Neural Markers of Vulnerability to Anxiety Outcomes after Traumatic Brain Injury.

Anxiety outcomes after traumatic brain injury (TBI) are complex, and the underlying neural mechanisms are poorly understood. Here, we developed a multi-dimensional behavioral profiling approach to investigate anxiety-like outcomes in mice that takes into account individual variability. Departing from the tradition of comparing outcomes in TBI versus sham groups, we identified a subgroup within the TBI group that is vulnerable to anxiety dysfunction, and present increased exploration of the anxiogenic zone compared to sham controls or resilient injured animals, by applying dimensionality reduction, clustering, and post hoc validation to behavioral data obtained from multiple assays for anxiety at several post-injury time points. These vulnerable animals expressed distinct molecular profiles in the corticolimbic network, with downregulation in gamma-aminobutyric acid and glutamate and upregulation in neuropeptide Y markers. Indeed, among vulnerable animals, not resilient or sham controls, severity of anxiety-related outcomes correlated strongly with expression of molecular markers. Our results establish a foundational approach, with predictive power, for reliably identifying maladaptive anxiety outcomes after TBI and uncovering neural signatures of vulnerability to anxiety.

Long-Term Effects of Traumatic Brain Injury on Anxiety-Like Behaviors in Mice: Behavioral and Neural Correlates.

Traumatic brain injury (TBI) has been frequently linked to affective disorders such as anxiety and depression. However, much remains to be understood about the underlying molecular and signaling mechanisms that mediate affective dysfunctions following injury. A lack of consensus in animal studies regarding what the affective sequelae of TBI are has been a major hurdle that has slowed progress, with studies reporting the full range of effects: increase, decrease, and no change in anxiety following injury. Here, we addressed this issue directly by investigating long-term anxiety outcomes in mice following a moderate to severe controlled cortical impact (CCI) injury using a battery of standard behavioral tests-the open field (OF), elevated zero maze (EZM), and elevated plus maze (EPM). Mice were tested on weeks 1, 3, 5 and 7 post-injury. Our results show that the effect of injury is time- and task-dependent. Early on-up to 3 weeks post-injury, there is an increase in anxiety-like behaviors in the elevated plus and zero mazes. However, after 5 weeks post-injury, anxiety-like behavior decreases, as measured in the OF and EZM. Immunostaining in the basolateral amygdala (BLA) for GAD, a marker for GABA, at the end of the behavioral testing showed the late decrease in anxiety behavior was correlated with upregulation of inhibition. The approach adopted in this study reveals a complex trajectory of affective outcomes following injury, and highlights the importance of comparing outcomes in different assays and time-points, to ensure that the affective consequences of injury are adequately assessed.

Non-pain-related CRF1 activation in the amygdala facilitates synaptic transmission and pain responses.

BACKGROUND: Corticotropin-releasing factor (CRF) plays an important role in affective states and disorders. CRF is not only a "stress hormone" but also a neuromodulator outside the hypothalamic-pituitary-adrenocortical (HPA) axis. The amygdala, a brain center for emotions, is a major site of extrahypothalamic expression of CRF and its G-protein-coupled receptors. Our previous studies showed that endogenous activation of CRF1 receptors in an arthritis pain model contributes to amygdala hyperactivity and pain-related behaviors. Here we examined the synaptic and behavioral effects of CRF in the amygdala of normal animals in the absence of tissue injury or disease. RESULTS: Whole-cell patch-clamp recordings of neurons in the latero-capsular division of the central nucleus of the amygdala (CeLC) in brain slices from normal rats showed that CRF (0.1-10 nM) increased excitatory postsynaptic currents (EPSCs) at the "nociceptive" parabrachio-amygdaloid (PB-CeLC) synapse and also increased neuronal output. Synaptic facilitation involved a postsynaptic action and was blocked by an antagonist for CRF1 (NBI27914, 1 µM) but not CRF2 (astressin-2B, 1 µM) and by an inhibitor of PKA (KT5720, 1 µM) but not PKC (GF109203X, 1 µM). CRF increased a latent NMDA receptor-mediated EPSC, and this effect also required CRF1 and PKA but not CRF2 and PKC. Stereotaxic administration of CRF (10 µM, concentration in microdialysis probe) into the CeLC by microdialysis in awake rats increased audible and ultrasonic vocalizations and decreased hindlimb withdrawal thresholds. Behavioral effects of CRF were blocked by a NBI27914 (100 µM) and KT5720 (100 µM) but not GF109203x (100 µM). CRF effects persisted when HPA axis function was suppressed by pretreatment with dexamethasone (50 µg/kg, subcutaneously). CONCLUSIONS: Non-pain-related activation of CRF1 receptors in the amygdala can trigger pain-responses in normal animals through a mechanism that involves PKA-dependent synaptic facilitation in CeLC neurons independent of HPA axis function. The results suggest that conditions of increased amygdala CRF levels can contribute to pain in the absence of tissue pathology or disease state.

