A Midbrain Circuit that Mediates Headache Aversiveness in Rats.

Migraines 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.

The Lateral Hypothalamic and BNST GABAergic Projections to the Anterior Ventrolateral Periaqueductal Gray Regulate Feeding.

Overeating is a serious issue in modern society, causing many health problems, including obesity. Although the hypothalamus has been previously identified as the key brain structure that regulates body weight homeostasis, the downstream pathways and non-canonical neural circuitry involved in feeding behavior remain largely uncharacterized. Here, we discover that suppressing the activity of GABAergic cells in the anterior ventrolateral periaqueductal gray (vlPAG), whether directly or through long-projection GABAergic inputs from either the bed nucleus of the stria terminalis (BNST) or the lateral hypothalamus (LH), is sufficient to promptly induce feeding behavior in well-fed mice. In contrast, optogenetic activation of these cells interrupts food intake in starved mice. Long-term chemogenetic manipulation of vlPAG GABAergic cell activity elicits a corresponding change in mouse body weight. Our studies reveal distinct midbrain GABAergic pathways and highlight an important role of GABAergic cells in the anterior vlPAG in feeding behavior.

Optogenetic Modulation of Locomotor Activity on Free-Behaving Rats.

The technology of optogenetics provides a new method to modulate neural activity with spatial specificity and millisecond-temporal scale. This nonelectrical modulation method also gives chance for simultaneous electrophysiological recording during stimulations. Here, we describe our locomotor activity modulation on free-behaving rats using optogenetic techniques. The target sites of the rat brain were dorsal periaqueductal gray (dPAG) and ventral tegmental area (VTA) for the modulation of defensive and reward behaviors, respectively.

Cellular circadian clocks in mood disorders.

Bipolar disorder (BD) and major depressive disorder (MDD) are heritable neuropsychiatric disorders associated with disrupted circadian rhythms. The hypothesis that circadian clock dysfunction plays a causal role in these disorders has endured for decades but has been difficult to test and remains controversial. In the meantime, the discovery of clock genes and cellular clocks has revolutionized our understanding of circadian timing. Cellular circadian clocks are located in the suprachiasmatic nucleus (SCN), the brain's primary circadian pacemaker, but also throughout the brain and peripheral tissues. In BD and MDD patients, defects have been found in SCN-dependent rhythms of body temperature and melatonin release. However, these are imperfect and indirect indicators of SCN function. Moreover, the SCN may not be particularly relevant to mood regulation, whereas the lateral habenula, ventral tegmentum, and hippocampus, which also contain cellular clocks, have established roles in this regard. Dysfunction in these non-SCN clocks could contribute directly to the pathophysiology of BD/MDD. We hypothesize that circadian clock dysfunction in non-SCN clocks is a trait marker of mood disorders, encoded by pathological genetic variants. Because network features of the SCN render it uniquely resistant to perturbation, previous studies of SCN outputs in mood disorders patients may have failed to detect genetic defects affecting non-SCN clocks, which include not only mood-regulating neurons in the brain but also peripheral cells accessible in human subjects. Therefore, reporters of rhythmic clock gene expression in cells from patients or mouse models could provide a direct assay of the molecular gears of the clock, in cellular clocks that are likely to be more representative than the SCN of mood-regulating neurons in patients. This approach, informed by the new insights and tools of modern chronobiology, will allow a more definitive test of the role of cellular circadian clocks in mood disorders.

Photobiomodulation enhances nigral dopaminergic cell survival in a chronic MPTP mouse model of Parkinson's disease.

We have shown previously that photobiomodulation or near-infrared light (NIr) treatment protects dopaminergic cells of the substantia nigra pars compacta (SNc) in an acute MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease (PD). In this study, we tested the protective and rescue action of NIr treatment in a chronic MPTP model, developed to resemble more closely the slow progressive degeneration in PD patients. We examined three regions of dopaminergic cells, the SNc, periaqueductal grey matter (PaG) and zona incerta-hypothalamus (ZI-Hyp). BALB/c mice had MPTP or saline injections over five weeks, followed by a three-week survival. NIr treatment was applied either at the same time as (simultaneous series) or after (post-treatment series) the MPTP insult. There were four groups within each series; Saline, Saline-NIr, MPTP and MPTP-NIr. Brains were processed for tyrosine hydroxylase (TH) immunochemistry and cell number was analysed using the optical fractionator method. In the SNc, there was a significant reduction (≈ 45%) in TH(+) cell number in the MPTP groups compared to the saline controls of both series. In the MPTP-NIr groups of both series, TH(+) cell number was significantly higher (≈ 25%) than in the MPTP groups, but lower than in the saline controls (≈ 20%). By contrast in the PaG and ZI-Hyp, there were no significant differences in TH(+) cell number between the MPTP an MPTP-NIr groups of either series. In summary, exposure to NIr either at the same time or well after chronic MPTP insult saved many SNc dopaminergic cells from degeneration.

