David B. Sprunger

Peripheral infection and aging interact to impair hippocampal memory consolidation

We report that a peripheral injection of Escherichia coli produces both anterograde and retrograde amnesia in 24 month old, but not 3 month old rats for memories that depend on the hippocampus, that is, memory of context, contextual fear, and place learning. The anterograde effect was restricted to measures of long-term memory. Short-term memory was not affected, nor did E. coli produce amnesia for auditory-cue fear conditioning. There were no age related effects on memory in vehicle-treated rats. In addition to these age-related cognitive effects of E.coli, we report that it produced a marked increased in IL-1beta levels in the hippocampus, but not in parietal cortex or serum. These findings support the hypothesis that age is a vulnerability factor that increases the likelihood that an immune challenge will produce a cognitive impairment. It is possible that this cognitive vulnerability is mediated by age-related changes in the glial environment that results in an exaggerated brain pro-inflammatory response to infection.

Proinflammatory cytokines oppose opioid-induced acute and chronic analgesia

Spinal proinflammatory cytokines are powerful pain-enhancing signals that contribute to pain following peripheral nerve injury (neuropathic pain). Recently, one proinflammatory cytokine, interleukin-1, was also implicated in the loss of analgesia upon repeated morphine exposure (tolerance). In contrast to prior literature, we demonstrate that the action of several spinal proinflammatory cytokines oppose systemic and intrathecal opioid analgesia, causing reduced pain suppression. In vitro morphine exposure of lumbar dorsal spinal cord caused significant increases in proinflammatory cytokine and chemokine release. Opposition of analgesia by proinflammatory cytokines is rapid, occurring < or =5 min after intrathecal (perispinal) opioid administration. We document that opposition of analgesia by proinflammatory cytokines cannot be accounted for by an alteration in spinal morphine concentrations. The acute anti-analgesic effects of proinflammatory cytokines occur in a p38 mitogen-activated protein kinase and nitric oxide dependent fashion. Chronic intrathecal morphine or methadone significantly increased spinal glial activation (toll-like receptor 4 mRNA and protein) and the expression of multiple chemokines and cytokines, combined with development of analgesic tolerance and pain enhancement (hyperalgesia, allodynia). Statistical analysis demonstrated that a cluster of cytokines and chemokines was linked with pain-related behavioral changes. Moreover, blockade of spinal proinflammatory cytokines during a stringent morphine regimen previously associated with altered neuronal function also attenuated enhanced pain, supportive that proinflammatory cytokines are importantly involved in tolerance induced by such regimens. These data implicate multiple opioid-induced spinal proinflammatory cytokines in opposing both acute and chronic opioid analgesia, and provide a novel mechanism for the opposition of acute opioid analgesia.

Memory for context is impaired by a post context exposure injection of interleukin-1 beta into dorsal hippocampus.

Prior research has revealed that treatments that elevate the level of the pro-inflammatory cytokine IL-1beta in the brain, if given after training, impair contextual but not auditory-cue fear conditioning. The present experiments add to these finding by showing that, (a) IL-1beta exerts its effect on contextual fear conditioning by impairing consolidation processes that support the storage of the memory representation of the context; (b) the dorsal hippocampus is a critical site for the effect of IL-1beta; (c) the effect of IL-1beta cannot be attributed to its effect on glucocorticoid levels; and (d) IL-1beta injected into dorsal hippocampus either, immediately, 3, or 24 h, but not 48 h, after training produces this impairment. At this time the mechanisms responsible for this impairment are not understood, but may involve late-phase protein synthesis processes associated with LTP, because later consolidation processes are being disrupted.

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.

BDNF mRNA expression in rat hippocampus following contextual learning is blocked by intrahippocampal IL-1β administration

The present study examined the modulating effects of an intrahippocampal injection of interleukin-1beta (IL-1beta) on brain-derived neurotrophic factor (BDNF) mRNA expression 0.5, 2, 4, and 6 h following contextual fear conditioning, a task known to increase BDNF mRNA, in rats. Contextual fear conditioning produced a time-dependent increase in BDNF mRNA that varied by region of hippocampus. IL-1beta blocked or reduced these increases in BDNF mRNA in the CA1, CA2, and dentate gyrus regions of the hippocampus, but had no effect in cortical regions. These data support the idea that IL-1beta-produced memory deficits may be mediated via BDNF mRNA reductions in hippocampus.

Amygdala Regulation of Immediate-Early Gene Expression in the Hippocampus Induced by Contextual Fear Conditioning

The basolateral nuclei of the amygdala (BLA) are thought to modulate memory storage in other brain regions (McGaugh, 2004). We reported that BLA modulates the memory for both an explored context and for contextual fear conditioning. Both of these memories depend on the hippocampus. Here, we examined the hypothesis that the BLA exerts its modulatory effect by regulating the expression of immediate-early genes (IEGs) in the hippocampus. The main findings of these experiments were: (1) Arc activity-regulated cytoskeletal protein (Arc), an immediate-early gene (also termed Arg 3.1) and c-fos mRNA are induced in the hippocampus after a context exposure, or context plus shock experience, but not after an immediate shock; and (2) BLA inactivation with muscimol attenuated the increase in Arc and c-fos mRNA in the hippocampus associated with contextual fear conditioning but did not influence Arc mRNA associated with context exploration. These results support the hypothesis that the amygdala modulates contextual fear memory by regulating expression of IEGs in the hippocampus.

