Periaqueductal Gray Stimulation Research Articles

Role of L- and T-type voltage-dependent calcium channels in the hierarchical organization of defensive responses to electrical stimulation of the rat dorsolateral periaqueductal gray

Stimulation of the dorsal half of the rat periaqueductal gray (DPAG) with 60-Hz pulses of increasing intensity, 30-μA pulses of increasing frequency, or increasing doses of an excitatory amino acid elicits sequential defensive responses of exophthalmia, immobility, trotting, galloping, and jumping. These responses may be controlled by voltage-gated calcium channel-specific firing patterns. Indeed, a previous study showed that microinjection of the DPAG with 15 nmol of verapamil, a putative blocker of L-type calcium channels, attenuated all defensive responses to electrical stimulation at the same site as the injection. Accordingly, here we investigated the effects of microinjection of lower doses (0.7 and 7 nmol) of both verapamil and mibefradil, a preferential blocker of T-type calcium channels, on DPAG-evoked defensive behaviors of the male rat. Behaviors were recorded either 24 h before or 10 min, 24 h, and 48 h after microinjection. Effects were analyzed by both threshold logistic analysis and repeated measures analysis of variance for treatment by session interactions. Data showed that the electrodes were all located within the dorsolateral PAG. Compared to the effects of saline, verapamil significantly attenuated exophthalmia, immobility, and trotting. Mibefradil significantly attenuated exophthalmia and marginally attenuated immobility while facilitating trotting. While galloping was not attenuated by either antagonist, jumping was unexpectedly attenuated by 0.7 nmol verapamil only. These results suggest that T-type calcium channels are involved in the low-threshold freezing responses of exophthalmia and immobility, whereas L-type calcium channels are involved in the trotting response that precedes the full-fledged escape responses of galloping and jumping.

Low-frequency (5-Hz) stimulation of ventrolateral periaqueductal gray modulates the descending serotonergic system in the peripheral neuropathic pain.

Neuropathic pain is a type of chronic pain that entails severe prolonged sensory dysfunctions caused by a lesion of the somatosensory system. Many of those suffering from the condition do not experience significant improvement with existing medications, resulting in various side effects. In this study, Sprague-Dawley male rats were used, and long-term deep brain stimulation of the ventrolateral periaqueductal gray was conducted in a rat model of spared nerve injury. We found that 5-Hz deep brain stimulation effectively modulated mechanical allodynia and induced neuronal activation in the rostral ventromedial medulla, restoring impaired descending serotonergic system. At the spinal level, glial cells were still activated but only the 5-HT1a receptor in the spinal cord was activated, implying its inhibitory role in mechanical allodynia. This study found that peripheral neuropathy caused dysfunction in the descending serotonergic system, and prolonged stimulation of ventrolateral periaqueductal gray can modulate the pathway in an efficient manner. This work would provide new opportunities for the development of targeted and effective treatments for this debilitating disease, possibly giving us lower chances of side effects from repeated high-frequency stimulation or long-term use of medication.

Decreased bladder contraction interval induced by periaqueductal grey stimulation is reversed by subthalamic stimulation in a Parkinson’s disease model rat

The medial prefrontal cortex (mPFC) regulates bladder contractions via the periaqueductal grey (PAG). Subthalamic nucleus deep brain stimulation (STN-DBS) modulates urinary afferent information from PAG in Parkinson’s disease (PD). We do not know how STN-DBS modulates the activities of mPFC induced by PAG stimulation. We aim to clarify how STN-DBS modulates the neuronal activity of mPFC induced by PAG stimulation and its effects on bladder contraction Experiments were conducted under urethane anesthesia in normal (n = 9) and 6-hydroxydopamine hemi-lesioned PD rats (n = 7). Left-sided PAG stimulation and STN-DBS were applied with simultaneous bladder contraction monitoring. Local field potential (LFP) recording and collection of extracellular fluid in the mPFC were performed before stimulation, during PAG stimulation, during PAG+STN stimulation, and after stimulation. The bladder inter-contraction intervals significantly decreased with PAG stimulation with a concomitant decrease in mPFC LFP power in PD rats. Adding STN stimulation to PAG stimulation significantly increased the bladder inter-contraction intervals with a concomitant increase in mPFC LFP power in PD rats. Several mPFC catecholamine levels were modulated by PAG or PAG+STN stimulation in PD rats. The present study revealed that STN-DBS modulate the activities of mPFC induced by PAG, thereby leading to normalization of bladder contraction.

Prefrontal and hippocampal theta rhythm show anxiolytic-like changes during periaqueductal-elicited "panic" in rats.

