CeA projects to the periaqueductal gray (PAG), a region critical for descending pain modulation, and may have a role in alcohol withdrawal hyperalgesia. Here, we tested the roles of (1) CeA projections to PAG, (2) CeA melanocortin signaling, and (3) PAG μ-opioid receptor signaling in mediating thermal nociception and alcohol withdrawal hyperalgesia in male Wistar rats. Our results demonstrate that alcohol dependence reduces GABAergic signaling from CeA terminals onto PAG neurons and alters the CeA melanocortin system, that CeA-PAG projections and CeA melanocortin signaling mediate alcohol withdrawal hyperalgesia, and that μ-opioid receptors in PAG filter CeA effects on thermal nociception.SIGNIFICANCE STATEMENT Hyperalgesia is commonly seen in individuals with alcohol use disorder during periods of withdrawal, but the neurological underpinnings behind this phenomenon are not completely understood. Here, we tested whether alcohol dependence exerts its influence on pain modulation via effects on the limbic system. Using behavioral, optogenetic, electrophysiological, and molecular biological approaches, we demonstrate that central nucleus of the amygdala (CeA) projections to periaqueductal gray mediate thermal hyperalgesia in alcohol-dependent and alcohol-naive rats. Using pharmacological approaches, we show that melanocortin receptor-4 signaling in CeA alters alcohol withdrawal hyperalgesia, but this effect is not mediated directly at synaptic inputs onto periaqueductal gray-projecting CeA neurons. Overall, our findings support a role for limbic influence over the descending pain pathway and identify a potential therapeutic target for treating hyperalgesia in individuals with alcohol use disorder .

(2R,6R)-hydroxynorketamine rescues chronic stress-induced depression-like behavior through its actions in the midbrain periaqueductal gray

It has been widely reported that ketamine rescues chronic stress-induced depression-like behavior, but the underlying cellular mechanisms of the rapid antidepressant actions of ketamine remain largely unclear. Both male and female Sprague-Dawley rats were used and received modified learned helplessness paradigm to induce depression-like behavior. Depression-like behavior was assayed and manipulated using forced swim tests, sucrose preference tests and pharmacological microinjection. We conducted whole-cell patch-clamp electrophysiological recordings in the midbrain ventrolateral periaqueductal gray (vlPAG) neurons. Surface and cytosolic glutamate receptor 1 (GluR1) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression were analyzed using Western blotting. Phosphorylated GluR1 expression was quantified using Western blotting analysis. The results showed that a single systemic administration of a ketamine metabolite (2R,6R)-hydroxynorketamine (2R,6R-HNK) rapidly rescued chronic stress-induced depression-like behavior and persisted for up to 21 days. Consistently, the chronic stress-induced diminished glutamatergic transmission and surface GluR1 expression in the vlPAG were also reversed by a single systemic injection of (2R,6R)-HNK. Furthermore, bath application of (2R,6R)-HNK increased the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in the vlPAG. Further evidence for the antidepressant action of (2R,6R)-HNK is provided by the finding that microinjection of (2R,6R)-HNK into the vlPAG exhibited a rapid-acting and long-lasting antidepressant effect. This antidepressant effect of (2R,6R)-HNK was prevented by the intra-vlPAG microinjection of AMPA receptor antagonist CNQX. Together, the current results provide evidence that (2R,6R)-HNK rescues chronic stress-induced depression-like behavior with rapid-acting and long-lasting antidepressant effects through enhancement of AMPA receptor-mediated transmission in the vlPAG.

Single unit activity of periaqueductal gray and deep mesencephalic nucleus neurons involved in sleep stage switching in the mouse.

A total of 668 single units were recorded in the mouse periaqueductal gray (PAG) and adjacent deep mesencephalic nucleus (DpMe) to determine their role in the switching of sleep-wake states, that is, wakefulness (W), slow-wave sleep (SWS) and paradoxical (or rapid eye movement) sleep (PS) in general, and, in particular, to determine whether PS-on and PS-off neurons involved in PS state switching are present in these structures and to identify neuronal substrates for the SWS-PS switching mediated by DpMe neurons. Both structures were found to contain similar percentages of W/PS-active neurons, which discharge at a higher rate during W and PS than during SWS, while W-active neurons, which discharge maximally during W, were found mainly in the PAG. Both also contained similar percentages of SWS/PS-active neurons, which discharge at higher rates during SWS and PS than during W, and PS-active neurons, which discharge maximally during PS, while SWS-active neurons, which discharge maximally during SWS, were found almost exclusively in the PAG. Both structures contained virtually no PS-on or PS-off neurons, which, respectively, discharge or cease firing selectively and tonically just prior to, and during, PS. Unlike the PAG, the DpMe contained many SWS/PS-on neurons, which discharge selectively at high rates during SWS and PS, but show a decrease in discharge rate at the transition from SWS to PS. Analysis of discharge profiles and trends in spike activity at the state transitions strongly suggests that PAG and DpMe neurons play an important role in the W-SWS, SWS-PS and/or PS-W switches.

