Roles of NMDA NR2B subtype receptor in prefrontal long-term potentiation and contextual fear memory.

Cortical plasticity is thought to be important for the establishment, consolidation, and retrieval of permanent memory. Hippocampal long-term potentiation (LTP), a cellular mechanism of learning and memory, requires the activation of glutamate N-methyl-D-aspartate (NMDA) receptors. In particular, it has been suggested that NR2A-containing NMDA receptors are involved in LTP induction, whereas NR2B-containing receptors are involved in LTD induction in the hippocampus. However, LTP in the prefrontal cortex is less well characterized than in the hippocampus. Here we report that the activation of the NR2B and NR2A subunits of the NMDA receptor is critical for the induction of cingulate LTP, regardless of the induction protocol. Furthermore, pharmacological or genetic blockade of the NR2B subunit in the cingulate cortex impaired the formation of early contextual fear memory. Our results demonstrate that the NR2B subunit of the NMDA receptor in the prefrontal cortex is critically involved in both LTP and contextual memory.

Long-term memory deficits in Pavlovian fear conditioning in Ca2+/calmodulin kinase kinase alpha-deficient mice.

Signaling by the Ca(2+)/calmodulin kinase (CaMK) cascade has been implicated in neuronal gene transcription, synaptic plasticity, and long-term memory consolidation. The CaM kinase kinase alpha (CaMKKalpha) isoform is an upstream component of the CaMK cascade whose function in different behavioral and learning and memory paradigms was analyzed by targeted gene disruption in mice. CaMKKalpha mutants exhibited normal long-term spatial memory formation and cued fear conditioning but showed deficits in context fear during both conditioning and long-term follow-up testing. They also exhibited impaired activation of the downstream kinase CaMKIV/Gr and its substrate, the transcription factor cyclic AMP-responsive element binding protein (CREB) upon fear conditioning. Unlike CaMKIV/Gr-deficient mice, the CaMKKalpha mutants exhibited normal long-term potentiation and normal levels of anxiety-like behavior. These results demonstrate a selective role for CaMKKalpha in contextual fear memory and suggest that different combinations of upstream and downstream components of the CaMK cascade may serve distinct physiological functions.

Alteration of cingulate long-term plasticity and behavioral sensitization to inflammation by environmental enrichment.

Exposure to an enriched environment (EE) has been shown to induce cortical plasticity. Considerable amount of research is focused on the effects of EE in the hippocampus; however, effects of EE on other brain regions and the mechanisms involved are not well known. To investigate this, we induced cortical plasticity by placing mice in an EE for one month and measured the effects of EE in the anterior cingulate cortex (ACC). Here, we show that EE enhanced the expression of the plasticity gene, egr-1, in the ACC of EE animals accompanied by enhanced cingulate long-term potentiation (LTP) and decreased cingulate long-term depression (LTD). The increased NMDA receptor NR2B/NR2A subunits current ratio is associated with the plasticity seen in the ACC while total protein levels remain unchanged. Furthermore, behavioral experiments show that these mice exposed to EE demonstrate enhanced responses to acute and long-term inflammation. Our findings suggest that exposure to EE alters physiological properties within the ACC which results in enhanced responses to inflammation.

Enhanced presynaptic neurotransmitter release in the anterior cingulate cortex of mice with chronic pain.

The anterior cingulate cortex (ACC) is a forebrain structure known for its roles in learning and memory. Recent studies show that painful stimuli activate the prefrontal cortex and that brain chemistry is altered in this area in patients with chronic pain. Components of the CNS that are involved in pain transmission and modulation, from the spinal cord to the ACC, are very plastic and undergo rapid and long-term changes after injury. Patients suffering from chronic pain often complain of memory and concentration difficulties, but little is known about the neural circuitry underlying these deficits. To address this question, we analyzed synaptic transmission in the ACC from mice with chronic pain induced by hindpaw injection of complete Freund's adjuvant (CFA). In vitro whole-cell patch-clamp recordings revealed a significant enhancement in neurotransmitter release probability in ACC synapses from mice with chronic pain. Trace fear memory, which requires sustained attention and the activity of the ACC, was impaired in CFA-injected mice. Using knock-out mice, we found that calmodulin-stimulated adenylyl cyclases, AC1 and/or AC8, were crucial in mediating the long-lasting enhanced presynaptic transmitter release in the ACC of mice with chronic pain. Our findings provide strong evidence that presynaptic alterations caused by peripheral inflammation contribute to memory impairments after injury.