Deep brain stimulation for neuropathic pain.

Deep brain stimulation (DBS) for pain was one of the earliest indications for the therapy. This study reports the outcome of DBS of the sensory thalamus and the periventricular and peri-aqueductal grey area (PVG/PAG) complex for different intractable neuropathic pain syndromes. Forty-seven patients (30 males and 17 females) were selected for surgery; they were suffering from any of the following types of pain: post-stroke neuropathic pain, phantom limb pain, post-herpetic neuralgia, anaesthesia dolorosa, brachial plexus injury and neuropathic pain secondary to neural damage from a variety of causes. Of the 47 patients selected for trial stimulation, 38 patients proceeded to permanent implantation. Patients suffering from post-stroke pain were the most likely to fail trial stimulation (33%), in contrast to individuals with phantom limb/post-brachial plexus injury pain and anaesthesia dolorosa, all of whom underwent permanent implantation. PVG stimulation alone was optimal in 17 patients (53%), whilst a combination of PVG and thalamic stimulation produced the greatest degree of analgesia in 11 patients (34%). Thalamic stimulation alone was optimal in 4 patients (13%). DBS of the PVG alone was associated with the highest degree of pain alleviation, with a mean improvement of 59% (p <0.001) and a > or =50% improvement in 66% of patients. Post-stroke pain responds in 70% of patients. We conclude that the outcomes of surgery appear to vary according to aetiology, but it would appear that the effects are best for phantom limb syndromes, head pain and anaesthesia dolorosa.

The periaqueductal grey area and the cardiovascular system.

In this chapter, we report that blood pressure can be increased or decreased depending on whether an electrode is in ventral or dorsal PAG. We also describe that it is theoretically possible to treat orthostatic hypotension. These are exciting developments not only because they provide an example of direct translational research from animal research to humans but also because they highlight a potential for future clinical therapies. The control of essential hypertension without drugs is attractive because of the side effects of medication such as precipitation of heart failure [10]. Similarly, drug treatment of orthostatic hypotension cannot differentiate between the supine and standing positions and can therefore lead to nocturnal hypertension [22, 29]. A stimulator could be turned off at night or contain a mercury switch that reacts to posture.

Midbrain control of spinal nociception discriminates between responses evoked by myelinated and unmyelinated heat nociceptors in the rat.

Descending control of spinal nociception is a major determinant of normal and chronic pain. Myelinated (A-fibre) and unmyelinated (C-fibre) nociceptors convey different qualities of the pain signal (first and second pain, respectively), and they play different roles in the development and maintenance of chronic pain states. It is of considerable importance, therefore, to determine whether descending control has differential effects on the central processing of A- vs. C-nociceptive input. In anaesthetised rats, biceps femoris EMG was recorded to monitor the thresholds and encoding properties of responses evoked by fast (7.5 degrees Cs(-1)) or slow (2.5 degrees Cs(-1)) rates of skin heating of the dorsal surface of a hindpaw to preferentially activate myelinated or unmyelinated heat nociceptors, respectively. Activation of neurones in the periaqueductal grey (PAG) by microinjection of dl-homocysteic acid (DLH) or bicuculline (BIC) significantly increased response thresholds to slow rates of heating (P<0.001), but not those to fast rates of heating (P>0.05). The ability of the EMG to encode the stimulus intensity of fast rates of skin heating remained intact and unaltered (r2=0.99, P<0.001) following BIC but not DLH injection. In contrast, encoding of the stimulus intensity of slow rates of skin heating was abolished following BIC and DLH injection. The functional significance of differential descending control of the central processing of C- and A-nociceptive inputs is discussed with respect to role of the PAG in mediating antinociception as part of active coping strategies in emergency situations and the role of C- and A-nociceptive inputs in animal models of chronic pain.

Prior electrical stimulation of dorsal periaqueductal grey matter or deep layers of the superior colliculus sensitizes rats to anxiety-like behaviors in the elevated T-maze test.

