S.F. Maier

Medial prefrontal cortex determines how stressor controllability affects behavior and dorsal raphe nucleus.

The degree of behavioral control that an organism has over a stressor is a potent modulator of the stressors impact; uncontrollable stressors produce numerous outcomes that do not occur if the stressor is controllable. Research on controllability has focused on brainstem nuclei such as the dorsal raphe nucleus (DRN). Here we find that the infralimbic and prelimbic regions of the ventral medial prefrontal cortex (mPFCv) in rats detect whether a stressor is under the organisms control. When a stressor is controllable, stress-induced activation of the DRN is inhibited by the mPFCv, and the behavioral sequelae of uncontrollable stress are blocked. This suggests a new function for the mPFCv and implies that the presence of control inhibits stress-induced neural activity in brainstem nuclei, in contrast to the prevalent view that such activity is induced by a lack of control.

Evidence for the involvement of spinal cord glia in subcutaneous formalin induced hyperalgesia in the rat

Abstract Subcutaneous (s.c.) injection of formalin induces a rapid and prolonged hyperalgesia across widespread areas of the body. This hyperalgesic state involves a brain‐to‐spinal cord pathway, likely arising from the nucleus raphe magnus. The present study examined whether subsequent activation of spinal cord glia may be critical for the hyperalgesic state to be observed in rats. Glia were considered candidates as they can, upon activation, release a variety of substances known to be critical for the mediation of subcutaneous formalin‐induced hyperalgesia including glutamate, aspartate, nitric oxide, arachidonic acid and cyclooxygenase products such as prostaglandins. This series of experiments demonstrate that formalin‐induced hyperalgesia in rats can be blocked by intrathecal administration of agents that: (a) disrupt glial function (using either 1 nmol fluorocitrate which is a glial metabolic inhibitor, or 9 &mgr;g CNI‐1493 which disrupts synthesis of nitric oxide and cytokines in monocyte‐derived cells; ANOVA revealed reliable group effects for each drug with P<0.0005); or (b) disrupt the action of glial products (using 10, 50, or 100 &mgr;g of a human recombinant interleukin‐1 receptor antagonist or 10 &mgr;l antibody directed against nerve growth factor; ANOVA revealed reliable group effects for each drug with P<0.001). Disruption appeared to be selective, as blockade of only select glial products was effective. That is, up to 120 &mgr;g of a functional antagonist of tumor necrosis factor‐&agr; (TNF binding protein) and 5 &mgr;l of an antibody against complement‐3 produced no statistically reliable reduction in formalin‐induced hyperalgesia. Taken together, the present series of experiments suggest an important role for spinal glial cells in the cascade of events that are initiated by descending signals following s.c. administration of formalin.

Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions

Fractalkine is a unique chemokine reported to be constitutively expressed by neurons. Its only receptor, CX3CR1, is expressed by microglia. Little is known about the expression of fractalkine and CX3CR1 in spinal cord. Given that peripheral nerve inflammation and/or injury gives rise to neuropathic pain, and neuropathic pain may be partially mediated by spinal cord glial activation and consequent glial proinflammatory cytokine release, there must be a signal released by affected neurons that triggers the activation of glia. We sought to determine whether there is anatomical evidence implicating spinal fractalkine as such a neuron‐to‐glia signal. We mapped the regional and cellular localization of fractalkine and CX3CR1 in the rat spinal cord and dorsal root ganglion, under basal conditions and following induction of neuropathic pain, employing both an inflammatory (sciatic inflammatory neuropathy; SIN) as well as a traumatic (chronic constriction injury; CCI) model. Fractalkine immunoreactivity and mRNA were observed in neurons, but not glia, in the rat spinal cord and dorsal root ganglia, and levels did not change following either CCI or SIN. By contrast, CX3CR1 was expressed by microglia in the basal state, and the microglial cellular concentration was up‐regulated in a regionally specific manner in response to neuropathy. CX3CR1‐expressing cells were identified as microglia by their cellular morphology and positive OX‐42 and CD4 immunostaining. The cellular distribution of fractalkine and CX3CR1 in the spinal circuit associated with nociceptive transmission supports a potential role in the mechanisms that contribute to the exaggerated pain state in these models of neuropathy.