Age is a determinant of leukocyte infiltration and loss of cortical volume after traumatic brain injury.

There is increasing evidence that the inflammatory response differs in the injured developing brain as compared to the adult brain. Here we compared cerebral blood flow and profiled the inflammatory response in mice that had been subjected to traumatic brain injury (TBI) at postnatal day (P)21 or at adulthood. Relative blood flow, determined by laser Doppler, revealed a 30% decrease in flow immediately after injury followed by prominent hyperemia between 7 and 35 days after injury in both age groups. The animals were euthanized at 1-35 days after injury and the brains prepared for the immunolocalization and quantification of CD45-, GR-1-, CD4- and CD8-positive (+) cells. On average, the number of CD45+ leukocytes in the cortex was significantly higher in the P21 as compared to the adult group. A similar trend was seen for GR-1+ granulocytes, whereas no age-related differences were noted for CD4+ and CD8+ cells. While CD45+ and GR-1+ cells in the P21 group remained elevated, relative to shams, over the first 2 weeks after injury, the adult group showed a time course limited to the first 3 days after injury. The loss of ipsilateral cortical volumes at 2 weeks after injury was significantly greater in the adult relative to the P21 group. While the adult group showed no further change in cortical volumes, there was a significant loss of cortical volumes between 2 and 5 weeks after injury in the P21 group, reaching values similar to that of the adult group by 5 weeks after injury. Together, these findings demonstrate age-dependent temporal patterns of leukocyte infiltration and loss of cortical volume after TBI.

Matrix metalloproteinases and neurotrauma: evolving roles in injury and reparative processes.

Matrix metalloproteinases (MMPs) are involved in a wide range of proteolytic events in fetal development and normal tissue remodeling as well as wound healing and inflammation. In the CNS, they have been implicated in a variety of neurodegenerative diseases ranging from multiple sclerosis to Alzheimer disease and are integral to stroke-related cell damage. Although studies implicate increased activity of MMPs in pathogenesis in the CNS, there is also a growing literature to support their participation in events that support recovery processes. Here the authors provide a brief overview of MMPs and their regulation, address their complex roles following traumatic injuries to the adult and developing CNS, and consider their time- and context-dependent signatures that influence both injury and reparative processes.

Facilitation of synaptic transmission and pain responses by CGRP in the amygdala of normal rats.