Electrical stimulation of the dorsal periaqueductal grey matter (DPAG) and deep layers of the superior colliculus (DLSC) of the rat elicits anxiety-like reactions such as freezing and flight. The temporal course of the effects of the aversive electrical stimulation of the DPAG (5, 15 and 30 min afterward) and DLSC (5, 10 and 15 min afterward) on the defensive response of rats exposed to elevated T-maze were determined. The elevated T-maze generates two defensive behaviors, inhibitory avoidance and one-way escape, which have been related, respectively, to generalized anxiety and panic disorders. Prior electrical stimulation of the DPAG (15 min) and DLSC (5 min) enhanced inhibitory avoidance when compared to no-operated and sham animals, although not affecting escape. Therefore, stimulation of the DPAG and DLSC causes a heightened responsivity to anxiogenic stimulus, but not to panicogenic stimulus, inherent to elevated T-maze. These findings support the participation of the DPAG and DLSC in the elaboration of adaptive responses to stressful situations. Besides, the data supports the view that prior electrical stimulation of DPAG and DLSC is selective in sensitizing rats to anxiety-like behaviors, but not to panic-like behaviors in the elevated T-maze test.

Pharmacological assessment of the freezing, antinociception, and exploratory behavior organized in the ventrolateral periaqueductal gray.

Opioid and serotonergic mechanisms of the ventrolateral periaqueductal gray (vlPAG) are recruited by conditioned freezing and antinociception. However, it is unclear whether freezing and antinociception induced by stimulation of the vlPAG are interrelated. To address this issue we looked at the effects of the opioid antagonist naltrexone, the 5-HT2 antagonist ketanserin, and the benzodiazepine agonist midazolam injected into the vlPAG on the freezing and antinociception induced by electrical stimulation of this region. This antinociception was evaluated by the tail-flick and formalin tests. To further characterize the involvement of the vlPAG in unconditioned fear, the effects of intra-vlPAG injections of midazolam on the exploratory behavior were also assessed in independent groups of rats submitted to the elevated plus-maze test (EPM). The data obtained showed that: (i) electrical stimulation of the vlPAG causes freezing blocked by midazolam but not by naltrexone and ketanserin; (ii) antinociception generated at the level of the vlPAG is inhibited by naltrexone, ketanserin, and midazolam; (iii) activation of benzodiazepine-mediated mechanisms in the vlPAG increased the exploratory behavior of rats in the closed arms but not the avoidance behavior of open arms of the EPM. Thus, freezing and antinociception generated in the vlPAG are dissociated pharmacologically. Whereas antinociception is a multimediated process sensitive to naltrexone, ketanserin, and midazolam, the freezing induced by vlPAG stimulation was reversed only by the benzodiazepine compound. As injections of midazolam into the vlPAG do not cause anxiolytic effects in the EPM, the aversive stimuli inherent of this test seem to bypass the vlPAG.

Projections of the ventrolateral pontine vocalization area in the squirrel monkey.

In four squirrel monkeys (Saimiri sciureus), the tracer biotin dextranamine (BDA) was injected into the ventrolateral pons at a site at which injection of the glutamate antagonist kynurenic acid blocked vocalization electrically elicited from the periaqueductal gray (PAG). Anterograde projections could be traced into all cranial motor and sensory nuclei involved in phonation, that is, the nucleus ambiguus, facial, hypoglossal and trigeminal motor nuclei, the motorneuron column in the ventral gray substance innervating the extrinsic laryngeal muscles, the nucleus retroambiguus, solitary tract and spinal trigeminal nuclei. Projections were also found into a number of auditory nuclei, namely the nucleus cochlearis-complex, superior olive, ventral and dorsal nuclei of the lateral lemniscus and inferior colliculus. Furthermore, there were projections into the reticular formation of the lateral and dorsocaudal medulla and lateral pons, into nucleus gracilis, inferior and medial vestibular nuclei, lateral reticular nucleus, ventral raphe, pontine gray, superior colliculus, PAG and mediodorsal thalamic nucleus. Injection of the tracer wheat germ agglutinin-conjugated horseradish peroxidase into the ventrolateral pontine vocalization-blocking area in one animal yielded retrograde labeling throughout the PAG. Injection of BDA into a vocalization-eliciting site of the PAG in another animal yielded projections into the ventrolateral pontine vocalization-blocking area. It is concluded that the ventral paralemniscal area in the ventrolateral pons represents a relay station of the descending periaqueductal vocalization-controlling pathway.