Anxiety and panic are both elicited by threat and co‐occur clinically. But, at the neural level, anxiety appears to inhibit the generation of panic; and vice versa. Anxiety and panic are thought to engage more anterior (a) and mid‐posterior (m) parts of the periaqueductal gray (PAG), respectively. Anxiety also engages the hippocampus and medial prefrontal cortex. Here, we tested if mPAG but not aPAG stimulation would suppress prefrontal and hippocampal theta rhythm as do anxiolytic drugs. Twelve male rats with implanted electrodes were stimulated alternately (30 s interval) in the left PAG or right reticular formation (reticularis pontis oralis [RPO]—as a positive control) with recording in the left prelimbic cortex and left and right hippocampus. PAG stimulation was set to produce freezing and RPO to produce 7–8 Hz theta rhythm before tests lasting 10 min on each of 5 days. mPAG stimulation decreased, and aPAG increased, theta power at all sites during elicited freezing. mPAG, but not aPAG, stimulation decreased prefrontal theta frequency. Stimulation did not substantially change circuit dynamics (pairwise phase consistency and partial directed coherence). Together with previous reports, our data suggest that panic‐ and anxiety‐control systems are mutually inhibitory, and neural separation of anxiety and panic extends down to the aPAG and mPAG, respectively. Our findings are consistent with recent proposals that fear and anxiety are controlled by parallel neural hierarchies extending from PAG to the prefrontal cortex.

Molecular Pathways Responsible for Adaptive Resistance to Single-Checkpoint Inhibition May Reveal Biomarkers and Immunotherapy Targets in Multiple Myeloma Patients

Immune checkpoints blocking antibodies represent a promising strategy to harness anti-tumor immune responses and improve the clinical outcome of patients with hematological malignancies. Early studies based on single-agent PD-1 blockade have fallen short of clinical expectations in multiple myeloma patients. Thus, several strategies are under consideration to implement and optimize the clinical efficacy of immune checkpoint blockade in multiple myeloma patients. We have focused on Vγ9Vδ2 T cells because we believe that these cells are very sensitive tools to assess the immune competence of multiple myeloma (MM) patients in the tumor microenvironment. By interrogating the bone marrow (BM) Vγ9Vδ2 T-cell immune reactivity to phosphoantigens, we have revealed a very early and unexpected tumor microenvironment commitment to immune suppression which is already detectable in monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted in clinical remission after autologous stem cell transplantation. Multiple cell subsets (myeloma cells, myeloid-derived suppressor cells, regulatory T cells, bone marrow stromal cells) are involved in Vγ9Vδ2 T-cell inhibition via several immune suppressive mechanisms including the immune checkpoint circuity.We have shown that there is a multifaceted immune checkpoint expression in the tumor microenvironment which is dependent on the clinical stage and cannot be neutralized with single-agent PD-1 blockade. To test our hypothesis that adaptive resistance to PD-1 blockade in MM patients may be due to the compensatory up-regulation of other immune checkpoints, we analyzed the expression of other inhibitory receptors (TIM-3, LAG-3, TIGIT and CTLA-4) in PB and BM Vg9Vd2 T cells from healthy donors and MM patients and observed that also TIM-3 beyond PD-1 is significantly upregulated in BM Vγ9Vδ2 T cells from MM patients at diagnosis. Unlike healthy immune cells, the attempt to activate functionally exhausted immune cells can further compromise their anti-tumor functions. pAg stimulation of PD-1+ and TIM-3+ BM Vγ9Vδ2 T cells further increases their inhibitory receptor expression. Interestingly, TIM-3 up-regulation is even more pronounced than PD-1 up-regulation in BM Vγ9Vδ2 T cells and it is specular to Vγ9Vδ2 T -cell proliferation, confirming that any inappropriate attempt to activate these cells leads further worsen their immune competence status. Our observations about the compensatory up-regulation of multiple inhibitory receptors (PD-1 and TIM-3) on BM Vγ9Vδ2 T cells and the further up-regulation of TIM-3 after anti-PD1 treatment suggested us to assess the combination of both immune checkpoint inhibitors (anti-PD1 and anti-TIM3) to overcome the PD-1 single blocking resistance and recover anti-myeloma reactivity. TIM-3 blockade alone was able to partially recover pAg-induced Vγ9Vδ2 T-cell proliferation but the best recovery was obtained when pAg stimulation is carried out in the presence of concurrent PD-1 and TIM-3 blockade. To investigate the molecular mechanisms behind the up-regulation of multiple immune checkpoints responsible for PD-1-mediated hyporesponsiveness and the effector cells exhaustion, we analyzed the main PD-1-mediated (PI3K/AKT) and TIM-3-mediated (JAK/STAT) signal transduction pathways. After pAg stimulation, purified Vγ9Vδ2 T cells from peripheral blood (PB) CTRL, PB MM and BM MM has been tested by western blot analysis for the expression of several intracellular proteins: Ras, phospho-ERK1/2, ERK1/2, phospho-Akt, Akt, phospho- JAK1, JAK1, phospho-STAT1, STAT1, phospho-STAT3, STAT3. Vγ9Vδ2 T cells isolated from BM MM and PB MM showed a remarkable down-modulation of pAKT, a key player of PD-1-mediated signal transduction, and pJAK-1 and pSTAT1, both involved in TIM-3-mediated signaling pathway. Morever, we observed a significantly reduced expression of the transcription factor T-bet, the downstream target gene of pSTAT1, selectively in Vγ9Vδ2 T cells isolated from the tumor site, suggesting that low T-bet expression is a further hallmark of BM Vγ9Vδ2 T cells exhaustion.Altogether, our findings allow to identify potential molecular mechanisms responsible for the poor efficacy of single immune checkpoint blockade in the tumor microenvironment and to design rational combination therapeutic strategy to be applied to the different clinical settings. DisclosuresBoccadoro:Celgene: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Massaia:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Novartis: Research Funding.