The role of dorsal hippocampus and basolateral amygdala NMDA receptors in the acquisition and retrieval of context and contextual fear memories.

The authors used 3-phase context preexposure facilitation methodology to study the contribution of N-methyl-D-aspartate (NMDA) receptors in dorsal hippocampus (DH) and the basal lateral region of the amygdala (BLA) to (a) acquisition of the context memory, (b) retrieval of the context memory, (c) acquisition of context-shock association, and (d) retrieval of the context-shock association. The NMDA receptor antagonist D-2-amino-5 phosphonopentanoic acid (D-AP5) was injected into either the DH or BLA prior to (a) the context preexposure phase, (b) the immediate shock phase, or (c) the test for contextual fear. Antagonizing NMDA receptors in the DH impaired the acquisition of the context memory but did not affect its retrieval or retrieval of the fear memory. Antagonizing NMDA receptors with D-AP5 in the BLA impaired acquisition of the context-shock association but had no effect on the expression of fear. However, both DL-AP5 and L-AP5 reduced the expression of fear when they were injected into the amygdala prior to testing for contextual fear.

Differential effects of neonatal handling on early life infection-induced alterations in cognition in adulthood

We have previously demonstrated that bacterial infection (Escherichia coli) in neonatal rats is associated with impaired memory in a fear-conditioning task in adulthood. This impairment, however, is only observed if a peripheral immune challenge (lipopolysaccharide; LPS) is administered around the time of learning. We used a brief separation/handling paradigm to determine if the adult memory impairment associated with neonatal-infection could be prevented. Naturally occurring variations in maternal care promote striking variations in offspring cognitive development, and handling paradigms are used to manipulate the quality and quantity of maternal care. Rats were injected on post natal (P) day 4 with E. coli or PBS, and half from each group were handled for 15 min/day from P4 to 20. All rats were then tested in adulthood. Neonatal handling of rats infected as neonates prevented the increase in microglial cell marker reactivity within the hippocampus, and the exaggerated brain IL-1beta production to LPS normally produced by the infection. Thus, these neural processes were now comparable to levels of non-infected PBS controls. Furthermore, handling completely prevented LPS-induced memory impairment in a context-fear task in adult rats infected as neonates. Finally, neonatal handling dramatically improved spatial learning and memory and decreased anxiety in rats treated early with PBS, but had no beneficial effect on these measures in rats infected as neonates. Taken together, these data suggest that maternal care may profoundly influence neuroinflammatory processes in adulthood, and that infection may also prevent maternal care influences on cognition later in life.

Indwelling supradural catheters for induction of facial allodynia: surgical procedures, application of inflammatory stimuli, and behavioral testing.

Migraine headaches are debilitatingly painful and poorly managed. Facial allodynia is often associated with migraine, and clinical evidence indicates that it is a critical point in migraine progression. That is, if the migraine can be treated prior to the onset of facial allodynia, the migraine can be halted using triptans, whereas if treatment is administered after facial allodynia has begun, the treatment is ineffective. The meninges and the immune cells therein have been implicated in migraine facial pain. Indeed, application of inflammatory mediators over the meninges has been used to study changes in pain responsive neurons in trigeminal complex, and changes in their receptive fields. Much of this research has been carried out in anesthetized rats, which limits the clinical application. Our indwelling supradural catheter model, in which inflammatory mediators can be administered to the meninges in awake and freely moving rats, allows for the assessment of behavioral changes shortly after injection. Following administration of inflammatory soup (histamine, serotonin, bradykinin, and prostaglandin E2) or the immunogenic HIV-1 coat protein gp120 results in reliable periorbital mechanical allodynia. This model provides an additional means to study the neurocircuitry and neuropharmacology of facial allodynia. Here, we describe detailed methods for the placement of the catheter, injection procedures, and assessment of facial allodynia.

Supradural inflammatory soup in awake and freely moving rats induces facial allodynia that is blocked by putative immune modulators

Facial allodynia is a migraine symptom that is generally considered to represent a pivotal point in migraine progression. Treatment before development of facial allodynia tends to be more successful than treatment afterwards. As such, understanding the underlying mechanisms of facial allodynia may lead to a better understanding of the mechanisms underlying migraine. Migraine facial allodynia is modeled by applying inflammatory soup (histamine, bradykinin, serotonin, prostaglandin E2) over the dura. Whether glial and/or immune activation contributes to such pain is unknown. Here we tested if trigeminal nucleus caudalis (Sp5C) glial and/or immune cells are activated following supradural inflammatory soup, and if putative glial/immune inhibitors suppress the consequent facial allodynia. Inflammatory soup was administered via bilateral indwelling supradural catheters in freely moving rats, inducing robust and reliable facial allodynia. Gene expression for microglial/macrophage activation markers, interleukin-1β, and tumor necrosis factor-α increased following inflammatory soup along with robust expression of facial allodynia. This provided the basis for pursuing studies of the behavioral effects of 3 diverse immunomodulatory drugs on facial allodynia. Pretreatment with either of two compounds broadly used as putative glial/immune inhibitors (minocycline, ibudilast) prevented the development of facial allodynia, as did treatment after supradural inflammatory soup but prior to the expression of facial allodynia. Lastly, the toll-like receptor 4 (TLR4) antagonist (+)-naltrexone likewise blocked development of facial allodynia after supradural inflammatory soup. Taken together, these exploratory data support that activated glia and/or immune cells may drive the development of facial allodynia in response to supradural inflammatory soup in unanesthetized male rats.