Calcitonin gene-related peptide (CGRP) plays an important role in peripheral and central sensitization. CGRP also is a key molecule in the spino-parabrachial-amygdaloid pain pathway. Blockade of CGRP1 receptors in the spinal cord or in the amygdala has antinociceptive effects in different pain models. Here we studied the electrophysiological mechanisms of behavioral effects of CGRP in the amygdala in normal animals without tissue injury.Whole-cell patch-clamp recordings of neurons in the latero-capsular division of the central nucleus of the amygdala (CeLC) in rat brain slices showed that CGRP (100 nM) increased excitatory postsynaptic currents (EPSCs) at the parabrachio-amygdaloid (PB-CeLC) synapse, the exclusive source of CGRP in the amygdala. Consistent with a postsynaptic mechanism of action, CGRP increased amplitude, but not frequency, of miniature EPSCs and did not affect paired-pulse facilitation. CGRP also increased neuronal excitability. CGRP-induced synaptic facilitation was reversed by an NMDA receptor antagonist (AP5, 50 microM) or a PKA inhibitor (KT5720, 1 microM), but not by a PKC inhibitor (GF109203X, 1 microM). Stereotaxic administration of CGRP (10 microM, concentration in microdialysis probe) into the CeLC by microdialysis in awake rats increased audible and ultrasonic vocalizations and decreased hindlimb withdrawal thresholds. Behavioral effects of CGRP were largely blocked by KT5720 (100 microM) but not by GF109203X (100 microM).The results show that CGRP in the amygdala exacerbates nocifensive and affective behavioral responses in normal animals through PKA- and NMDA receptor-dependent postsynaptic facilitation. Thus, increased CGRP levels in the amygdala might trigger pain in the absence of tissue injury.

Models of traumatic cerebellar injury.

Traumatic brain injury (TBI) is a major cause of morbidity and mortality worldwide. Studies of human TBI demonstrate that the cerebellum is sometimes affected even when the initial mechanical insult is directed to the cerebral cortex. Some of the components of TBI, including ataxia, postural instability, tremor, impairments in balance and fine motor skills, and even cognitive deficits, may be attributed in part to cerebellar damage. Animal models of TBI have begun to explore the vulnerability of the cerebellum. In this paper, we review the clinical presentation, pathogenesis, and putative mechanisms underlying cerebellar damage with an emphasis on experimental models that have been used to further elucidate this poorly understood but important aspect of TBI. Animal models of indirect (supratentorial) trauma to the cerebellum, including fluid percussion, controlled cortical impact, weight drop impact acceleration, and rotational acceleration injuries, are considered. In addition, we describe models that produce direct trauma to the cerebellum as well as those that reproduce specific components of TBI including axotomy, stab injury, in vitro stretch injury, and excitotoxicity. Overall, these models reveal robust characteristics of cerebellar damage including regionally specific Purkinje cell injury or loss, activation of glia in a distinct spatial pattern, and traumatic axonal injury. Further research is needed to better understand the mechanisms underlying the pathogenesis of cerebellar trauma, and the experimental models discussed here offer an important first step toward achieving that objective.

PKA and ERK, but not PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior.

The laterocapsular division of the central nucleus of the amygdala (CeLC) has emerged as an important site of pain-related plasticity and pain modulation. Glutamate and neuropeptide receptors in the CeLC contribute to synaptic and behavioral changes in the arthritis pain model, but the intracellular signaling pathways remain to be determined. This study addressed the role of PKA, PKC, and ERK in the CeLC. Adult male Sprague-Dawley rats were used in all experiments. Whole-cell patch-clamp recordings of CeLC neurons were made in brain slices from normal rats and from rats with a kaolin/carrageenan-induced monoarthritis in the knee (6 h postinduction). Membrane-permeable inhibitors of PKA (KT5720, 1 microM; cAMPS-Rp, 10 microM) and ERK (U0126, 1 microM) activation inhibited synaptic plasticity in slices from arthritic rats but had no effect on normal transmission in control slices. A PKC inhibitor (GF109203x, 1 microM) and an inactive structural analogue of U0126 (U0124, 1 microM) had no effect. The NMDA receptor-mediated synaptic component was inhibited by KT5720 or U0126; their combined application had additive effects. U0126 did not inhibit synaptic facilitation by forskolin-induced PKA-activation. Administration of KT5720 (100 microM, concentration in microdialysis probe) or U0126 (100 microM) into the CeLC, but not striatum (placement control), inhibited audible and ultrasonic vocalizations and spinal reflexes of arthritic rats but had no effect in normal animals. GF109203x (100 microM) and U0124 (100 microM) did not affect pain behavior. The data suggest that in the amygdala PKA and ERK, but not PKC, contribute to pain-related synaptic facilitation and behavior by increasing NMDA receptor function through independent signaling pathways.

