Opioid activation of toll-like receptor 4 contributes to drug reinforcement.

Opioid action was thought to exert reinforcing effects solely via the initial agonism of opioid receptors. Here, we present evidence for an additional novel contributor to opioid reward: the innate immune pattern-recognition receptor, toll-like receptor 4 (TLR4), and its MyD88-dependent signaling. Blockade of TLR4/MD2 by administration of the nonopioid, unnatural isomer of naloxone, (+)-naloxone (rats), or two independent genetic knock-outs of MyD88-TLR4-dependent signaling (mice), suppressed opioid-induced conditioned place preference. (+)-Naloxone also reduced opioid (remifentanil) self-administration (rats), another commonly used behavioral measure of drug reward. Moreover, pharmacological blockade of morphine-TLR4/MD2 activity potently reduced morphine-induced elevations of extracellular dopamine in rat nucleus accumbens, a region critical for opioid reinforcement. Importantly, opioid-TLR4 actions are not a unidirectional influence on opioid pharmacodynamics, since TLR4(-/-) mice had reduced oxycodone-induced p38 and JNK phosphorylation, while displaying potentiated analgesia. Similar to our recent reports of morphine-TLR4/MD2 binding, here we provide a combination of in silico and biophysical data to support (+)-naloxone and remifentanil binding to TLR4/MD2. Collectively, these data indicate that the actions of opioids at classical opioid receptors, together with their newly identified TLR4/MD2 actions, affect the mesolimbic dopamine system that amplifies opioid-induced elevations in extracellular dopamine levels, therefore possibly explaining altered opioid reward behaviors. Thus, the discovery of TLR4/MD2 recognition of opioids as foreign xenobiotic substances adds to the existing hypothesized neuronal reinforcement mechanisms, identifies a new drug target in TLR4/MD2 for the treatment of addictions, and provides further evidence supporting a role for central proinflammatory immune signaling in drug reward.

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 stressor's 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 organism's 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.

Prostaglandins are necessary and sufficient to induce contextual fear learning impairments after interleukin-1 beta injections into the dorsal hippocampus.

The intra-hippocampal administration of interleukin-1beta (IL-1beta) as well as the induction of elevated but physiological levels of IL-1beta within the hippocampus interferes with the formation of long-term memory. There is evidence suggesting that the induction of prostaglandin (PG) formation by IL-1beta is involved in impairments in working and spatial memory following IL-1beta. The present experiments extend these findings by showing that PGs are responsible for memory deficits in contextual fear conditioning that occur following IL-1beta injection into the dorsal hippocampus of Sprague-Dawley rats. Cyclooxygenase (COX) inhibition blocked the disruption in contextual fear conditioning produced by IL-1beta and COX inhibition alone also disrupted contextual memory, suggesting an inverted U-shaped relationship between PG levels and memory. In addition to demonstrating the necessity of PGs in IL-1beta-mediated memory deficits, we also show that PGs injected directly into the dorsal hippocampus are sufficient to impair context memory and significantly reduce post-conditioning levels of BDNF within the hippocampus, suggesting a possible mechanism for the memory-impairing effects of PGs.

Expression of fibroblast growth factor-2 and brain-derived neurotrophic factor mRNA in the medial prefrontal cortex and hippocampus after uncontrollable or controllable stress.

Neurotrophic factors, including basic fibroblast growth factor (FGF-2) and brain-derived neurotrophic factor (BDNF) are known to be affected by exposure to stressful experiences. Here, we examine the effects of behaviorally controllable (escapable tailshock, ES) or uncontrollable (inescapable tailshock, IS) stress on the expression of FGF-2 and BDNF mRNA in subregions of the medial prefrontal cortex (mPFC) and the hippocampal formation (HF) of male Sprague-Dawley rats. ES rats were placed in Plexiglas boxes equipped with a free spinning wheel and IS rats were placed in identical boxes with the wheels fixed. ES and IS rats were yoked such that they received the same tailshocks, but the ES rat could terminate each shock for both rats. No stress controls (NS) remained in their home cages. Rats were killed 0, 2, 24, or 72 h after termination of the stress session. In situ hybridization was performed to measure FGF-2 and BDNF mRNA in the mPFC and HF. In the mPFC, ES produced a significant increase in FGF-2 mRNA expression at 0 and 2 h post-stress. In the HF, ES produced a greater increase in FGF-2 mRNA expression than IS and NS only in CA2. ES also produced an increase in BDNF mRNA expression in the anterior cingulate at 0 h post-stress. No effects of stressor controllability on BDNF were observed in the HF, although both ES and IS decreased BDNF mRNA in the DG. FGF-2 in the mPFC may be involved in emotional regulation ("coping") during stressful experiences.

CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.