Deep brain stimulation can regulate arterial blood pressure in awake humans.

The periaqueductal grey matter is known to play a role in cardiovascular control in animals. Cardiovascular responses to electrical stimulation of the periventricular/periaqueductal grey matter were measured in 15 awake human study participants following implantation of deep brain stimulating electrodes for treatment of chronic pain. We found that stimulation of the ventral periventricular/periaqueductal grey matter caused a mean reduction in systolic blood pressure of 14.2+/-3.6 mmHg in seven patients and stimulation of the dorsal periventricular/periaqueductal grey matter caused a mean increase of 16.7+/-5.9 mmHg in six patients. A comparison between ventral and dorsal electrodes demonstrated significant differences (P<0.05). These changes were accompanied by analogous changes in diastolic blood pressure, pulse pressure, maximum dP/dt but not in the time interval between each R wave on the electrocardiogram.

Deep brain stimulation for phantom limb pain.

Phantom limb pain is an often severe and debilitating phenomenon that has been reported in up to 85% of amputees. Its pathophysiology is poorly understood. Peripheral and spinal mechanisms are thought to play a role in pain modulation in affected individuals; however central mechanisms are also likely to be of importance. The neuromatrix theory postulates a genetically determined representation of body image, which is modified by sensory input to create a neurosignature. Persistence of the neurosignature may be responsible for painless phantom limb sensations, whereas phantom limb pain may be due to abnormal reorganisation within the neuromatrix. This study assessed the clinical outcome of deep brain stimulation of the periventricular grey matter and somatosensory thalamus for the relief of chronic neuropathic pain associated with phantom limb in three patients. These patients were assessed preoperatively and at 3 month intervals postoperatively. Self-rated visual analogue scale pain scores assessed pain intensity, and the McGill Pain Questionnaire assessed the quality of the pain. Quality of life was assessed using the EUROQOL EQ-5D scale. Periventricular gray stimulation alone was optimal in two patients, whilst a combination of periventricular gray and thalamic stimulation produced the greatest degree of relief in one patient. At follow-up (mean 13.3 months) the intensity of pain was reduced by 62% (range 55-70%). In all three patients, the burning component of the pain was completely alleviated. Opiate intake was reduced in the two patients requiring morphine sulphate pre-operatively. Quality of life measures indicated a statistically significant improvement. This data supports the role for deep brain stimulation in patients with phantom limb pain. The medical literature relating to the epidemiology, pathogenesis, and treatment of this clinical entity is reviewed in detail.

Potentiating role of interleukin-1beta (IL-1beta) and IL-1beta type 1 receptors in the medial hypothalamus in defensive rage behavior in the cat.

Recently, this laboratory provided evidence that interleukin-1beta (IL-1beta), an immune and brain-derived cytokine, microinjected into the medial hypothalamus, potentiates defensive rage behavior in the cat elicited from the midbrain periaqueductal gray (PAG), and that such effects are blocked by a 5-HT2 receptor antagonist. Since this finding represents the first time that a brain cytokine has been shown to affect defensive rage behavior, the present study replicated and extended these findings by documenting the specific potentiating role played by IL-1beta Type 1 receptor (IL-1RI), and the anatomical relationship between IL-1beta and 5-HT2 receptors in the medial hypothalamus. IL-1beta (10 ng) microinjected into the medial hypothalamus induced two separate phases of facilitation, one at 60 min and another at 180 min, post-injection. In turn, these effects were blocked with pretreatment of the selective IL-1 Type I receptor antagonist (IL-1ra) (10 ng), demonstrating the selectivity of the effects of IL-1beta on medial hypothalamic neurons upon PAG-elicited defensive rage behavior. The next stage of the study utilized immunohistochemical methods to demonstrate that IL-1beta and 5-HT2 receptors were present on the same neurons within regions of the medial hypothalamus where IL-1beta and the IL-1beta receptor antagonists were administered. This provided anatomical evidence suggesting a relationship between IL-1RI and 5-HT2 receptors in the medial hypothalamus that is consistent with the previous pharmacological observations in our laboratory. The overall findings show that activation of IL-1RI in the medial hypothalamus potentiates defensive rage behavior in the cat and that these effects may also be linked to the presence of 5-HT2 receptors on the same groups of neurons in this region of hypothalamus.