Immune regulation of central nervous system functions: from sickness responses to pathological pain

Classically, the central nervous system (CNS) and the immune system are thought to operate independently of each other. This simplistic view has been corrected in recent years, first with the recognition that the brain dynamically modulates the immune system, and later with the reverse; that is, that the immune system modulates the CNS as well. The evidence that the immune system regulates CNS functions is first reviewed. This immune‐to‐brain communication pathway triggers the production of a constellation of CNS‐mediated phenomena, collectively referred to as ‘sickness responses’. These sickness responses are created by immune‐to‐brain signals activating CNS glia to release glial proinflammatory cytokines. The most recently recognized member of this constellation of changes is enhanced pain responsivity. The hypothesis is then developed that pathological, chronic pain may result from ‘tapping into’ this ancient survival‐oriented circuitry, including the activation of immune and glial cells and the release of immune/glial proinflammatory cytokines. This can occur at the level of peripheral nerves, dorsal root ganglia, spinal cord, and likely at higher brain areas. The implications of this model for human chronic pain syndromes and clinical resolution of these chronic pain states are then discussed.

Evidence that exogenous and endogenous fractalkine can induce spinal nociceptive facilitation in rats

Recent evidence suggests that spinal cord glia can contribute to enhanced nociceptive responses. However, the signals that cause glial activation are unknown. Fractalkine (CX3C ligand‐1; CX3CL1) is a unique chemokine expressed on the extracellular surface of spinal neurons and spinal sensory afferents. In the dorsal spinal cord, fractalkine receptors are primarily expressed by microglia. As fractalkine can be released from neurons upon strong activation, it has previously been suggested to be a neuron‐to‐glial signal that induces glial activation. The present series of experiments provide an initial investigation of the spinal pain modulatory effects of fractalkine. Intrathecal fractalkine produced dose‐dependent mechanical allodynia and thermal hyperalgesia. In addition, a single injection of fractalkine receptor antagonist (neutralizing antibody against rat CX3C receptor‐1; CX3CR1) delayed the development of mechanical allodynia and/or thermal hyperalgesia in two neuropathic pain models: chronic constriction injury (CCI) and sciatic inflammatory neuropathy. Intriguingly, anti‐CX3CR1 reduced nociceptive responses when administered 5–7 days after CCI, suggesting that prolonged release of fractalkine may contribute to the maintenance of neuropathic pain. Taken together, these initial investigations of spinal fractalkine effects suggest that exogenous and endogenous fractalkine are involved in spinal sensitization, including that induced by peripheral neuropathy.

Activation of serotonin-immunoreactive cells in the dorsal raphe nucleus in rats exposed to an uncontrollable stressor.

The dorsal raphe nucleus (DRN) and its serotonergic terminal regions have been suggested to be part of the neural substrate by which exposure to uncontrollable stressors produces poor escape responding and enhanced conditioned fear expression. Such stressor exposure is thought to selectively activate DRN serotonergic neurons in such a way as to render them transiently sensitized to further input. As a result of this sensitized state, behavioral testing procedures are thought to cause excess serotonergic activity in brain regions that control these behaviors. The present studies were conducted to investigate activity in the DRN following exposure to escapable and yoked, inescapable tailshock. Neural activity was characterized using immunohistochemistry to detect the immediate early gene product Fos in serotonin-immunoreactive cells in the DRN. Inescapable tailshock led to greater serotonergic neural activity than did escapable tailshock, supporting the hypothesis that uncontrollable stressors preferentially activate serotonergic neurons in the DRN.

Mechanisms of tumor necrosis factor-α (TNF-α) hyperalgesia

Abstract Activation of immune cells by pathogens induces the release of a variety of proinflammatory cytokines, including IL-1β and TNF-α. Previous studies using IL-1β have demonstrated that this cytokine can alter brain function, resulting in a variety of ‘illness responses’ including increased sleep, decreased food intake, fever, etc. We have recently demonstrated that i.p. IL-1β also produces hyperalgesia and that this hyperalgesia (as well as most illness responses) is mediated via activation of subdiaphragmatic vagal afferents. The present series of studies were designed to provide an initial examination of the generality of proinflammatory cytokine-induced hyperalgesia by examining the effects of i.p. TNF-α on pain responsivity. These studies demonstrate that: (a) i.p. TNF-α produces dose-dependent hyperalgesia as measured by the tailflick test, (b) this hyperalgesia is mediated via the induced release of IL-1β, (c) hyperalgesia is mediated via activation of subdiaphragmatic vagal afferents, and (d) the effects of subdiaphragmatic vagotomy cannot be explained by a generalized depression of neural excitability.