Serotonergic treatment normalizes midbrain dopaminergic neuron increase after periaqueductal gray stimulation.

Electrical stimulation of the dorsolateral periaqueductal gray (dlPAG) in rats has been shown to elicit panic-like behaviour and can be a useful as an unconditioned stimulus for modelling anticipatory fear and agoraphobia in a contextual fear conditioning paradigm. In this study, we further analysed our previous data on the effects of escitalopram (a selective serotonin reuptake inhibitor, SSRI) and buspirone (a 5-HT1A receptor partial agonist) on dlPAG-induced anticipatory fear behaviour in a rat model using freezing as a measure. We then attempted to unravel some of the interactions with dopamine signalling using tyrosine hydroxylase (TH) immunohistochemistry to probe the effects on dopaminergic neurons. We showed that acute treatment of escitalopram, but not buspirone, was effective in reducing anticipatory freezing behaviour, while chronic administrations of both drugs were effective. We found that the dlPAG stimulation induced increase number of dopaminergic neurons in the ventral tegmental area (VTA) which was reversed in both chronic buspirone and escitalopram groups. We further found a strong positive correlation between the number of dopaminergic neurons and freezing in the VTA and showed positive correlations between dopaminergic neurons in the VTA and substantia nigra pars compacta (SNpc) in escitalopram and buspirone groups, respectively. Overall, we showed that chronic treatment with an SSRI and a 5-HT1A agonist reduced anticipatory freezing behaviour which seems to be associated, through correlative studies, with a reversal of dlPAG stimulation induced increase in number of dopaminergic neurons in the VTA and/or SNpc.

Central Nervous System Electrical Stimulation for Neuroprotection in Acute Cerebral Ischemia: Meta-Analysis of Preclinical Studies.

Background and Purpose- Brain electrical stimulation, widely studied to facilitate recovery from stroke, has also been reported to confer direct neuroprotection in preclinical models of acute cerebral ischemia. Systematic review of controlled preclinical acute cerebral ischemia studies would aid in planning for initial human clinical trials. Methods- A systematic Medline search identified controlled, preclinical studies of central nervous system electrical stimulation in acute cerebral ischemia. Studies were categorized among 6 stimulation strategies. Three strategies applied different stimulation types to tissues within the ischemic zone (cathodal hemispheric stimulation [CHS], anodal hemispheric stimulation, and pulsed hemispheric stimulation), and 3 strategies applied deep brain stimulation to different neuronal targets remote from the ischemic zone (fastigial nucleus stimulation, subthalamic vasodilator area stimulation, and dorsal periaqueductal gray stimulation). Random-effects meta-analysis assessed electrical stimulation modification of final infarct volume. Study-level risk of bias and intervention-level readiness-for-translation were assessed using formal rating scales. Results- Systematic search identified 28 experiments in 21 studies, including a total of 350 animals, of electrical stimulation in preclinical acute cerebral ischemia. Overall, in animals undergoing electrical stimulation, final infarct volumes were reduced by 37% (95% CI, 34%-40%; P<0.001), compared with control. There was evidence of heterogeneity of efficacy among stimulation strategies (I2=93.1%, Pheterogeneity<0.001). Among the within-ischemic zone stimulation strategies, only CHS significantly reduced the infarct volume (27 %; 95% CI, 22%-33%; P<0.001); among the remote-from ischemic zone approaches, all (fastigial nucleus stimulation, subthalamic vasodilator area stimulation, and dorsal periaqueductal gray stimulation) reduced infarct volumes by approximately half. On formal rating scales, CHS studies had the lowest risk of bias, and CHS had the highest overall quality of intervention-level evidence supporting readiness to proceed to clinical testing. Conclusions- Electrical stimulation reduces final infarct volume across preclinical studies. CHS shows the most robust evidence and is potentially appropriate for progression to early-stage human clinical trial testing as a promising neuroprotective intervention.