Altered behavioral responses to noxious stimuli and fear in glutamate receptor 5 (GluR5)- or GluR6-deficient mice.

Different kainate receptor (KAR) subtypes contribute to the regulation of both excitatory and inhibitory transmission. However, no study has reported a role for KAR subtypes in behavioral responses to persistent pain and fear memory. Here we show that responses to capsaicin or inflammatory pain were significantly reduced in mice lacking glutamate receptor 5 (GluR5) but not GluR6 subunits. In classic fear-memory tests, mice lacking GluR6 but not GluR5 showed a significant reduction in fear memory when measured 3, 7, or 14 d after training. Additionally, synaptic potentiation was significantly reduced in the lateral amygdala of GluR6 but not GluR5 knock-out mice. Our findings provide evidence that distinct KAR subtypes contribute to chemical/inflammatory pain and fear memory. Selectively targeting different KAR subtypes may provide a useful strategy for treating persistent pain and fear-related mental disorders.

Deficits in trace fear memory and long-term potentiation in a mouse model for fragile X syndrome.

Trace fear memory requires the activity of the anterior cingulate cortex (ACC) and is sensitive to attention-distracting stimuli. Fragile X syndrome is the most common form of mental retardation with many patients exhibiting attention deficits. Previous studies in fragile X mental retardation 1 (FMR1) knock-out (KO) mice, a mouse model for fragile X, focused mainly on hippocampal-dependent plasticity and spatial memory. We demonstrate that FMR1 knock-out mice show a defect in trace fear memory without changes in locomotion, anxiety, and pain sensitivity. Whole-cell path-clamp recordings in the ACC show that long-term potentiation (LTP) was completely abolished. A similar decrease in LTP was found in the lateral amygdala, another structure implicated in fear memory. No significant changes were found in basal synaptic transmission. This suggests that synaptic plasticity in the ACC and amygdala of FMR1 KO mice plays an important role in the expression of behavioral phenotypes similar to the symptoms of fragile X syndrome.

Upregulation of forebrain NMDA NR2B receptors contributes to behavioral sensitization after inflammation.

Transgenic overexpression of NMDA NR2B receptors in forebrain regions increased behavioral responses to persistent inflammatory pain. However, it is not known whether inflammation leads to the upregulation of NR2B receptors in these regions. Here, we show that peripheral inflammation increased the expression of NMDA NR2B receptors and NR2B receptor-mediated synaptic currents in the anterior cingulate cortex (ACC). In freely moving mice, the increase in NR2B receptors after inflammation contributed to enhanced NMDA receptor-mediated responses in the ACC. Inhibition of NR2B receptors in the ACC selectively reduced behavioral sensitization related to inflammation. Our results demonstrate that the upregulation of NR2B receptors in the ACC contributes to behavioral sensitization caused by inflammation.

CREB activity maintains the survival of cingulate cortical pyramidal neurons in the adult mouse brain.

Cyclic AMP-responsive element binding protein (CREB) activity is known to contribute to important neuronal functions, such as synaptic plasticity, learning and memory. Using a microelectroporation technique to overexpress dominant negative mutant CREB (mCREB) in the adult mouse brain, we found that overexpression of mCREB in the forebrain cortex induced neuronal degeneration. Our findings suggest that constitutively active CREB phosphorylation is important for the survival of mammalian cells in the brain.

Hot receptors in the brain.

