Substance P, opioid, and catecholamine systems in the mouse central nervous system (CNS).

I an article in this issue of PNAS, Jasmin et al. (1) provide new evidence that noradrenaline is a key neurotransmitter in the endogenous pain inhibitory systems in the central nervous system (CNS) of the mouse. They show that this adrenergic inhibitory system interacts with that part of the sensory nociceptive system by using the neuropeptide substance P in a mutually antagonistic manner. They conclude that substance P, when unopposed by tonic release of noradrenaline, is the major factor underlying thermal hyperalgesia. Jasmin et al. also present evidence that the reduced opioid efficacy seen in the absence of noradrenaline is the result of increased NK1 receptor stimulation by endogenous substance P. Their paper (1) is a good example of the way in which critical, and well controlled, experiments on transgenic animals can help to elucidate complex problems in neurobiology. This fascinating study supports other recently published work suggesting that substance P has a key role in pain perception in the mouse by way of critical interactions with other systems, e.g., the PAR2 proteasedependent receptor (2, 3). It has been accepted for many years that noradrenaline is a key neurotransmitter in the descending inhibitory systems by which the brainstem controls the sensitivity of the dorsal horn of the spinal cord to nociceptive sensory inputs (4). Synaptically released noradrenaline acts through 2 adrenoceptors to reduce the sensitivity of dorsal horn relay neurons to noxious but not to nonnoxious stimuli and it also potentiates the effects of opioid drugs, such as morphine (5, 6). Investigations using agonist and antagonist drugs to examine interactions between effects mediated by opioid receptors and adrenoceptors have sometimes given confusing results, as have studies where pathways have been lesioned or chemically depleted of their transmitter content. It has thus been difficult to investigate the putative noradrenergic dysfunction believed to underlie some chronic pain conditions in humans (7). This variability fits with current ideas on the plastic nature of pain but underlines the need for new animal models in which chronic pain states can be studied. Accordingly, to avoid the problems that plagued earlier studies, Jasmin et al. (1) chose to do experiments in mice in which the gene for dopamine-hydroxylase (DBH, the enzyme that converts dopamine to noradrenaline) had been genetically deleted (8). These mice can have their normal noradrenergic function restored by the ingenious expedient of dosing them with L-threo-3,4-dihydroxyphenylserine (DOPS), a synthetic amino acid precursor of noradrenaline that can be converted to noradrenaline by aromatic amino acid decarboxylase (AADC, which is present in both control and DBH mice). A further refinement is that, if the mice are dosed with carbidopa, an AADC inhibitor which does not cross the blood– brain barrier, and then given DOPS, it is possible to replace the brain noradrenaline in the DBH knockout mice, leaving peripheral noradrenaline absent (9). The DBH knockout mice have normal baseline sensory and nociceptive behaviors and are viable for long-term experiments. This latter point is important as the lack of a severe phenotype in the knockout animals renders the conclusions from experiments less open to misinterpretation. Nociceptive testing of the DBH knockout mice revealed that they were thermally hyperalgesic compared with control animals but had normal responses to mechanical stimuli. When inf lammation was produced in a paw, then the DBH knockout animals displayed mechanical hyperalgesia, but to a similar extent to control animals. Restoring noradrenaline in the CNS, by giving DOPS plus carbidopa, abolished the thermal hyperalgesia in DBH knockout mice, but this treatment had no effect on control mice. The antinociceptive effect of DOPS carbidopa, in the DBH knockout mice, was reversed by the 2-adrenoreceptor blocker, SKF-86466, but not by the 1adrenoreceptor blocker HEAT. CNS noradrenergic neurons and their projections were preserved in DBH knockout mice, thereby providing the circuitry from which noradrenaline could be released once its synthesis was established by giving DOPS carbidopa. As Jasmin et al. (1) believed that part of the antinociceptive effect of 2-adrenoreceptor stimulation might be the result of the inhibition of substance P release from primary afferent fibers, they studied the effects of drugs that blocked the action of substance P at its target NK1 receptors on the thermal hyperalgesia seen in DBH knockout mice. All three antagonists used (RP-67580, CP-96345, and L-733060) reversed the hyperalgesia in the knockout mice but not in control mice, and inactive enantiomers of two of these blockers were without effect, confirming that the responses studied were indeed operated through NK1 receptors. It was found that the amounts of NK1 receptor in spinal cord dorsal horn were similar in DBH knockout and control mice, but that the amount of substance P immunoreactivity in DBH knockout mice was lower than in controls, suggesting increased release turnover leading to depletion of stores. It was also found that the DBH knockout mice were less sensitive to the antinociceptive effects of morphine than were control mice, consistent with the idea that part of the action of morphine is through stimulation of descending inhibitory circuits by using noradrenaline as a transmitter (5). This observation was confirmed by giv-