Vγ9Vδ2 T Cells as Strategic Weapons to Improve the Potency of Immune Checkpoint Blockade and Immune Interventions in Human Myeloma.

The advent of immune checkpoint (ICP) blockade has introduced an unprecedented paradigm shift in the treatment of cancer. Though very promising, there is still a substantial proportion of patients who do not respond or develop resistance to ICP blockade. In vitro and in vivo models are eagerly needed to identify mechanisms to maximize the immune potency of ICP blockade and overcome primary and acquired resistance to ICP blockade. Vγ9Vδ2 T cells isolated from the bone marrow (BM) from multiple myeloma (MM) are excellent tools to investigate the mechanisms of resistance to PD-1 blockade and to decipher the network of mutual interactions between PD-1 and the immune suppressive tumor microenvironment (TME). Vγ9Vδ2 T cells can easily be interrogated to dissect the progressive immune competence impairment generated in the TME by the long-lasting exposure to myeloma cellss. BM MM Vγ9Vδ2 T cells are PD-1+ and anergic to phosphoantigen (pAg) stimulation; notably, single agent PD-1 blockade is insufficient to fully recover their anti-tumor activity in vitro indicating that additional players are involved in the anergy of Vγ9Vδ2 T cells. In this mini-review we will discuss the value of Vγ9Vδ2 T cells as investigational tools to improve the potency of ICP blockade and immune interventions in MM.

Deficient post-ictal cardiorespiratory compensatory mechanisms mediated by the periaqueductal gray may lead to death in a mouse model of SUDEP

Post-ictal cardiorespiratory failure is implicated as a major cause of sudden unexpected death in epilepsy (SUDEP) in patients. The DBA/1 mouse model of SUDEP is abnormally susceptible to fatal seizure-induced cardiorespiratory failure (S-CRF) induced by convulsant drug, hyperthermia, electroshock, and acoustic stimulation. Clinical and pre-clinical studies have implicated periaqueductal gray (PAG) abnormalities in SUDEP. Recent functional neuroimaging studies observed that S-CRF resulted in selective changes in PAG neuronal activity in DBA/1 mice. The PAG plays a critical compensatory role for respiratory distress caused by numerous physiological challenges in non-epileptic individuals. These observations suggest that abnormalities in PAG-mediated cardiorespiratory modulation may contribute to S-CRF in DBA/1 mice. To evaluate this, electrical stimulation (20 Hz, 20–100 μA, 10 s) was presented in the PAG of anesthetized DBA/1 and C57BL/6 (non-epileptic) control mice, and post-stimulus changes in respiration [inter-breath interval (IBI)] and heart rate variability (HRV) were examined. The post-stimulus period was considered analogous to the post-ictal period when S-CRF occurred in previous DBA/1 mouse studies. PAG stimulation caused significant intensity-related decreases in IBI in both mouse strains. However, this effect was significantly reduced in DBA/1 vis-a-vis C57BL/6 mice. These changes began immediately following cessation of stimulation and remained significant for 10 s. This time period is critical for initiating resuscitation to successfully prevent seizure-induced death in previous DBA/1 mouse experiments. Significant post-stimulus increases in HRV were also seen at ≥60 μA in the PAG in C57BL/6 mice, which were absent in DBA/1 mice. These data along with previous neuroimaging findings suggest that compensatory cardiorespiratory modulation mediated by PAG is deficient, which may be important to the susceptibility of DBA/1 mice to S-CRF. These observations suggest that correcting this deficit pharmacologically or by electrical stimulation may help to prevent S-CRF. These findings further support the potential importance of PAG abnormalities to human SUDEP.

Daily maternal separations during stress hyporesponsive period decrease the thresholds of panic-like behaviors to electrical stimulation of the dorsal periaqueductal gray of the adult rat

