The Periaqueductal Gray Orchestrates Sensory and Motor Circuits at Multiple Levels of the Neuraxis.

The periaqueductal gray (PAG) coordinates behaviors essential to survival, including striking changes in movement and posture (e.g., escape behaviors in response to noxious stimuli vs freezing in response to fear-evoking stimuli). However, the neural circuits underlying the expression of these behaviors remain poorly understood. We demonstrate in vivo in rats that activation of the ventrolateral PAG (vlPAG) affects motor systems at multiple levels of the neuraxis through the following: (1) differential control of spinal neurons that forward sensory information to the cerebellum via spino-olivo-cerebellar pathways (nociceptive signals are reduced while proprioceptive signals are enhanced); (2) alterations in cerebellar nuclear output as revealed by changes in expression of Fos-like immunoreactivity; and (3) regulation of spinal reflex circuits, as shown by an increase in α-motoneuron excitability. The capacity to coordinate sensory and motor functions is demonstrated in awake, behaving rats, in which natural activation of the vlPAG in fear-conditioned animals reduced transmission in spino-olivo-cerebellar pathways during periods of freezing that were associated with increased muscle tone and thus motor outflow. The increase in spinal motor reflex excitability and reduction in transmission of ascending sensory signals via spino-olivo-cerebellar pathways occurred simultaneously. We suggest that the interactions revealed in the present study between the vlPAG and sensorimotor circuits could form the neural substrate for survival behaviors associated with vlPAG activation. SIGNIFICANCE STATEMENT: Neural circuits that coordinate survival behaviors remain poorly understood. We demonstrate in rats that the periaqueductal gray (PAG) affects motor systems at the following multiple levels of the neuraxis: (1) through altering transmission in spino-olivary pathways that forward sensory signals to the cerebellum, reducing and enhancing transmission of nociceptive and proprioceptive information, respectively; (2) by alterations in cerebellar output; and (3) through enhancement of spinal motor reflex pathways. The sensory and motor effects occurred at the same time and were present in both anesthetized animals and behavioral experiments in which fear conditioning naturally activated the PAG. The results provide insights into the neural circuits that enable an animal to be ready and able to react to danger, thus assisting in survival.

Periaqueductal grey cyclooxygenase-dependent facilitation of C-nociceptive drive and encoding in dorsal horn neurons in the rat.

The experience of pain is strongly affected by descending control systems originating in the brainstem ventrolateral periaqueductal grey (VL-PAG), which control the spinal processing of nociceptive information. A- and C-fibre nociceptors detect noxious stimulation, and have distinct and independent contributions to both the perception of pain quality (fast and slow pain, respectively) and the development of chronic pain. Evidence suggests a separation in the central processing of information arising from A- vs. C-nociceptors; for example, inhibition of the cyclooxygenase-1 (COX-1)-prostaglandin system within the VL-PAG alters spinal nociceptive reflexes evoked by C-nociceptor input in vivo via descending pathways, leaving A-nociceptor-evoked reflexes largely unaffected. As the spinal neuronal mechanisms underlying these different responses remain unknown, we determined the effect of inhibition of VL-PAG COX-1 on dorsal horn wide dynamic-range neurons evoked by C- vs. A-nociceptor activation. Inhibition of VL-PAG COX-1 in anaesthetised rats increased firing thresholds of lamina IV-V wide dynamic-range dorsal horn neurons in response to both A- and C-nociceptor stimulation. Importantly, wide dynamic-range dorsal horn neurons continued to faithfully encode A-nociceptive information, even after VL-PAG COX-1 inhibition, whereas the encoding of C-nociceptor information by wide dynamic-range spinal neurons was significantly disrupted. Dorsal horn neurons with stronger C-nociceptor input were affected by COX-1 inhibition to a greater extent than those with weak C-fibre input. These data show that the gain and contrast of C-nociceptive information processed in individual wide dynamic-range dorsal horn neurons is modulated by prostanergic descending control mechanisms in the VL-PAG.

Neural substrates underlying fear-evoked freezing: the periaqueductal grey-cerebellar link.