Spinal CGRP1 receptors contribute to supraspinally organized pain behavior and pain-related sensitization of amygdala neurons.

CGRP receptor activation has been implicated in peripheral and central sensitization. The role of spinal CGRP receptors in supraspinal pain processing and higher integrated pain behavior is not known. Here we studied the effect of spinal inhibition of CGRP1 receptors on supraspinally organized vocalizations and activity of amygdala neurons. Our previous studies showed that pain-related audible and ultrasonic vocalizations are modulated by the central nucleus of the amygdala (CeA). Vocalizations in the audible and ultrasonic range and hindlimb withdrawal thresholds were measured in awake adult rats before and 5-6h after induction of arthritis by intra-articular injections of kaolin and carrageenan into one knee. Extracellular single-unit recordings were made from neurons in the latero-capsular division of the CeA (CeLC) in anesthetized rats before and after arthritis induction. CGRP1 receptor antagonists were applied to the lumbar spinal cord intrathecally (5 microl/min) 6h postinduction of arthritis. Spinal administration of peptide (CGRP8-37, 1 microM) and non-peptide (BIBN4096BS, 1 microM) CGRP1 receptor antagonists significantly inhibited the increased responses of CeLC neurons to mechanical stimulation of the arthritic knee but had no effect under normal conditions. In arthritic rats, the antagonists also inhibited the audible and ultrasonic components of vocalizations evoked by noxious stimuli and increased the threshold of hindlimb withdrawal reflexes. The antagonists had no effect on vocalizations and spinal reflexes in normal rats. These data suggest that spinal CGRP1 receptors are not only important for spinal pain mechanisms but also contribute significantly to the transmission of nociceptive information to the amygdala and to higher integrated behavior.

Techniques for assessing knee joint pain in arthritis.

The assessment of pain is of critical importance for mechanistic studies as well as for the validation of drug targets. This review will focus on knee joint pain associated with arthritis. Different animal models have been developed for the study of knee joint arthritis. Behavioral tests in animal models of knee joint arthritis typically measure knee joint pain rather indirectly. In recent years, however, progress has been made in the development of tests that actually evaluate the sensitivity of the knee joint in arthritis models. They include measurements of the knee extension angle struggle threshold, hind limb withdrawal reflex threshold of knee compression force, and vocalizations in response to stimulation of the knee. A discussion of pain assessment in humans with arthritis pain conditions concludes this review.

Pain-related synaptic plasticity in spinal dorsal horn neurons: role of CGRP.

BACKGROUND: The synaptic and cellular mechanisms of pain-related central sensitization in the spinal cord are not fully understood yet. Calcitonin gene-related peptide (CGRP) has been identified as an important molecule in spinal nociceptive processing and ensuing behavioral responses, but its contribution to synaptic plasticity, cellular mechanisms and site of action in the spinal cord remain to be determined. Here we address the role of CGRP in synaptic plasticity in the spinal dorsal horn in a model of arthritic pain. RESULTS: Whole-cell current- and voltage-clamp recordings were made from substantia gelatinosa (SG) neurons in spinal cord slices from control rats and arthritic rats (> 6 h postinjection of kaolin/carrageenan into the knee). Monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of afferents in the dorsal root near the dorsal root entry zone. Neurons in slices from arthritic rats showed increased synaptic transmission and excitability compared to controls. A selective CGRP1 receptor antagonist (CGRP8-37) reversed synaptic plasticity in neurons from arthritic rats but had no significant effect on normal transmission. CGRP facilitated synaptic transmission in the arthritis pain model more strongly than under normal conditions where both facilitatory and inhibitory effects were observed. CGRP also increased neuronal excitability. Miniature EPSC analysis suggested a post- rather than pre-synaptic mechanism of CGRP action. CONCLUSION: This study is the first to show synaptic plasticity in the spinal dorsal horn in a model of arthritic pain that involves a postsynaptic action of CGRP on SG neurons.

Impaired inflammatory pain and thermal hyperalgesia in mice expressing neuron-specific dominant negative mitogen activated protein kinase kinase (MEK).