The present study examined whether early life maternal separation (MS), a model of childhood separation anxiety, predisposes to panic at adulthood. For this purpose, male pups were submitted to 3-h daily maternal separations along postnatal (PN) days of either the ‘stress hyporesponsive period’ (SHRP) from PN4 to PN14 (MS11) or throughout lactation from PN2 to PN21 (MS20). Pups were further reunited to conscious (CM) or anesthetized (AM) mothers to assess the effect of mother-pup interaction upon reunion. Controls were subjected to brief handling (15 s) once a day throughout lactation (BH20). As adults (PN60), rats were tested for the thresholds to evoke panic-like behaviors upon electrical stimulation of dorsal periaqueductal gray matter and exposed to an elevated plus-maze, an open-field, a forced swim and a sucrose preference test. A factor analysis was also performed to gain insight into the meaning of behavioral tests. MS11-CM rather than MS20-CM rats showed enhanced panic responses and reductions in both swimming and sucrose preference. Panic facilitations were less intense in mother-neglected rats. Although MS did not affect anxiety, MS11-AM showed robust reductions of defecation in an open-field. Factor analysis singled out anxiety, hedonia, exploration, coping and gut activity. Although sucrose preference and coping loaded on separate factors, appetite (adult weight) correlated with active coping in both forced swim and open-field (central area exploration). Concluding, whereas 3h-daily maternal separations during SHRP increased rat’s susceptibility to experimental panic attacks, separations throughout lactation had no effects on panic and enhanced active coping.

Some Autonomic Deficits of Acute or Chronic Cervical Spinal Contusion Reversed by Interim Brainstem Stimulation.

Prolonged electrical stimulation of the hindbrain's nucleus raphe magnus (NRM) or of its major midbrain input region, the periaqueductal gray (PAG), was previously found in rats to promote recovery from sensory-motor and histological deficits of acute thoracic spinal cord injury (SCI). Here, some visceral deficits of acute and chronic midline cervical (C5) contusion are similarly examined. Cranially implanted wireless stimulators delivered intermittent 8 Hz, 30-70 μA cathodal pulse trains to a brainstem microelectrode. Injured controls were given inactive stimulators; rats without injuries or implants were also compared. Rectal distension or squeezing of the forepaws caused an exaggerated rise in mean arterial pressure in injured, untreated rats under anesthesia on post-injury week 6, probably reflecting autonomic dysreflexia (AD). These pressor responses became normal when 7 days of unilateral PAG stimulation was started on the injury day. Older untreated injuries (weeks 18-19) showed normal pressor responses, but unexpectedly had significant resting and nociceptive bradycardia, which was reversed by 3 weeks of PAG stimulation started on weeks 7 or 12. Subsequent chronic studies examined gastric emptying (GE), as indicated by intestinal transit of gavaged dye, and serum chemistry. GE and fasting serum insulin were reduced on injury weeks 14-15, and were both normalized by ∼5 weeks of PAG stimulation begun in weeks 7-8. Increases in calcitonin gene-related peptide, a prominent visceral afferent neurotransmitter, measured near untreated injuries (first thoracic segment) in superficial dorsal laminae were reversed by acutely or chronically initiated PAG stimulation. The NRM, given 2-3 weeks of stimulation beginning 2 days after SCI, prevented abnormalities in both pressor responses and GE on post-injury week 9, consistent with its relaying of repair commands from the PAG. The descending PAG-NRM axis thus exhibits broadly restorative influences on visceral as well as sensory-motor deficits, improving chronic as well as acute signs of injury.

Modulation of Synaptic Reactions of the “Nociceptive” Neurons in the Cat Cortex upon Stimulation of the Periaqueductal Gray and Application of Some Pharmacological Agents

We examined the effects of electrical stimulation of the periaqueductal gray (PAG) and systemic morphine injections on postsynaptic processes in neurons of the cat somatosensory cortex, which were activated by nociceptive influences. Stimulation of the PAG resulted in long-lasting suppression of synaptic reactions induced by stimulation of dental pulp nociceptors. There was certain parallelism within the conditioning effects of PAG stimulation and effects of systemic introduction of morphine. Ionophoretic application of strychnine on such pyramidal cortical neurons did not evoke paroxysmal depolarization shifts (PDSh) of the membrane potential in such neurons. At the same time, surface application of this agent on the cortex (which influenced extensive populations of cortical neurons) resulted in the development of considerable PDShs. This observation confirms the synaptic nature of the above shifts. Applications of strychnine using both techniques resulted in the blockade of, first of all, early IPSP components in pyramidal neurons but did not affect significantly late components of these potentials; this is indicative of different geneses of the above components. It is supposed that the early IPSP component is generated due to activation of axo-somatic inhibitory synapses, while the late component of such reactions is related to activation of inhibitory synapses localized in the dendrites. The mechanisms of modulation of postsynaptic reactions of somatosensory cortex neurons developing upon activation of high-threshold (nociceptive) afferent inputs are discussed. Such modulation is probably based on changes in both pre- and post-synaptic intracortical cell structures.

Electroacupuncture Inhibits the Activation of p38MAPK in the Central Descending Facilitatory Pathway in Rats with Inflammatory Pain.