The central neural pathways involved in fear-evoked behaviour are highly conserved across mammalian species, and there is a consensus that understanding them is a fundamental step towards developing effective treatments for emotional disorders in man. The ventrolateral periaqueductal grey (vlPAG) has a well-established role in fear-evoked freezing behaviour. The neural pathways underlying autonomic and sensory consequences of vlPAG activation in fearful situations are well understood, but much less is known about the pathways that link vlPAG activity to distinct fear-evoked motor patterns essential for survival. In adult rats, we have identified a pathway linking the vlPAG to cerebellar cortex, which terminates as climbing fibres in lateral vermal lobule VIII (pyramis). Lesion of pyramis input-output pathways disrupted innate and fear-conditioned freezing behaviour. The disruption in freezing behaviour was strongly correlated to the reduction in the vlPAG-induced facilitation of α-motoneurone excitability observed after lesions of the pyramis. The increased excitability of α-motoneurones during vlPAG activation may therefore drive the increase in muscle tone that underlies expression of freezing behaviour. By identifying the cerebellar pyramis as a critical component of the neural network subserving emotionally related freezing behaviour, the present study identifies novel neural pathways that link the PAG to fear-evoked motor responses.

Spinal processing of noxious and innocuous cold information: differential modulation by the periaqueductal gray.

In addition to cold being an important behavioral drive, altered cold sensation frequently accompanies pathological pain states. However, in contrast to peripheral mechanisms, central processing of cold sensory input has received relatively little attention. The present study characterized spinal responses to noxious and innocuous intensities of cold stimulation in vivo and established the extent to which they are modulated by descending control originating from the periaqueductal gray (PAG), a major determinant of acute and chronic pain. In lightly anesthetized rats, hindpaw cooling with ethyl chloride, but not acetone, was sufficiently noxious to evoke withdrawal reflexes, which were powerfully inhibited by ventrolateral (VL)-PAG stimulation. In a second series of experiments, subsets of spinal dorsal horn neurons were found to respond to innocuous and/or noxious cold. Descending control from the VL-PAG distinguished between activity in nociceptive versus non-nociceptive spinal circuits in that innocuous cold information transmitted by non-nociceptive class 1 and wide-dynamic-range class 2 neurons remained unaltered. In contrast, noxious cold information transmitted by class 2 neurons and all cold-evoked activity in nociceptive-specific class 3 neurons was significantly depressed. We therefore demonstrate that spinal responses to cold can be powerfully modulated by descending control systems originating in the PAG, and that this control selectively modulates transmission of noxious versus innocuous information. This has important implications for central processing of cold somatosensation and, given that chronic pain states are dependent on dynamic alterations in descending control, will help elucidate mechanisms underlying aberrant cold sensations that accompany pathological pain states.

A rapamycin-sensitive signaling pathway is essential for the full expression of persistent pain states.

Translational control through the mammalian target of rapamycin (mTOR) is critical for synaptic plasticity, cell growth, and axon guidance. Recently, it was also shown that mTOR signaling was essential for the maintenance of the sensitivity of subsets of adult sensory neurons. Here, we show that persistent pain states, but not acute pain behavior, are substantially alleviated by centrally administered rapamycin, an inhibitor of the mTOR pathway. We demonstrate that rapamycin modulates nociception by acting on subsets of primary afferents and superficial dorsal horn neurons to reduce both primary afferent sensitivity and central plasticity. We found that the active form of mTOR is present in a subpopulation of myelinated dorsal root axons, but rarely in unmyelinated C-fibers, and heavily expressed in the dorsal horn by lamina I/III projection neurons that are known to mediate the induction and maintenance of pain states. Intrathecal injections of rapamycin inhibited the activation of downstream targets of mTOR in dorsal horn and dorsal roots and reduced the thermal sensitivity of A-fibers. Moreover, in vitro studies showed that rapamycin increased the electrical activation threshold of Adelta-fibers in dorsal roots. Together, our results imply that central rapamycin reduces neuropathic pain by acting both on an mTOR-positive subset of A-nociceptors and lamina I projection neurons and suggest a new pharmacological route for therapeutic intervention in persistent pain states.

Cyclooxygenase-1-derived prostaglandins in the periaqueductal gray differentially control C- versus A-fiber-evoked spinal nociception.