Periaqueductal efferents to dopamine and GABA neurons of the VTA.

Neurons in the periaqueductal gray (PAG) modulate threat responses and nociception. Activity in the ventral tegmental area (VTA) on the other hand can cause reinforcement and aversion. While in many situations these behaviors are related, the anatomical substrate of a crosstalk between the PAG and VTA remains poorly understood. Here we describe the anatomical and electrophysiological organization of the VTA-projecting PAG neurons. Using rabies-based, cell type-specific retrograde tracing, we observed that PAG to VTA projection neurons are evenly distributed along the rostro-caudal axis of the PAG, but concentrated in its posterior and ventrolateral segments. Optogenetic projection targeting demonstrated that the PAG-to-VTA pathway is predominantly excitatory and targets similar proportions of Ih-expressing VTA DA and GABA neurons. Taken together, these results set the framework for functional analysis of the interplay between PAG and VTA in the regulation of reward and aversion.

The involvement of the pathway connecting the substantia nigra, the periaqueductal gray matter and the retrotrapezoid nucleus in breathing control in a rat model of Parkinson's disease

Parkinson's disease (PD) is characterized by a reduction in the number of dopaminergic neurons of the substantia nigra (SNpc), accompanied by motor and non-motor deficiencies such as respiratory failure. Here, our aim was to investigate possible neuronal communications between the SNpc and chemoreceptor neurons within the retrotrapezoid nucleus (RTN), in order to explain neurodegeneration and the loss of breathing function in the 6-OHDA PD animal model. Male Wistar rats received tracer injections in the SNpc, RTN and periaqueductal gray (PAG) regions to investigate the projections between those regions. The results showed that neurons of the SNpc project to the RTN by an indirect pathway that goes through the PAG region. In different groups of rats, reductions in the density of neuronal markers (NeuN) and the number of catecholaminergic varicosities in PAG, as well as reductions in the number of CO2-activated PAG neurons with RTN projections, were observed in a 6-OHDA model of PD. Physiological experiments showed that inhibition of the PAG by bilateral injection of muscimol did not produce resting breathing disturbances but instead reduced genioglossus (GGEMG) and abdominal (AbdEMG) muscle activity amplitude induced by hypercapnia in control rats that were urethane-anesthetized, vagotomized, and artificially ventilated. However, in a model of PD, we found reductions in resting diaphragm muscle activity (DiaEMG) and GGEMG frequencies, as well as in hypercapnia-induced DiaEMG, GGEMG and AbdEMG frequencies and GGEMG and AbdEMG amplitudes. Therefore, we can conclude that there is an indirect pathway between neurons of the SNpc and RTN that goes through the PAG and that there is a defect of this pathway in an animal model of PD.

Brief optogenetic inhibition of rat lateral or ventrolateral periaqueductal gray augments the acquisition of Pavlovian fear conditioning.

The midbrain periaqueductal gray (PAG) coordinates the expression and topography of defensive behaviors to threat and also plays an important role in Pavlovian fear learning itself. Whereas the role of PAG in the expression of defensive behavior is well understood, the relationship between the activity of PAG neurons and fear learning, the exact timing of PAG contributions to learning during the conditioning trial, and the contributions of different PAG columns to fear learning are poorly understood. We assessed the effects of optogenetic inhibition of lateral (LPAG) and ventrolateral PAG (VLPAG) neurons on fear learning. Using adenoassociated viral vectors expressing halorhodopsin, we show that brief optogenetic inhibition of LPAG or VLPAG during delivery of the shock unconditioned stimulus (US) augments acquisition of contextual or cued fear conditioning, and we also show that this inhibition augments postencounter defensive responses to a nonnoxious threat. Taken together, these results show that LPAG and VLPAG serve a key role in the regulation of Pavlovian fear learning at the time of US delivery. These findings provide strong support for existing models that state that LPAG and VLPAG contribute to a fear prediction error signal determining variations in the effectiveness of the aversive US in supporting learning. (PsycINFO Database Record

Monoaminergic descending pathways contribute to modulation of neuropathic pain by increasing-intensity treadmill exercise after peripheral nerve injury

This study characterizes the impact of increasing-intensity treadmill exercise (iTR) on noradrenergic (NE) and serotonergic (5HT) modulation of neuropathic pain. Following sciatic nerve transection and repair (SNTR) rats developed significant mechanical and thermal hyperalgesia that was partially prevented by iTR performed during the first 2weeks after injury.Marked decrease in the expression of 5HT2A and α1A and β-, but not α2A adrenergic receptors in the spinal cord dorsal horn was associated to SNTR and recovered by iTR, particularly in lamina II. iTR significantly increased 5HT2A in periaqueductal grey (PAG), raphe magnus (RM) and dorsal raphe nucleus (DRN), with a pattern suggesting reorganization of serotonergic excitatory interconnections between PAG and DRN. iTR also increased the expression of α1A in locus coeruleus (LC) and DRN, and β2 in LC, indicating that exercise enhanced activity of NE neurons, likely by activating autologous projections from DRN and PAG.iTR hypoalgesia was antagonized by blockade of β2 and 5HT2A receptors with administration of butoxamine and ketanserin. The neurotoxin DSP4 was injected to induce depletion of NE projections from LC before starting iTR. DSP4 treatment worsened mechanical hyperalgesia, but iTR hypoalgesia was similarly produced. Moreover, 5HT2A expression in LC further increased after DSP4 injection, all these results suggesting an intrinsic regulation of 5HT and NE activity between PAG, DRN and LC neurons activated by iTR.Finally, iTR significantly reduced microglial reactivity in LC and increased non-microglial BDNF expression, an effect that was reverted by butoxamine, implicating BDNF regulation in central 5HT/NE actions on neuropathic pain.