The mitogen-activated protein kinases (MAPKs), especially p38MAPK, play a pivotal role in chronic pain. Electroacupuncture (EA) relieves inflammatory pain underlying the descending pathway, that is, the periaqueductal gray (PAG), the rostral ventromedial medulla (RVM), and the spinal cord dorsal horn (SCDH). However, whether EA antagonizes inflammatory pain through regulation of p38MAPK in this descending facilitatory pathway is unclear. Complete Freund's adjuvant (CFA) was injected into the hind paw of rats to establish inflammatory pain model. EA was administrated for 30 min at Zusanli and Kunlun acupoints at 0.5, 24.5, 48.5, and 72.5 h, respectively. The paw withdrawal threshold (PWT), paw edema, and Phosphor-p38MAPK-Immunoreactivity (p-p38MAPK-IR) cells were measured before (0 h) and at 1, 3, 5, 7, 25, and 73 h after CFA or saline injection. EA increased PWT at 1, 3, 25, and 73 h and inhibited paw edema at 25 and 73 h after CFA injection. Moreover, the increasing number of p-p38MAPK-IR cells which was induced by CFA was suppressed by EA stimulation in PAG and RVM at 3 and 5 h and in SCDH at 5, 7, 25, and 73 h. These results suggest that EA suppresses inflammation-induced hyperalgesia probably through inhibiting p38MAPK activation in the descending facilitatory pathway.

Immune Checkpoint Blockade Combinations As Promising Strategy for Cancer Immunotherapy in Multiple Myeloma Patients

Tumor cells exploit immune checkpoints to withstand immune recognition and effector cells’ onslaught. Pre-clinical findings, corroborated by initial results of clinical studies, indicate that immune checkpoint blockade is a promising strategy to harness anti-tumor immune responses and improve the clinical outcome of patients with hematological malignancies. Multiple myeloma (MM) is a prototypic disease in which immune checkpoints significantly contribute to the immune suppressive contexture that myeloma cells establish in the bone marrow (BM) in cooperation with regulatory T cells (Tregs), myeloid derived suppressor cells (MDSC), and BM stromal cells (BMSC). Vγ9Vδ2 T cells are among the immune effector cells strategically victimized by suppressor cells. These are non-conventional T cells halfway between innate and adaptive immunity with a natural inclination to react against malignant B cells, including myeloma cells. Vγ9Vδ2 T cells are equipped with a peculiar array of receptors for stress-induced self-ligands and a unique TCR-dependent recognition ability of phosphoantigens (pAgs) generated in the mevalonate (Mev) pathway. Recently, we have shown that BM Vγ9Vδ2 T cells are anergic to pAg stimulation and that the programmed death 1(PD-1)/programmed death ligand 1 (PD-L1) immune checkpoint pair contributes to their dysfunction. This is an early event already detectable in individuals with monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted even when MM patients achieve clinical remission after autologous stem cell transplantation (auto-SCT). Anti-PD-1 treatment partially recovers the ability of BM Vγ9Vδ2 T cells to proliferate and exert cytotoxic activity after pAg stimulation, but early studies based on single-agent PD-1 blockade have fallen short of clinical expectations in MM. Thus, several strategies are under consideration to implement the clinical efficacy of immune checkpoint blockade like the association with lenalidomide and/or concurrent tumor vaccination.Our results indicate that TIM-3 is significantly upregulated in BM Vγ9Vδ2 T cells from MM patients at diagnosis. We have previously shown that pAg stimulation of PD-1+ BM Vγ9Vδ2 T cells further increase PD-1 expression and preliminary data suggest that this stimulation also increases TIM-3 expression. Interestingly, TIM-3 up-regulation is even more pronounced than PD-1 up-regulation in BM Vγ9Vδ2 T cells and it occurs also in peripheral blood (PB) Vγ9Vδ2 T cells from anergic MM patients. We have recently shown that pAg reactivity of BM Vγ9Vδ2 T cells from MM at diagnosis can be partially recovered by PD-1 blockade. Our results reveal that TIM-3 blockade is also able to partially recover pAg-induced Vγ9Vδ2 T-cell proliferation. The best recovery is obtained when pAg stimulation is carried out in the presence of concurrent PD-1 and TIM-3 blockade. BM Vγ9Vδ2 T cells from MM patients who are in remission (MM-rem) after auto-SCT are still PD1+ and anergic to pAg stimulation. Remarkably, percentages of PD-L1+ MDSC in the BM of MM-rem are also unchanged compared to MM at diagnosis (MM-dia) and MM in relapse (MM-rel). These data indicate that the immune suppressive contexture is still operative at the tumor site even when most of myeloma cells have been cleared by chemotherapy. Interestingly, chemoresistant residual myeloma cells after auto-SCT have been reported to be PD-L1+, and circulating exhausted PD-1+ CD8+ T cells have been described in the PB after auto-SCT. This may explain why our previous idiotype vaccination studies in MM patients have failed.We have initiated to investigate the effect of immune checkpoint blockade in different phases of the disease and preliminary results suggest that the functional outcome of PD-1 blockade can be very different according to the disease status: the most signifcant recovery of Vγ9Vδ2 T-cell proliferation is observed after PD-1 blockade in MM-rem, while the anergy of Vγ9Vδ2 T cells from MM-rel is totally refractory to immune checkpoint blockade.In conclusion, our results suggest that recovery of pAg reactivity by PB Vγ9Vδ2 T cells is a reliable biomarker to predict or assess the clinical efficacy of immune checkpoint in vivo and provide scientific groundwork to optimize anti-PD1 treatment as single agent or in combination with other antibodies (i.e, anti-TIM-3) to maximize the efficacy of immune checkpoint blockade according to the disease status. DisclosuresBoccadoro:Amgen: Honoraria, Research Funding; SANOFI: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Mundipharma: Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; Abbivie: Honoraria. Massaia:Roche: Other: advisory board, research support; Janssen: Other: advisory board; Gilead: Other: advisory board.