We have reviewed a battery of useful tests for evaluating sensorimotor function and plasticity acutely and chronically in unilateral rat models of central nervous system injury. These tests include forelimb use for weight shifting during vertical exploration in a cylindrical enclosure, an adhesive removal test of sensory function, and forelimb placing. These tests monitor recovery of sensorimotor function independent of the extent of test experience. Data are presented for four models, including permanent focal ischemia, focal injury to the forelimb area of sensorimotor cortex, dopaminergic neurodegeneration of the nigrostriatal system, and cervical spinal cord injury. The effect of the dendrite growth promoting factor, Osteogenic Protein-1 (OP-1) on outcome following permanent middle cerebral artery (MCA) occlusion was used as an example to illustrate how the tests can be applied preclinically. OP-1 showed a beneficial effect on limb use asymmetry in the cylinder test.

Microinjection of urocortin 2 into the dorsal raphe nucleus activates serotonergic neurons and increases extracellular serotonin in the basolateral amygdala.

The intra dorsal raphe nucleus (DRN) administration of corticotropin releasing hormone (CRF) inhibits serotonergic (5-HT) activity in this structure, an effect blocked by antagonists selective for the type 1 CRF receptor (CRF1). The DRN has a high density of the type 2 receptor (CRF2), and so the present experiments explored the impact of CRF2 activation within the DRN on 5-HT function. The intra-DRN administration of the selective CRF2 agonist urocortin 2 (Ucn 2) dose dependently increased 5-HT efflux in the basolateral amygdala, a projection region of the DRN. Intra-DRN Ucn 2 also increased c-fos expression in labeled 5-HT neurons. Both of these effects of Ucn 2 were completely blocked by intra-DRN antisauvagine-30 (ASV-30), a relatively selective CRF2 antagonist. These data suggest that CRF1 and CRF2 activation within the DRN affect 5-HT neurons in opponent fashion. Implications of these results for understanding the behavioral effects of CRF and other CRF-like ligands are discussed.

Stress potentiation of morphine-induced dopamine efflux in the nucleus accumbens shell is dependent upon stressor uncontrollability and is mediated by the dorsal raphe nucleus.

A single session of uncontrollable (inescapable tailshock, IS), but not controllable (escapable tailshock, ES), stress is known to selectively potentiate subsequent morphine-conditioned place preference in a dorsal raphe nucleus (DRN) serotonin (5-HT) dependent manner. Here, in vivo microdialysis is used to test the hypothesis that prior IS, but not ES, will potentiate morphine-induced dopamine (DA) efflux in the nucleus accumbens (NAc) shell and that this will occur by a pathway involving DRN 5-HT neurons. Male Sprague-Dawley rats were exposed to yoked IS, ES, or no stress. Twenty-four hours later, morphine (3 mg/kg s.c.) or saline was administered during microdialysis. As predicted, prior IS selectively potentiated morphine-induced DA, but not 5-HT, efflux in the NAc. This potentiation was due to morphine's action in the DRN because it was blocked by intra-DRN microinjection of the opioid antagonist naltrexone (10 microg). IS potentiation of morphine-induced DA efflux in the NAc was also dependent upon activation of 5-HT neurons in the DRN because it was blocked by intra-DRN microinjection of the 5-HT1A autoreceptor agonist 8-hydroxy-2-di-n-(propylamino)-tetralin (1 microg). No effect of IS was found on morphine-induced 5-HT or DA efflux in the ventral tegmental area. These results suggest a neural substrate for stress potentiation of morphine reward involving 5-HT neurotransmission in the DRN.

Early overuse and disuse of the affected forelimb after moderately severe intraluminal suture occlusion of the middle cerebral artery in rats.

We have previously shown that early forced overuse of the affected forelimb worsens outcome following moderately severe transient focal cortical ischemic stroke in rats using a distal middle cerebral artery occlusion (MCAo) model. This effect may be site-dependent, because we have also found that early forced use of the affected limb after unilateral 6-OHDA induced degeneration of ascending nigrostriatal dopamine neurons markedly enhanced functional outcome and is neuroprotective. The present study examines the effects of early overuse and disuse following a moderately severe proximal MCAo model, by means of intraluminal suture occlusion. Ischemia was produced in male Long-Evans rats with 60 min of occlusion, or sham surgery was performed. Early overuse or disuse of the affected forelimb was forced by immobilizing either the ipsilateral or contralateral forelimb, respectively, in a plaster cast or the animal was left uncasted. Casts were removed on day 10 and sensorimotor testing was performed weekly during days 17-38. Animals were sacrificed on day 45 and brains were fixed for later cresyl violet staining. The MCAo+contralateral cast group performed worse than all other groups on tests of forelimb sensorimotor function. All MCAo groups regardless of cast condition had significant atrophy of the ischemic striatum, but there was no significant atrophy of the ischemic cortex in any group. Forced disuse, but not overuse, of the affected forelimb immediately following proximal ischemia using the intraluminal suture model has detrimental effects on functional outcome, without exaggerating anatomical damage. The effects of disuse and overuse during the first 10 days after stroke differ depending on cortical or subcortical involvement.

Movement-related glutamate levels in rat hippocampus, striatum, and sensorimotor cortex.

