mGluR5 from Primary Sensory Neurons Promotes Opioid-Induced Hyperalgesia and Tolerance by Interacting with and Potentiating Synaptic NMDA Receptors.

Aberrant activation of presynaptic NMDARs in the spinal dorsal horn is integral to opioid-induced hyperalgesia and analgesic tolerance. However, the signaling mechanisms responsible for opioid-induced NMDAR hyperactivity remain poorly identified. Here, we show that repeated treatment with morphine or fentanyl reduced monomeric mGluR5 protein levels in the dorsal root ganglion (DRG) but increased levels of mGluR5 monomers and homodimers in the spinal cord in mice and rats of both sexes. Coimmunoprecipitation analysis revealed that monomeric and dimeric mGluR5 in the spinal cord, but not monomeric mGluR5 in the DRG, directly interacted with GluN1. By contrast, mGluR5 did not interact with µ-opioid receptors in the DRG or spinal cord. Repeated morphine treatment markedly increased the mGluR5-GluN1 interaction and protein levels of mGluR5 and GluN1 in spinal synaptosomes. The mGluR5 antagonist MPEP reversed morphine treatment-augmented mGluR5-GluN1 interactions, GluN1 synaptic expression, and dorsal root-evoked monosynaptic EPSCs of dorsal horn neurons. Furthermore, CRISPR-Cas9-induced conditional mGluR5 knockdown in DRG neurons normalized mGluR5 levels in spinal synaptosomes and NMDAR-mediated EPSCs of dorsal horn neurons increased by morphine treatment. Correspondingly, intrathecal injection of MPEP or conditional mGluR5 knockdown in DRG neurons not only potentiated the acute analgesic effect of morphine but also attenuated morphine treatment-induced hyperalgesia and tolerance. Together, our findings suggest that opioid treatment promotes mGluR5 trafficking from primary sensory neurons to the spinal dorsal horn. Through dimerization and direct interaction with NMDARs, presynaptic mGluR5 potentiates and/or stabilizes NMDAR synaptic expression and activity at primary afferent central terminals, thereby maintaining opioid-induced hyperalgesia and tolerance.SIGNIFICANCE STATEMENT Opioids are essential analgesics for managing severe pain caused by cancer, surgery, and tissue injury. However, these drugs paradoxically induce pain hypersensitivity and tolerance, which can cause rapid dose escalation and even overdose mortality. This study demonstrates, for the first time, that opioids promote trafficking of mGluR5, a G protein-coupled glutamate receptor, from peripheral sensory neurons to the spinal cord; there, mGluR5 proteins dimerize and physically interact with NMDARs to augment their synaptic expression and activity. Through dynamic interactions, the two distinct glutamate receptors mutually amplify and sustain nociceptive input from peripheral sensory neurons to the spinal cord. Thus, inhibiting mGluR5 activity or disrupting mGluR5-NMDAR interactions could reduce opioid-induced hyperalgesia and tolerance and potentiate opioid analgesic efficacy.

NMDA Receptors at Primary Afferent-Excitatory Neuron Synapses Differentially Sustain Chemotherapy- and Nerve Trauma-Induced Chronic Pain.

The spinal dorsal horn contains vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons and vesicular GABA transporter (VGAT)-expressing inhibitory neurons, which normally have different roles in nociceptive transmission. Spinal glutamate NMDAR hyperactivity is a crucial mechanism of chronic neuropathic pain. However, it is unclear how NMDARs regulate primary afferent input to spinal excitatory and inhibitory neurons in neuropathic pain. Also, the functional significance of presynaptic NMDARs in neuropathic pain has not been defined explicitly. Here we showed that paclitaxel treatment or spared nerve injury (SNI) similarly increased the NMDAR-mediated mEPSC frequency and dorsal root-evoked EPSCs in VGluT2 dorsal horn neurons in male and female mice. By contrast, neither paclitaxel nor SNI had any effect on mEPSCs or evoked EPSCs in VGAT neurons. In mice with conditional Grin1 (gene encoding GluN1) KO in primary sensory neurons (Grin1-cKO), paclitaxel treatment failed to induce pain hypersensitivity. Unexpectedly, SNI still caused long-lasting pain hypersensitivity in Grin1-cKO mice. SNI increased the amplitude of puff NMDA currents in VGluT2 neurons and caused similar depolarizing shifts in GABA reversal potentials in WT and Grin1-cKO mice. Concordantly, spinal Grin1 knockdown diminished SNI-induced pain hypersensitivity. Thus, presynaptic NMDARs preferentially amplify primary afferent input to spinal excitatory neurons in neuropathic pain. Although presynaptic NMDARs are required for chemotherapy-induced pain hypersensitivity, postsynaptic NMDARs in spinal excitatory neurons play a dominant role in traumatic nerve injury-induced chronic pain. Our findings reveal the divergent synaptic connectivity and functional significance of spinal presynaptic and postsynaptic NMDARs in regulating cell type-specific nociceptive input in neuropathic pain with different etiologies.SIGNIFICANCE STATEMENT Spinal excitatory neurons relay input from nociceptors, whereas inhibitory neurons repress spinal nociceptive transmission. Chronic nerve pain is associated with aberrant NMDAR activity in the spinal dorsal horn. This study demonstrates, for the first time, that chemotherapy and traumatic nerve injury preferentially enhance the NMDAR activity at primary afferent-excitatory neuron synapses but have no effect on primary afferent input to spinal inhibitory neurons. NMDARs in primary sensory neurons are essential for chemotherapy-induced chronic pain, whereas nerve trauma causes pain hypersensitivity predominantly via postsynaptic NMDARs in spinal excitatory neurons. Thus, presynaptic and postsynaptic NMDARs at primary afferent-excitatory neuron synapses are differentially engaged in chemotherapy- and nerve injury-induced chronic pain and could be targeted respectively for treating these painful conditions.