Chronic morphine reduces the readily releasable pool of GABA, a presynaptic mechanism of opioid tolerance.

Chronic treatment with opioids, such as morphine, leads to analgesic tolerance. While postsynaptic opioid tolerance is well documented, the involvement of presynaptic mechanisms remains unclear. We show that chronic morphine reduces the ability of periaqueductal grey (PAG) neurons to maintain GABAergic transmission. This depression of GABAergic transmission was due to a reduction in the effective size of the readily releasable pool. This also led to a reduction in opioid presynaptic inhibition; these presynaptic adaptations need to be considered in the development of strategies to reduce opioid tolerance. The midbrain periaqueductal grey (PAG) plays a critical role in tolerance to the analgesic actions of opioids such as morphine. While numerous studies have identified the postsynaptic adaptations induced by chronic morphine treatment in this and other brain regions, the presence of presynaptic adaptations remains uncertain. We examined GABAergic synaptic transmission within rat PAG brain slices from animals which underwent a low dose morphine treatment protocol which produces tolerance, but not withdrawal. Evoked GABAergic IPSCs (inhibitory postsynaptic currents) were less in morphine compared to control saline treated animals. Postsynaptic GABAA receptor mediated currents and desensitization, presynaptic release probability (Pr ), and inhibition by endogenous neurotransmitters were similar in morphine and saline treated animals. By contrast, the effective size of the readily releasable pool (RRP) was smaller in morphine treated animals. While the μ-opioid agonist DAMGO produced a reduction in Pr and RRP size in saline treated animals, it only reduced Pr in morphine treated animals. Consequently, DAMGO-induced inhibition of evoked IPSCs during short burst stimulation was less in morphine, compared to saline treated animals. These results indicate that low dose chronic morphine treatment reduces presynaptic μ-opioid inhibition by reducing the size of the pool of vesicles available for action potential dependent release. This novel presynaptic adaptation may provide important insights into the development of efficacious pain therapies that can circumvent the development of opioid tolerance.

A leptin-regulated circuit controls glucose mobilization during noxious stimuli.

Adipocytes secrete the hormone leptin to signal the sufficiency of energy stores. Reductions in circulating leptin concentrations reflect a negative energy balance, which augments sympathetic nervous system (SNS) activation in response to metabolically demanding emergencies. This process ensures adequate glucose mobilization despite low energy stores. We report that leptin receptor-expressing neurons (LepRb neurons) in the periaqueductal gray (PAG), the largest population of LepRb neurons in the brain stem, mediate this process. Application of noxious stimuli, which often signal the need to mobilize glucose to support an appropriate response, activated PAG LepRb neurons, which project to and activate parabrachial nucleus (PBN) neurons that control SNS activation and glucose mobilization. Furthermore, activating PAG LepRb neurons increased SNS activity and blood glucose concentrations, while ablating LepRb in PAG neurons augmented glucose mobilization in response to noxious stimuli. Thus, decreased leptin action on PAG LepRb neurons augments the autonomic response to noxious stimuli, ensuring sufficient glucose mobilization during periods of acute demand in the face of diminished energy stores.

Effects of chemogenetic excitation or inhibition of the ventrolateral periaqueductal gray on the acquisition and extinction of Pavlovian fear conditioning

The midbrain periaqueductal gray (PAG) has been implicated in the generation and transmission of a prediction error signal that instructs amygdala-based fear and extinction learning. However, the PAG also plays a key role in the expression of conditioned fear responses. The evidence for a role of the PAG in fear learning and extinction learning has been obtained almost exclusively using PAG-dependent fear responses. It is less clear whether the PAG regulates fear learning when other measures of learned fear are used. Here we combined a chemogenetic approach, permitting excitation or inhibition of neurons in the ventrolateral PAG (VLPAG), with conditioned suppression as the measure of learned fear to assess the role of VLPAG in the acquisition and extinction of fear learning. We show that chemogenetic excitation of VLPAG (with some encroachment on lateral PAG [LPAG]) impairs acquisition of fear and, conversely, chemogenetic inhibition impairs extinction of fear. These effects on fear and extinction learning were specific to the combination of DREADD expression and injection of CNO because they were observed relative to both eYFP controls injected with CNO as well as DREADD expressing controls injected with vehicle. Taken together, these results show that activity of L/VLPAG neurons regulates both the acquisition and extinction of Pavlovian fear learning.