Micturition-related neuronal firing in the periaqueductal gray area in cats.

The midbrain periaqueductal gray (PAG) is the area promoting emotional motor responses, reproductive behaviors and analgesia. Recent studies suggest that neurons in the PAG may be crucial for regulating the micturition reflex in both experimental animals and humans. We examined single neuronal activities in the PAG and the adjacent area in response to isovolumetric spontaneous micturition reflexes in 20 supracollicular decerebrated cats. In total, 84 neurons were recorded in the PAG that were related to urinary storage/micturition cycles. Of the neurons recorded, the most common were tonic storage neurons (43%), followed by tonic micturition neurons (29%), phasic storage neurons (15%) and phasic micturition neurons (13%). In addition to the tonic/phasic as well as storage/micturition classification, the neurons showed diverse discharge patterns: augmenting, constant and decrementing, with the constant discharge pattern being most common. Of the 16 neurons located within the PAG that had similar discharge patterns to those just ventral to the PAG, the micturition neurons were distributed in a broader area, whereas the storage neurons seemed to be concentrated in the middle part of the PAG (P0-1, Horsley-Clarke coordinate). High-frequency stimulation (HFS; 0.2-ms duration, 100 Hz) applied in the PAG elicited inhibition of the micturition reflex. Effective amplitude of the electrical stimulation for evoking inhibitory responses was less than 50 microA. In conclusion, the results of the present study showed that HFS of the PAG inhibited the micturition reflex and there were micturition-related neuronal firings in the PAG in cats, suggesting that the PAG is involved in neural control of micturition.

Electrical stimulation of the dorsal periaqueductal gray decreases volume of the brain infarction independently of accompanying hypertension and cerebrovasodilation.

We investigated whether selective stimulation of neurons of the sympathoinhibitory ventral periaqueductal gray (VPAG), or sympathoexcitatory dorsal periaqueductal gray (DPAG), differentially modulates CBF and EEG and exerts neuroprotection. Electrical stimulation of either regions of PAG comparably elevated AP and CBF, whereas chemical stimulation with the D,L-homocysteine produced either sympathoinhibition accompanied by decrease in CBF from ventral region or sympathoexcitation accompanied by increase in CBF from dorsal region in nonspinalized rats. The CBF effects evoked from DPAG and VPAG by chemical stimulation were preserved in spinalized rats supporting that the evoked CBF responses result directly from stimulation and are not secondary to AP changes. Stimulation of either region, whether chemical or electrical, synchronized the EEG. To explore whether PAG stimulation might protect the brain against ischemic injury, in other rats the VPAG or DPAG were stimulated for 1 h (50 Hz, 1 s on/1 s off, 75-100 microA) and the middle cerebral artery occluded 72 h later. Stimulation of the DPAG, but not VPAG, significantly reduced infarction volumes relative to sham-stimulated controls as determined 24 h after occlusion. Elevations of AP and CBF did not differ between groups. We conclude: (a). intrinsic neurons of D- and VPAG differentially regulate CBF; (b). neurons of DPAG are neuroprotective independently of changes in CBF and/or AP. The DPAG effect on infarct volume may be related to the central neuroprotective pathway evoked by stimulation of the cerebellar FN.

Strain differences to the effects of aversive frequency ultrasound on behaviour and brain topography of c-fos expression in the rat.

Previous studies have shown that ultrasound at 20 kHz produces an escape (defence) response in the hooded Lister rat. This study compares the ultrasound-induced behavioural response in the hooded Lister and albino Wistar rat. Ultrasound (continuous tone, square wave, 20 kHz) produced an initial characteristic startle response (brisk running) in the hooded Lister rat that was followed immediately after cessation of the ultrasound by a period of freezing behaviour. In contrast, Wistar rats showed no initial escape response but a prolonged period of freezing that started during the ultrasound and continued for a period after the end of the ultrasound. Immunohistochemical assessment of c-fos expression also showed a difference between the two strains with preferential expression in the dorsal region of the rostral and caudal periaqueductal grey (PAG) in the hooded Lister rat, while the expression occurred in the ventral PAG in the Wistar rats. In summary, the two strains exhibit distinct defensive behaviours and patterns of neuronal activation in response to the same aversive signal. It remains to be determined whether these differences relate to neuronal circuitry or perception of the signal, but analysis of the mechanisms involved may help our understanding of the heterogeneity of anxiety disorders.