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert analgesic effects by inhibiting peripheral cyclooxygenases (COXs). It is now clear that these drugs also have central actions that include the modulation of descending control of spinal nociception from the midbrain periaqueductal gray (PAG). Descending control is a powerful determinant of the pain experience and is thus a potential target for analgesic drugs, including COX inhibitors. Noxious information from the periphery is conveyed to the spinal cord in A- and C-fiber nociceptors, which convey different qualities of the pain signal and have different roles in chronic pain. This in vivo study used different rates of skin heating to preferentially activate A- or C-heat nociceptors to further investigate the actions of COX inhibitors and prostaglandins in the PAG on spinal nociceptive processing. The results significantly advance our understanding of the central mechanisms underlying the actions of NSAIDs and prostaglandins by demonstrating that (1) in the PAG, it is COX-1 and not COX-2 that is responsible for acute antinociceptive effects of NSAIDs in vivo; (2) these effects are only evoked from the opioid-sensitive ventrolateral PAG; and (3) prostaglandins in the PAG exert tonic facilitatory control that targets C- rather than A-fiber-mediated spinal nociception. This selectivity of control is of particular significance given the distinct roles of A- and C-nociceptors in acute and chronic pain. Thus, effects of centrally acting prostaglandins are pivotal, we suggest, to both the understanding of nociceptive processing and the development of new analgesic drugs.

Development of a novel high-throughput assay for the investigation of GlyT-1b neurotransmitter transporter function.

The glycine transporter (GlyT-1b) is a Na(+)/Cl(-)-dependent electrogenic transporter which mediates the rapid re-uptake of glycine from the synaptic cleft. Based on its tissue distribution, GlyT-1 has been suggested to co-localise with the NMDA receptor where it may modulate the concentration of glycine at its co-agonist binding site. This data has led to GlyT-1 inhibitors being proposed as targets for disorders such as schizophrenia and cognitive dysfunction. Radiolabelled uptake assays (e.g. [(3)H]glycine) have been traditionally used in compound screening to identify glycine transporter inhibitors. While such an assay format is useful for testing limited numbers of compounds, the identification of novel glycine uptake inhibitors requires a functional assay compatible with high-throughput screening (HTS) of large compound libraries. Here, the authors present the development of a novel homogenous cell-based assay using the FLIPR membrane potential blue dye (Molecular Devices) and FLEXstation. Pharmacological data for the GlyT-1 inhibitors Org 24598 and ALX 5407 obtained using this novel electrogenic assay correlated well with the conventional [(3)H]-glycine uptake assay format. Furthermore, the assay has been successfully miniaturised using FLIPR(3) and therefore has the potential to be used for high-throughput screening.

Local translation in primary afferent fibers regulates nociception.

Recent studies have demonstrated the importance of local protein synthesis for neuronal plasticity. In particular, local mRNA translation through the mammalian target of rapamycin (mTOR) has been shown to play a key role in regulating dendrite excitability and modulating long-term synaptic plasticity associated with learning and memory. There is also increased evidence to suggest that intact adult mammalian axons have a functional requirement for local protein synthesis in vivo. Here we show that the translational machinery is present in some myelinated sensory fibers and that active mTOR-dependent pathways participate in maintaining the sensitivity of a subpopulation of fast-conducting nociceptors in vivo. Phosphorylated mTOR together with other downstream components of the translational machinery were localized to a subset of myelinated sensory fibers in rat cutaneous tissue. We then showed with electromyographic studies that the mTOR inhibitor rapamycin reduced the sensitivity of a population of myelinated nociceptors known to be important for the increased mechanical sensitivity that follows injury. Behavioural studies confirmed that local treatment with rapamycin significantly attenuated persistent pain that follows tissue injury, but not acute pain. Specifically, we found that rapamycin blunted the heightened response to mechanical stimulation that develops around a site of injury and reduced the long-term mechanical hypersensitivity that follows partial peripheral nerve damage--a widely used model of chronic pain. Our results show that the sensitivity of a subset of sensory fibers is maintained by ongoing mTOR-mediated local protein synthesis and uncover a novel target for the control of long-term pain states.