BACKGROUND: Numerous studies have implicated spinal extracellular signal-regulated kinases (ERKs) as mediators of nociceptive plasticity. These studies have utilized pharmacological inhibition of MEK to demonstrate a role for ERK signaling in pain, but this approach cannot distinguish between effects of ERK in neuronal and non-neuronal cells. The present studies were undertaken to test the specific role of neuronal ERK in formalin-induced inflammatory pain. Dominant negative MEK (DN MEK) mutant mice in which MEK function is suppressed exclusively in neurons were tested in the formalin model of inflammatory pain. RESULTS: Formalin-induced second phase spontaneous pain behaviors as well as thermal hyperalgesia measured 1 - 3 hours post-formalin were significantly reduced in the DN MEK mice when compared to their wild type littermate controls. In addition, spinal ERK phosphorylation following formalin injection was significantly reduced in the DN MEK mice. This was not due to a reduction of the number of unmyelinated fibers in the periphery, since these were almost double the number observed in wild type controls. Further examination of the effects of suppression of MEK function on a downstream target of ERK phosphorylation, the A-type potassium channel, showed that the ERK-dependent modulation of the A-type currents is significantly reduced in neurons from DN MEK mice compared to littermate wild type controls. CONCLUSION: Our results demonstrate that the neuronal MEK-ERK pathway is indeed an important intracellular cascade that is associated with formalin-induced inflammatory pain and thermal hyperalgesia.

Inflammation persistently enhances nocifensive behaviors mediated by spinal group I mGluRs through sustained ERK activation.

Group I metabotropic glutamate receptors (mGluRs) and their downstream signaling pathways, which involve the extracellular signal-regulated kinases (ERKs), have been implicated as mediators of plasticity in several pain models. In this study, we report that inflammation leads to a long-lasting enhancement of behavioral responses induced by activation of spinal group I mGluRs. Thus, the nocifensive response to intrathecal injection of the group I mGluR agonist (RS)-3,5-Dihydroxyphenylglycine (DHPG) is significantly potentiated seven days following Complete Freund's Adjuvant (CFA)-induced inflammation of the hind paw. This potentiation is not associated with increased mGlu1 or mGlu5 receptor expression but is associated with increased levels of phosphorylated ERK in dorsal horn neurons. We also tested whether the increased behavioral response to DHPG following inflammation may be explained by enhanced coupling of the group I mGluRs to ERK activation. DHPG-induced ERK phosphorylation in the dorsal horn is not potentiated following inflammation. However, inhibiting ERK activation using a MEK inhibitor, U0126, following inflammation attenuates the intrathecal DHPG-induced behavioral responses to a greater extent than in control animals. The results from this study indicate that persistent ERK activation is required for the enhanced behavioral responses to spinal group I mGluR activation following inflammation and suggest that tonic modulation of ERK activity may underlie a component of central sensitization in dorsal horn neurons.

Modulation of presynaptic calcium transients by metabotropic glutamate receptor activation: a differential role in acute depression of synaptic transmission and long-term depression.

Activation of group I metabotropic glutamate receptors (mGluRs) can induce acute depression of excitatory synaptic transmission and long-term depression (LTD) in area CA1 of the rat hippocampus. The underlying mechanisms for both forms of depression are unknown. By measuring presynaptic calcium transients, we show that a reduction in the stimulation-induced presynaptic calcium rise that triggers vesicular release causes the acute depression of transmission by group I mGluRs. In contrast, the mechanism underlying mGluR-induced LTD does not involve a persistent change in stimulation-induced calcium influx. However, analysis of paired-pulse facilitation experiments suggests a presynaptic location for expression of this form of LTD. Furthermore, we show that mGluR-induced LTD can be completely blocked by a specific mGluR5 antagonist, whereas mGluR1 antagonists strongly attenuate the acute depression of transmission. These results support the hypothesis that the acute depression of transmission caused by activation of group I mGluRs involves regulation of stimulation-induced presynaptic calcium transients, whereas mGluR-induced LTD involves a distinct presynaptic modulation downstream of calcium influx.