Brain-derived neurotrophic factor mRNA downregulation produced by social isolation is blocked by intrahippocampal interleukin-1 receptor antagonist

Manipulations that increase the expression of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) in the hippocampus (e.g. peripheral administration of lipopolysaccharide, i.c.v. glycoprotein 120, social isolation) as well as the intrahippocampal injection of IL-1beta following a learning experience, dramatically impair the memory of that experience if the formation of the memory requires the hippocampus. Here we employed social isolation to further study this phenomenon, as well as its relation to brain-derived neurotrophic factor (BDNF). BDNF was studied because of its well-documented role in the formation of hippocampally based memory. A 6 h period of social isolation immediately after contextual fear conditioning impaired memory for context fear measured 48 h later, and decreased BDNF mRNA in the dentate gyrus and the CA3 region of the hippocampus assessed immediately after the isolation. Moreover, an intrahippocampal injection of the IL-1 receptor antagonist prior to the isolation period prevented both the BDNF downregulation and the memory impairments produced by the isolation. These data suggest that hippocampal-dependent memory impairments induced by elevated levels of brain IL-1beta may occur via an IL-1beta-induced downregulation in hippocampal BDNF.

An initial investigation of spinal mechanisms underlying pain enhancement induced by fractalkine, a neuronally released chemokine.

Fractalkine is a chemokine that is tethered to the extracellular surface of neurons. Fractalkine can be released, forming a diffusible signal. Spinal fractalkine (CX3CL1) is expressed by sensory afferents and intrinsic neurons, whereas its receptor (CX3CR1) is predominantly expressed by microglia. Pain enhancement occurs in response both to intrathecally administered fractalkine and to spinal fractalkine endogenously released by peripheral neuropathy. The present experiments examine whether fractalkine‐induced pain enhancement is altered by a microglial inhibitor (minocycline) and/or by antagonists/inhibitors of three putative glial products implicated in pain enhancement: interleukin‐1 (IL1), interleukin‐6 (IL6) and nitric oxide (NO). In addition, it extends a prior study that demonstrated that intrathecal fractalkine‐induced mechanical allodynia is blocked by a neutralizing antibody to the rat fractalkine receptor, CX3CR1. Here, intrathecal anti‐CX3CR1 also blocked fractalkine‐induced thermal hyperalgesia. Furthermore, blockade of microglial activation with minocycline prevented both fractalkine‐induced mechanical allodynia (von Frey test) and thermal hyperalgesia (Hargreaves test). Microglial activation appears to lead to the release of IL1, given that pretreatment with IL1 receptor antagonist blocked both fractalkine‐induced mechanical allodynia and thermal hyperalgesia. IL1 is not the only proinflammatory cytokine implicated, as a neutralizing antibody to rat IL6 also blocked fractalkine‐induced pain facilitation. Lastly, NO appears to be importantly involved, as l‐NAME, a broad‐spectrum NO synthase inhibitor, also blocked fractalkine‐induced effects. Taken together, these data support that neuronally released fractalkine enhances pain via activation of spinal cord glia. Thus, fractalkine may be a neuron‐to‐glia signal triggering pain facilitation.

The role of the habenular complex in the elevation of dorsal raphe nucleus serotonin and the changes in the behavioral responses produced by uncontrollable stress

Previous research indicates that the serotonergic neurons of the caudal dorsal raphe nucleus (DRN) are activated to a greater degree by inescapable shock (IS) as compared to escapable shock (ES), causing a greater release of serotonin (5-HT) in the DRN and in target regions. This differential activation is necessary for the behavioral changes that occur after exposure to IS, but not to ES (i.e. learned helplessness/behavioral depression). Although the critical role of the DRN in learned helplessness is clear, the neural inputs to the caudal DRN which result in this selective activation are unknown. One structure that may be involved in the activation of the DRN and the induction of learned helplessness/behavioral depression is the habenular complex. In experiment 1, habenula lesions eliminated the differential rise in DRN extracellular 5-HT levels in response to IS and ES exposure by severely attenuating the rise in 5-HT for both groups. In experiment 2, sham operated and habenula lesioned rats were exposed to either ES, IS or no stress (home cage control; HCC). Twenty-four hours later, sham rats previously exposed to IS exhibited longer escape latencies as compared to both ES and HCC rats (i.e. learned helplessness). The habenular lesion eliminated the differences in escape latency between groups, thus eliminating the induction of learned helplessness/behavioral depression. These results suggest that the habenula is necessary for the differential activation of the DRN and the escape deficits produced by IS.