Deep brain stimulation of the periaqueductal gray releases endogenous opioids in humans

Deep brain stimulation (DBS) of the periaqueductal gray (PAG) is used in the treatment of severe refractory neuropathic pain. We tested the hypothesis that DBS releases endogenous opioids to exert its analgesic effect using [11C]diprenorphine (DPN) positron emission tomography (PET). Patients with de-afferentation pain (phantom limb pain or Anaesthesia Dolorosa (n=5)) who obtained long-lasting analgesic benefit from DBS were recruited. [11C]DPN and [15O]water PET scanning was performed in consecutive sessions; first without, and then with PAG stimulation. The regional cerebral tracer distribution and kinetics were quantified for the whole brain and brainstem. Analysis was performed on a voxel-wise basis using statistical parametric mapping (SPM) and also within brainstem regions of interest and correlated to the DBS-induced improvement in pain score and mood. Brain-wide analysis identified a single cluster of reduced [11C]DPN binding (15.5% reduction) in the caudal, dorsal PAG following DBS from effective electrodes located in rostral dorsal/lateral PAG. There was no evidence for an accompanying focal change in blood flow within the PAG. No correlation was found between the change in PAG [11C]DPN binding and the analgesic effect or the effect on mood (POMSSV) of DBS. The analgesic effect of DBS in these subjects was not altered by systemic administration of the opioid antagonist naloxone (400ug). These findings indicate that DBS of the PAG does indeed release endogenous opioid peptides focally within the midbrain of these neuropathic pain patients but we are unable to further resolve the question of whether this release is responsible for the observed analgesic benefit.

Characterising the Analgesic Effect of Different Targets for Deep Brain Stimulation in Trigeminal Anaesthesia Dolorosa

Background: Several deep brain stimulation (DBS) targets have been explored for the alleviation of trigeminal anaesthesia dolorosa. We aimed to characterise the analgesia produced from the periaqueductal grey (PAG) and centromedian-parafascicular (CmPf) nucleus using a within-subject design. Method: We report a case series of 3 subjects implanted with PAG and CmPf DBS systems for the treatment of anaesthesia dolorosa. At follow-up, testing of onset and offset times, magnitude, and thermal and mechanical sensitivity was performed. Results: The mean pain score of the cohort was acutely reduced by 56% (p < 0.05) with PAG and 67% (p < 0.01) with CmPf stimulation at mean time intervals of 38 and 16 min, respectively. The onset time was 12.5 min (p < 0.05) for PAG stimulation and 2.5 min (p < 0.01) for CmPf. The offset time was 2.5 min (p < 0.05) for PAG and 12.5 min (p < 0.01) for CmPf. The two targets were effective at different stimulation frequencies and were not antagonistic in effect. Conclusion: The mechanisms by which stimulation at these two targets produces analgesia are likely to be different. Certain pain qualities may respond more favourably to specific targets. Knowledge of onset and offset times for the targets can guide optimisation of stimulation settings. The use of more than one stimulation target may be beneficial and should be considered in anaesthesia dolorosa patients.