Changes in brain extracellular glutamate during movement stress were studied using in vivo microdialysis. Male Long-Evans rats were placed in a clear cylinder designed to elicit behavioral activation while undergoing microdialysis sampling from either the hippocampus, striatum or sensorimotor cortex. Glutamate levels were determined by high performance liquid chromatography with fluorescence detection in the dialysates taken before, during, and after exposure to the cylinder. Animals were in a behaviorally quiescent state before exposure to the cylinder, but they demonstrated increases in rearing, locomotion, and turning while in the cylinder. Dialysate glutamate levels were significantly enhanced in the samples taken while the rat was in the cylinder compared with samples taken before or after exposure to the cylinder. In a second study, rats were implanted with bilateral probes in the forelimb sensorimotor cortex, and one forelimb was immobilized by means of a plaster of paris cast. Glutamate, aspartate, serine, and taurine levels were quantified in casted animals. In casted animals, dialysate glutamate levels were lower on the side contralateral to the immobilized limb during both quiescence and movement stress. Aspartate and taurine, but not serine levels increased during movement stress in both the side contralateral and the side ipsilateral to the immobilized limb. These results suggest that there is extracellular overflow of glutamate and other neuroactive amino acids during spontaneous movement, and chronic disuse can suppress extracellular glutamate levels.

Corpus callosum damage and interhemispheric transfer of information following closed head injury in children.

We evaluated the relationship of corpus callosum atrophy and/or lesions on magnetic resonance imaging (MRI) to functional hemispheric disconnection following closed head injury (CHI) in 51 pediatric patients, including mild CHI, moderate to severe CHI with extracallosal lesions, and moderate to severe CHI with callosal atrophy and/or lesions. Interhemispheric transfer of information was assessed using auditory, motor, tactile, and visual tests in patients and in 16 uninjured children. Total and regional callosal areas were measured from the midsagittal MRI slice by morphometry. The corpus callosum lesion group demonstrated a greater right ear advantage on verbal dichotic listening than all other groups. Areas of the posterior corpus callosum were negatively correlated with laterality indices of verbal dichotic listening performance and tachistoscopic identification of verbal material. The relationship of corpus callosum atrophy and/or lesions to asymmetry in dichotic listening is consistent with previous investigation of posttraumatic hemispheric disconnection effects in adults.

Use-dependent exaggeration of brain injury: is glutamate involved?

Extreme overreliance on the impaired forelimb following unilateral lesions of the forelimb representation area of the rat sensorimotor cortex (FL-SMC) leads to exaggeration of the initial cortical injury. Glutamate has repeatedly been implicated in the secondary processes leading to neuronal death following traumatic insult, chiefly because of the neuroprotective properties of excitatory amino acid antagonists in a variety of animal models of brain injury. The present study investigated the possibility that NMDA receptor-mediated processes are involved in use-dependent exaggeration of neuronal injury. Rats were fitted with one-sleeved casts that immobilized the intact forelimb for the first 7 days following FL-SMC lesion, a procedure previously shown to result in use-dependent exaggeration of injury and more severe and persistent limb-use deficits. In the present investigation, administration of MK-801 (1 mg/kg ip once daily on alternate days) during the casting period spared neural tissue surrounding the lesion and enhanced functional recovery of the impaired forelimb. These results suggest a role for NMDA receptor-mediated processes in use-dependent exaggeration of injury.

Early exclusive use of the affected forelimb after moderate transient focal ischemia in rats : functional and anatomic outcome.

BACKGROUND AND PURPOSE: Previous work by researchers in our laboratory has shown that in the rat, the exclusive use of the affected forelimb during an early critical period exaggerates lesion volume and retards functional recovery after electrolytic lesions of the forelimb sensorimotor cortex. In the present study, we examined the effects of exclusive use of the affected forelimb after middle cerebral artery occlusion (MCAO). METHODS: Ischemia of moderate severity was produced in male Long-Evans rats through 45 minutes of occlusion of the left middle cerebral and both common carotid arteries. Exclusive use of either the affected or unaffected forelimb was forced through immobilization of either the ipsilateral (MCAO+ipsi) or contralateral (MCAO+contra) forelimb, respectively, for 10 days in a plaster cast, or the animal was left uncasted (MCAO+nocast). Sham surgeries were performed, and animals were also casted for 10 days or left uncasted. Sensorimotor testing was performed during days 17 to 38. At the end of sensorimotor testing, cognitive performance was tested with use of the Morris water maze. In a separate experiment, temperatures and corticosterone levels were measured during the 10-day period after 45-minute ischemia and casting. RESULTS: The MCAO+ipsi group performed worse on sensorimotor tasks than the MCAO+contra, MCAO+nocast, and sham groups. Infarct volume was significantly larger in the MCAO+ipsi group than in the sham and MCAO+contra groups but not in the MCAO+nocast group. No group differences were found with the Morris water maze, and no group differences were found in either temperature or plasma corticosterone level. CONCLUSIONS: The exclusive use of the affected forelimb immediately after focal ischemia has detrimental effects on sensorimotor function that cannot be attributed to hyperthermia or stress.