Two major approaches have been employed for the development of novel drugs to treat chronic pain. The most traditional approach identifies molecules involved in pain as potential therapeutic targets and has focused mainly on the periphery and spinal cord. A more recent approach identifies molecules that are involved in long-term plasticity. Drugs developed through the latter approach are predicted to treat chronic, but not physiological or acute, pain. The TRPV1 (transient receptor potential vanilloid-1) receptor is involved in nociceptive processing, and is a candidate therapeutic target for pain. While most research on TRPV1 receptors has been conducted at the level of the spinal cord and peripheral structures, considerably less research has focused on supraspinal structures. This short paper summarizes progress made on TRPV1 receptors, and reviews research on the expression and function of TRPV1 receptors in supraspinal structures. We suggest that the TRPV1 receptor may be involved in pain processing in higher brain structures, such as the anterior cingulate cortex. In addition, some regions of the brain utilize the TRPV1 receptor for functions apparently unrelated to pain.

Genetic reduction of chronic muscle pain in mice lacking calcium/calmodulin-stimulated adenylyl cyclases.

BACKGROUND: The Ca2+/calmodulin-stimulated adenylyl cyclase (AC) isoforms AC1 and AC8, couple NMDA receptor activation to cAMP signaling pathways in neurons and are important for development, learning and memory, drug addiction and persistent pain. AC1 and AC8 in the anterior cingulate cortex (ACC) and the spinal cord were previously shown to be important in subcutaneous inflammatory pain. Muscle pain is different from cutaneous pain in its characteristics as well as conducting fibers. Therefore, we conducted the present work to test the role of AC1 and AC8 in both acute persistent and chronic muscle pain. RESULTS: Using an acute persistent inflammatory muscle pain model, we found that the behavioral nociceptive responses of both the late phase of acute muscle pain and the chronic muscle inflammatory pain were significantly reduced in AC1 knockout (KO) and AC1&8 double knockout (DKO) mice. Activation of other adenylyl cyclases in these KO mice by microinjection of forskolin into the ACC or spinal cord, but not into the peripheral tissue, rescued the behavioral nociceptive responses. Additionally, intra-peritoneal injection of an AC1 inhibitor significantly reduced behavioral responses in both acute persistent and chronic muscle pain. CONCLUSION: The results of the present study demonstrate that neuronal Ca2+/calmodulin-stimulated adenylyl cyclases in the ACC and spinal cord are important for both late acute persistent and chronic inflammatory muscle pain.

Calmodulin regulates synaptic plasticity in the anterior cingulate cortex and behavioral responses: a microelectroporation study in adult rodents.

We developed a microelectroporation method for the transfer of genes into neurons in the cerebral cortex of adult rodents, both rats and mice. We selectively expressed either green-fluorescent protein (GFP) or a Ca2+-binding deficient calmodulin (CaM) mutant in the anterior cingulate cortex (ACC). In mice that expressed GFP, positive neuronal cell bodies were found specifically at the injection site in the ACC. Mice that expressed CaM12, a mutant CaM with two impaired Ca2+ binding sites in the N-terminal lobe, exhibited significant changes in vocalization, locomotion, and sensory functions. Long-term potentiation and long-term depression, two major forms of central plasticity, were completely abolished by expression of CaM12. Mice that expressed CaM34, a mutant CaM with two impaired Ca2+ binding sites in the C-terminal lobe, did not show any significant behavioral or electrophysiological alterations. These findings provide strong evidence that CaM is critical for bidirectional synaptic plasticity. This new method will be useful for investigating gene function in specific brain regions of freely moving animals. Furthermore, this approach also may facilitate gene therapy in adult human brains.

Contribution of CaMKIV to injury and fear-induced ultrasonic vocalizations in adult mice.

Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that activates the transcription factor CREB. Our previous work demonstrated that mice lacking CaMKIV had a defect in fear memory while behavioral responses to noxious stimuli were unchanged. Here, we measured ultrasonic vocalizations (USVs) before and after fear conditioning and in response to a noxious injection of capsaicin to measure behavioral responses to emotional stimuli. Consistent with previous findings, behavioral nociceptive responses to capsaicin were undistinguishable between wild-type and CaMKIV-/- mice. Wild-type animals showed a selective increase in 50 kHz USVs in response to capsaicin while such an increase was absent in CaMKIV-/- mice. The foot shock given during fear conditioning caused an increase in 30 kHz USVs in both wild-type and CaMKIV-/- mice. When returned to the context one hour later, USVs from the wild-type were significantly decreased. Additionally, the onset of a tone, which had previously been paired with the foot shock, caused a significant decrease in USVs during auditory conditioning. CaMKIV-/- mice showed significantly less reduction in USVs when placed in the same context three days after receiving the shock, consistent with the decrease in freezing reported previously. Our results provide a new approach for investigating the molecular mechanism for emotional vocalization in mice and suggest that CaMKIV dependent signaling pathways play an important role in the emotional response to pain and fear.

