CB1 receptor mediated analgesia from the Nucleus Reticularis Gigantocellularis pars alpha is activated in an animal model of neuropathic pain.

Cannabinoids are known to suppress responses to noxious stimulation in animals and man. Recent research has suggested a role for endogenous cannabinoids in the descending inhibition of dorsal horn cells via a supraspinal site of action. We have recently demonstrated [J. Physiol. 506(2) (1998) 459] that the nucleus reticularis gigantocellularis pars alpha (GiA) is a major source of such descending modulation, and importantly, that this system is activated in response to noxious stimulation. We have therefore investigated the role of CB1 receptor activation in mediating the antinociceptive effects of activation of GiA in models of acute and chronic pain. Microinjections (0.5 microl 60% DMSO) of either WIN 55,212-2 (5 microg, selective CB1 agonist), SR141716A (50 microg, competitive CB1 antagonist), both compounds together, or vehicle alone into GiA were performed prior to these tests in a randomised, blind manner. In control animals, WIN 55,212-2 markedly increased withdrawal latencies in the tail flick test and reduced responses to subcutaneous formalin. These effects were blocked by co-administration of SR141716A. These data suggest that activation of cannabinoid CB1 receptor subtypes in GiA leads to behavioural analgesia. In animals with partial sciatic nerve ligation, microinjection of drugs and injection of formalin were performed contralaterally to the site of ligation. Partial sciatic nerve ligation significantly reduced behavioural responses to contralaterally applied formalin. Microinjection of SR141716A to GiA reversed this inhibition of responses to formalin in animals with partial sciatic nerve ligation. These data provide evidence that endogenous CB1 receptor ligands are involved in GiA mediated antinociception, and that this system is important for the modulation of nociceptive transmission in an animal model of chronic neuropathic pain.

The behavioural importance of dynamically activated descending inhibition from the nucleus reticularis gigantocellularis pars alpha.

We have recently demonstrated (J Physiol 506 (1998) 459) that the dynamic activation of descending inhibition of the nociceptive response of spinal multireceptive cells occurs in the nucleus reticularis gigantocellularis pars alpha (GiA). In the same paper we have shown that Lamina I dorsal horn cells are responsible for activating this inhibition via a pathway which runs in the contralateral dorsolateral funiculus. The effects of dynamically activating this system by noxious stimulation on behavioural responses to noxious stimuli have not been established. Here we demonstrate the effects of GiA on the behavioural response during application of standardized noxious stimuli. As this system is activated in response to noxious stimulation (J Physiol 506 (1998) 459), it is possible that chronic pain states may also activate GiA. We have therefore investigated this possibility in animals following partial sciatic nerve ligation (an animal model of chronic pain; Pain 43 (1990) 205). Male Wistar rats (280-310 g) were anaesthetized with halothane (0.5-2% in O(2)). Guide cannulae for microinjections were stereotaxically placed above GiA. In one group of animals the sciatic nerve was partially ligated. Animals were allowed to recover for 4-6 days. The responses of each animal during the formalin test (Pain 4 (1977) 161) and the tail flick test (Pain 12 (1982) 229) were recorded on different days. Microinjections (0.5 microl) of either gamma-aminobutyric acid (GABA, 200 mM), D-L homocysteic acid (DLH, 25 mM) or 0.9% saline (as control) into GiA were performed during these tests in a randomized, blind manner. In animals without sciatic nerve ligation, microinjection of GABA to GiA did not significantly affect the animal's response during the tail flick test. However microinjection of DLH significantly increased the latency of tail flick from 6.2 +/- 0.8 to 8.4 +/- 0.5 s for up to 15 min (n = 7, P < 0.01, Mann-Whitney U-test). Microinjection of GABA to GiA increased the behavioural response to formalin between 10 and 20 min post-injection, while microinjection of DLH reduced this response at all time points except 10 min post-injection (n = 8, P < 0.05, Mann-Whitney U-test). In animals with sciatic nerve ligation, microinjections (0.5 microl) of either GABA (200 mM), or saline (as control) into GiA contralateral to the partial sciatic ligation were performed during these tests in a randomized, blind manner. Partial sciatic ligation significantly reduced the behavioural response to contralaterally applied formalin from 15 min post-injection onwards, compared to controls without sciatic nerve ligation. Microinjection of GABA to GiA significantly increased the behavioural response to formalin from 20 to 50 min post-injection. The inactivation of GiA only causes behavioural effects in nociceptive tests of a long enough duration to activate the system (i.e. the formalin test but not the tail flick test). Chemical activation of the system affects both tests. These data strongly support the concept of an important analgesic system which is activated in response to noxious stimulation, and subsequently acts to reduce behavioural responses to noxious stimuli.

Periaqueductal grey mediated inhibition of responses to noxious stimulation is dynamically activated in a rat model of neuropathic pain.