Connections from the rat dorsal column nuclei (DCN) to the periaqueductal gray matter (PAG)

Electrical stimulation of the dorsal columns (DCs; spinal cord stimulation; SCS) has been proposed to treat chronic neuropathic pain. SCS may activate a dual mechanism that would affect both the spinal cord and supraspinal levels. Stimulation of DCs or DC nuclei (DCN) in animals where neuropathic pain has been induced causes activation of brainstem centers including the periaqueductal gray (PAG), which is involved in the endogenous pain suppression system. Biotinylated dextran-amine (BDA) was iontophoretically injected into the DCN to analyze the ascending projection directed to the PAG. Separate injections into the gracile nucleus (GrN) and the cuneate nucleus (CunN) showed BDA-positive fibers terminating in different regions of the contralateral PAG. GrN-PAG afferents terminated in the caudal and middle portions of PAG-l, whereas CunN-PAG fibers terminated in the middle and rostral portions of PAG-l. Based on the DCN somatotopic map, the GrN sends information to the PAG from the contralateral hindlimb and the tail and the CunN from the contralateral forelimb, shoulder, neck and ear. This somatotopic organization is consistent with earlier electrophysiological and PAG stimulation studies. These fibers could form part of the DCs-brainstem-spinal cord loop, which may be involved in the inhibitory effects of SCS on neuropathic pain.

Anergic bone marrow Vγ9Vδ2 T cells as early and long-lasting markers of PD-1-targetable microenvironment-induced immune suppression in human myeloma

Vγ9Vδ2 T cells have a natural inclination to recognize malignant B cells in vitro via receptors for stress-induced self-ligands and TCR-dependent recognition of phosphoantigens (pAgs) generated in the mevalonate (Mev) pathway. This inclination is continuously challenged in vivo by the immune suppression operated by tumor cells. Multiple myeloma (MM) is a prototypic B-cell malignancy in which myeloma cells subvert the local microenvironment to reshape antitumor immune responses. In this study, we have investigated the immune competence of bone marrow (BM) Vγ9Vδ2 T cells in a large series of MM patients. We have found that the BM microenvironment significantly hampers the pAg-reactivity of BM Vγ9Vδ2 T cells, which become largely PD-1+ and are surrounded by PD-L1+ myeloma cells and increased numbers of PD-L1+ myeloid-derived suppressor cells (MDSC). Vγ9Vδ2 T-cell dysfunction is an early event that can be already detected in individuals with monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted even when MM patients achieve clinical remission. Anti-PD-1 treatment increases the cytotoxic potential of Vγ9Vδ2 T cells by almost 5-fold after pAg stimulation, and appears to be a promising strategy for effective immune interventions in MM.

Role of endocannabinoid signalling in the dorsolateral periaqueductal grey in the modulation of distinct panic-like responses.

Panic attacks, a major feature of panic disorder, can be modelled in rats by exposing animals to stimuli that induce escape reactions, such as the elevated T-maze or the activation of the dorsolateral periaqueductal grey. Since the cannabinoid CB1 receptor modulates various types of aversive responses, this study tested the hypothesis that enhancement of endocannabinoid signalling in the dorsolateral periaqueductal grey inhibits panic-like reactions in rats. Local injection of the CB1 agonist, arachidonoyl 2-Chloroethylamide (0.005-0.5 pmol), attenuated the escape response from the open arm of the elevated T-maze, a panicolytic effect. The anandamide hydrolysis inhibitor, URB597 (0.3-3 nmol), did not induce consistent results. In the test of dorsolateral periaqueductal grey stimulation with d,l-homocysteic acid, arachidonoyl 2-Chloroethylamide, at the lowest dose, attenuated the escape reaction. The highest dose of URB597 also inhibited this response, contrary to the result obtained in the elevated T-maze. This effect was reversed by the CB1 antagonist, AM251 (100 pmol). The present results confirm the anti-aversive property of direct CB1 receptor activation in the dorsolateral periaqueductal grey. The effect of the anandamide hydrolysis inhibitor, however, could be detected only in a model employing direct stimulation of this structure. Altogether, these results suggest that anandamide signalling is recruited only under certain types of aversive stimuli.

In a rat model of panic, corticotropin responses to dorsal periaqueductal gray stimulation depend on physical exertion

Panic disorder patients are exquisitely and specifically sensitive to hypercapnia. The demonstration that carbon dioxide provokes panic in fear-unresponsive amygdala-calcified Urbach-Wiethe patients emphasizes that panic is not fear nor does it require the activation of the amygdala. This is consonant with increasing evidence suggesting that panic is mediated caudally at midbrain's dorsal periaqueductal gray matter (DPAG). Another startling feature of the apparently spontaneous clinical panic is the counterintuitive lack of increments in corticotropin, cortisol and prolactin, generally considered 'stress hormones'. Here we show that the stress hormones are not changed during DPAG-evoked panic when escape is prevented by stimulating the rat in a small compartment. Neither did the corticotropin increase when physical exertion was statistically adjusted to the same degree as non-stimulated controls, as measured by lactate plasma levels. Conversely, neuroendocrine responses to foot-shocks were independent from muscular effort. Data are consonant with DPAG mediation of panic attacks.