Feed-forward inhibition: a novel cellular mechanism for the analgesic effect of substance P.

Substance P (SP) is a neuropeptide well known for its contribution to pain transmission in the spinal cord, however, less is known about the possible modulatory effects of SP. A new study by Gu and colleagues, published in Molecular Pain (2005, 1:20), describes its potential role in feed-forward inhibition in lamina V of the dorsal horn of the spinal cord. This inhibition seems to function through a direct excitation of GABAergic interneurons by substance P released from primary afferent fibers and has a distinct temporal phase of action from the well-described glutamate-dependent feed-forward inhibition. It is believed that through this inhibition, substance P can balance nociceptive output from the spinal cord.

Pavlovian fear memory induced by activation in the anterior cingulate cortex.

Identifying higher brain central region(s) that are responsible for the unpleasantness of pain is the focus of many recent studies. Here we show that direct stimulation of the anterior cingulate cortex (ACC) in mice produced fear-like freezing responses and induced long-term fear memory, including contextual and auditory fear memory. Auditory fear memory required the activation of N-methyl-D-aspartate (NMDA) receptors in the amygdala. To test the hypothesis that neuronal activity in the ACC contributes to unpleasantness, we injected a GABAA receptor agonist, muscimol bilaterally into the ACC. Both contextual and auditory memories induced by foot shock were blocked. Furthermore, activation of metabotropic glutamate receptors in the ACC enhanced behavioral escape responses in a noxious hot-plate as well as spinal nociceptive tail-flick reflex. Our results provide strong evidence that the excitatory activity in the ACC contribute to pain-related fear memory as well as descending facilitatory modulation of spinal nociception.

Genetic alteration of anxiety and stress-like behavior in mice lacking CaMKIV.

Calcium-calmodulin-dependent protein kinase IV (CaMKIV) phosphorylates the major transcription factor cyclic AMP-response element binding protein (CREB), which plays a role in emotional behavior. Here, CaMKIV knockout mice (CaMKIV(-/-)) were tested in a battery of stress and anxiety-related behavioral tests, to determine if CaMKIV plays a role in emotional behavior. CaMKIV(-/-) exhibited a decrease in anxiety-like behavior in both the elevated plus maze and dark-light emergence tests when compared to wild-type mice. Both the acoustic startle response and prepulse inhibition of startle were decreased with the deletion of CaMKIV. In addition, CaMKIV(-/-) mice displayed a lack of stress-induced analgesia following restraint or cold swim stress. Our results demonstrate a key role for CaMKIV in anxiety and stress-related behavior.

A new assay of thermal-based avoidance test in freely moving mice.

UNLABELLED: The hot plate test is a standard way to measure nociceptive response latencies to a noxious thermal stimulus. Here we have modified the classic hot plate by allowing animals to escape to an adjacent chamber after exposure to the heated surface. In this test, the animals escape to the adjacent chamber after exposure to the hot plate set at 50 degrees C. Repeated exposure to the hot plate resulted in a facilitation of escape responses, as measured by a reduced latency to escape from the noxious thermal stimulus. Signs of nociceptive behavior, such as licking or jumping, were not affected in animals that received hot plate training. The reduction of escape latencies after repeated hot plate exposure might be a useful measure for studying the facilitation of escape responses. In addition, the modified hot plate described here might be useful in studying performance and memory deficits related to noxious thermal stimuli. PERSPECTIVE: We modified a hot plate to measure facilitation of escape responses to a noxious thermal stimulus. The measure of escape responses might be useful in the assessment of memory defects, evaluation of drug therapies, and the behavioral characterization of transgenic mice.

Selective contribution of Egr1 (zif/268) to persistent inflammatory pain.