The periaqueductal grey (PAG) has been shown to be a major source of descending inhibition of dorsal horn cells (Textbook of Pain (1999) 309). However, few studies have demonstrated alterations in behavioural responses to noxious stimulation following inactivation of this nucleus. Many behavioural studies have looked for effects on nociceptive withdrawal thresholds in acute nociceptive tests. These tests would not reveal the presence of inhibition which is activated in response to noxious input. We have therefore investigated this possibility by studying behavioural responses to subcutaneous formalin injection in control animals, and in animals following partial sciatic nerve ligation (an animal model of neuropathic pain (Pain 43(2) (1990) 205). In control animals, microinjection of gamma-aminobutyric acid (GABA) to PAG did not significantly alter behavioural responses to formalin, while microinjection of D,L-homocysteic acid (DLH) reduced these responses. Responses to contralaterally applied formalin were significantly reduced in animals with partial sciatic ligation. Microinjection of GABA to PAG significantly increased these behavioural responses to formalin. We conclude that a component of PAG mediated inhibition of nociception is inactive under normal conditions. This inhibition may be activated by persistent nociceptive input, and possibly reflects long term changes in nociceptive circuitry which occur in neuropathic pain states.

Supraspinal inhibition of nociceptive dorsal horn neurones in the anaesthetized rat: tonic or dynamic?

1. Tonic inhibition of sensory spinal neurones is well known to descend from the rostroventral medulla. It is not clear if this inhibition is dynamically activated by peripheral noxious stimuli. 2. Transection of the ipsilateral dorsolateral funiculus (DLF) removed a descending inhibition of multireceptive spinal neurones and disproportionally prolonged the after-discharge component of their response to a noxious cutaneous stimulus. 3. Microinjection of GABA or tetracaine into the medullary nucleus gigantocellularis pars alpha (GiA) similarly prolonged the after-discharge in response to noxious stimuli. 4. Recordings of GiA cells, initially using minimal surgery, revealed that many had low levels of spontaneous activity and responded vigorously to noxious stimuli applied to any part of the body surface. One hour after the surgery necessary to expose the spinal cord, GiA cells had a high firing rate but responded weakly to noxious stimuli. 5. The response of GiA cells to noxious stimuli was abolished by transection of only the DLF contralateral to the stimulus. 6. It is concluded that the inhibition of multireceptive dorsal horn neurones from GiA is dynamically activated by noxious cutaneous stimuli via a projection in the contralateral DLF. Surgical exposure of the spinal cord tonically activates this inhibition and masks the dynamic component.

Functional activation of somatostatin- and neuropeptide Y-containing neurons in the entorhinal cortex of chronically epileptic rats.

The in vitro release of somatostatin and neuropeptide Y, their tissue concentration and immunocytochemical pattern were examined in the entorhinal cortex of chronically epileptic rats. A systemic administration of 12 mg/kg kainic acid causing generalized tonic-clonic seizures for at least 3 h after injection was used to induce, 60 days later, a chronically enhanced susceptibility to seizures in the rats. The release of both peptides under depolarizing conditions was significantly reduced by 15% on average from slices of the entorhinal cortex two days after kainic acid-induced status epilepticus. At 60 days, the spontaneous and 30 mM KCl-induced release of somatostatin was significantly enhanced by 30% on average. The release induced by 100 mM KCl was raised by 70%. The spontaneous, 30 mM and 100 mM KCl-induced release of neuropeptide Y from the same slices was increased, respectively, by 120%, 76% and 36%. The late changes were associated with an increased tissue concentration of neuropeptide Y but not of somatostatin. This was confirmed by immunocytochemical evidence showing that neuropeptide Y-, but not somatostatin-immunoreactive neurons were increased in the entorhinal cortex of kainic acid-treated rats. These results indicate that neurotransmission mediated by somatostatin and neuropeptide Y, two peptides previously shown to play a role in limbic epileptogenesis, is enhanced in the entorhinal cortex of chronically epileptic rats.

Cellular localization of neuropeptide-Y receptors in the rat hippocampus: long-term effects of limbic seizures.

To clarify the cellular localization of neuropeptide-Y receptor subtypes in the dentate gyrus and CA3 sector of the rat dorsal hippocampus and their changes after kainic acid-induced seizures, we used receptor autoradiography to measure [125I]PYY binding to Y1 and Y2 receptors after colchicine treatment. Fifteen days after colchicine infusion in the dorsal hippocampus granule cells and their mossy fibres degenerated while the hilar interneurons and CA3 pyramidal cells were spared. This treatment markedly decreased [125I]PYY binding to Y1 receptors in the molecular layer of the dentate gyrus (-82%) and in the hilus (-70%). [125I]PYY binding to Y2 receptors was reduced by 40% and 48%, respectively, in the CA3 region and in the hilus. Thirty days after kainic acid treatment, [125I]PYY binding to Y1 receptors was decreased by 35% in the molecular layer of the dentate gyrus whereas the binding to Y2 receptors was increased by 116% in the hilus. The effect of colchicine in kainic acid-treated rats indicates that these plastic changes occur selectively on granule cell projections.