UNLABELLED: The zinc finger transcription factor Egr1 is critical for coupling extracellular signals to changes in cellular gene expression. Expression of Egr1, as well as other immediate early genes, is up-regulated in response to a number of noxious stimuli. Activity-dependent activation of Egr1 has been reported in forebrain regions, including the anterior cingulate cortex (ACC), after peripheral injury. However, no study has reported a direct contribution of Egr1 to behavioral nociceptive responses. Here, we use Egr1 knockout mice to show that Egr1 is selectively required for behavioral responses to persistent inflammatory pain. Behavioral responses to peripheral inflammation were significantly reduced in Egr1 knockout mice, whereas responses to acute noxious stimuli were normal. In addition, inflammation triggered an up-regulation of Egr1 expression in the ACC of wild-type mice. Last, synaptic potentiation induced by theta (theta) burst stimulation in the ACC was significantly reduced or blocked in Egr1 knockout mice. Our study suggests that the transcription factor Egr1 plays a selective role in nociceptive behavioral responses to persistent inflammatory pain but not to acute noxious stimuli. PERSPECTIVE: Chronic pain diminishes the quality of life. Here, we show that the immediate early gene Egr1 plays a role in chronic inflammatory, but not acute, pain. Egr1 knockout mice showed reduced nociceptive behaviors to persistent inflammatory pain and inflammation increased Egr1 expression in the anterior cingulate cortex of wild-type mice.

Transcription factor Egr-1 is required for long-term fear memory and anxiety.

The zinc finger transcription factor Egr-1 is critical for coupling extracellular signals to changes in cellular gene expression. In the hippocampus and amygdala, two major central regions for memory formation and storage, Egr-1 is up-regulated by long-term potentiation (LTP) and learning paradigms. Using Egr-1 knockout mice, we showed that Egr-1 was selectively required for late auditory fear memory while short term, trace and contextual memory were not affected. Additionally, synaptic potentiation induced by theta burst stimulation in the amygdala and auditory cortex was significantly reduced or blocked in Egr-1 knockout mice. Our study suggests that the transcription factor Egr-1 plays a selective role in late auditory fear memory.

Cingulate NMDA NR2B receptors contribute to morphine-induced analgesic tolerance.

Morphine is widely used to treat chronic pain, however its utility is hindered by the development of tolerance to its analgesic effects. While N-methyl-D-aspartate (NMDA) receptors are known to play roles in morphine tolerance and dependence, less is known about the roles of individual NMDA receptor subtypes. In this study, Ro 256981, an antagonist of the NMDA receptor subunit NR2B, was used to reduce the expression of analgesic tolerance to morphine. The mechanisms altered with chronic drug use share similarities with those underlying the establishment of long-tem potentiation (LTP) and behavioral memory. Since NMDA NR2B receptors in the anterior cingulate cortex (ACC) play roles in the establishment of LTP and fear memory, we explored their role in changes that occur in this region after chronic morphine. Both systemic and intra-ACC inhibition of NR2B in morphine-tolerant animals inhibited the expression of analgesic tolerance. Electrophysiological recordings revealed a significant increase in the NR2B component of NMDA receptor mediated excitatory postsynaptic currents (EPSCs), at both synaptic and extra-synaptic sites. However, there was no change in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor mediated EPSCs. This study suggests that selective inhibition of NMDA NR2B receptors may prove useful in combating the development of analgesic tolerance to morphine and proposes a novel role for the ACC in opioid tolerance and morphine induced changes in synaptic plasticity.

Kainate receptors and pain: from dorsal root ganglion to the anterior cingulate cortex.

Ionotropic glutamate receptors contain three subtypes: NMDA, AMPA and kainate receptors. The former two receptor subtypes have well defined roles in nociception, while the role of kainate receptors in pain is not as well characterized. Kainate receptors are expressed in nociceptive pathways, including the dorsal root ganglion, spinal cord, thalamus and cortex. Electrophysiological studies show that functional kainate receptors are located postsynaptically, where they mediate a portion of excitatory synaptic transmission, or are located presynaptically, where they modulate excitatory or inhibitory neurotransmission. Recent genetic and pharmacological studies suggest that kainate receptors can regulate nociceptive responses. These results highlight kainate receptors as a target for the development of new treatments for chronic pain.