Brief Opioid Exposure Paradoxically Augments Primary Afferent Input to Spinal Excitatory Neurons via α2δ-1-Dependent Presynaptic NMDA Receptors.

Treatment with opioids not only inhibits nociceptive transmission but also elicits a rebound and persistent increase in primary afferent input to the spinal cord. Opioid-elicited long-term potentiation (LTP) from TRPV1-expressing primary afferents plays a major role in opioid-induced hyperalgesia and analgesic tolerance. Here, we determined whether opioid-elicited LTP involves vesicular glutamate transporter-2 (VGluT2) or vesicular GABA transporter (VGAT) neurons in the spinal dorsal horn of male and female mice and identified underlying signaling mechanisms. Spinal cord slice recordings revealed that µ-opioid receptor (MOR) stimulation with DAMGO initially inhibited dorsal root-evoked EPSCs in 87% VGluT2 neurons and subsequently induced LTP in 49% of these neurons. Repeated morphine treatment increased the prevalence of VGluT2 neurons displaying LTP with a short onset latency. In contrast, DAMGO inhibited EPSCs in 46% VGAT neurons but did not elicit LTP in any VGAT neurons even in morphine-treated mice. Spinal superficial laminae were densely innervated by MOR-containing nerve terminals and were occupied by mostly VGluT2 neurons and few VGAT neurons. Furthermore, conditional Grin1 knockout in dorsal root ganglion neurons diminished DAMGO-elicited LTP in lamina II neurons and attenuated hyperalgesia and analgesic tolerance induced by repeated treatment with morphine. In addition, DAMGO-elicited LTP in VGluT2 neurons was abolished by protein kinase C inhibition, gabapentin, Cacna2d1 knockout, or disrupting the α2δ-1-NMDA receptor interaction with an α2δ-1 C terminus peptide. Thus, brief MOR stimulation distinctively potentiates nociceptive primary afferent input to excitatory dorsal horn neurons via α2δ-1-coupled presynaptic NMDA receptors, thereby causing hyperalgesia and reducing analgesic actions of opioids.SIGNIFICANCE STATEMENT Opioid drugs are potent analgesics for treating severe pain and are commonly used during general anesthesia. However, opioid use often induces pain hypersensitivity, rapid loss of analgesic efficacy, and dose escalation, which can cause dependence, addiction, and even overdose fatality. This study demonstrates for the first time that brief opioid exposure preferentially augments primary sensory input to genetically identified glutamatergic excitatory, but not GABAergic/glycinergic inhibitory, neurons in nociceptive dorsal horn circuits. This opioid-elicited synaptic plasticity is cell type specific and mediated by protein kinase C-dependent and α2δ-1-dependent activation of NMDA receptors at primary sensory nerve terminals. These findings elucidate how intraoperative use of opioids for preemptive analgesia paradoxically aggravates postoperative pain and increases opioid consumption and suggest new strategies to improve opioid analgesic efficacy.

Theta-Burst Stimulation of Primary Afferents Drives Long-Term Potentiation in the Spinal Cord and Persistent Pain via α2δ-1-Bound NMDA Receptors.

Long-term potentiation (LTP) and long-term depression (LTD) in the spinal dorsal horn reflect activity-dependent synaptic plasticity and central sensitization in chronic pain. Tetanic high-frequency stimulation is commonly used to induce LTP in the spinal cord. However, primary afferent nerves often display low-frequency, rhythmic bursting discharges in painful conditions. Here, we determined how theta-burst stimulation (TBS) of primary afferents impacts spinal cord synaptic plasticity and nociception in male and female mice. We found that TBS induced more LTP, whereas tetanic stimulation induced more LTD, in mouse spinal lamina II neurons. TBS triggered LTP, but not LTD, in 50% of excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2). By contrast, TBS induced LTD and LTP in 12-16% of vesicular GABA transporter (VGAT)-expressing inhibitory neurons. Nerve injury significantly increased the prevalence of TBS-induced LTP in VGluT2-expressing, but not VGAT-expressing, lamina II neurons. Blocking NMDARs, inhibiting α2δ-1 with gabapentin, or α2δ-1 knockout abolished TBS-induced LTP in lamina II neurons. Also, disrupting the α2δ-1-NMDAR interaction with α2δ-1Tat peptide prevented TBS-induced LTP in VGluT2-expressing neurons. Furthermore, TBS of the sciatic nerve induced long-lasting allodynia and hyperalgesia in wild-type, but not α2δ-1 knockout, mice. TBS significantly increased the α2δ-1-NMDAR interaction and synaptic trafficking in the spinal cord. In addition, treatment with NMDAR antagonists, gabapentin, or α2δ-1Tat peptide reversed TBS-induced pain hypersensitivity. Therefore, TBS-induced primary afferent input causes a neuropathic pain-like phenotype and LTP predominantly in excitatory dorsal horn neurons via α2δ-1-dependent NMDAR activation. α2δ-1-bound NMDARs may be targeted for reducing chronic pain development at the onset of tissue/nerve injury.SIGNIFICANCE STATEMENT Spinal dorsal horn synaptic plasticity is a hallmark of chronic pain. Although sensory nerves display rhythmic bursting discharges at theta frequencies during painful conditions, the significance of this naturally occurring firing activity in the induction of spinal synaptic plasticity is largely unknown. In this study, we found that theta-burst stimulation (TBS) of sensory nerves induced LTP mainly in excitatory dorsal horn neurons and that the prevalence of TBS-induced LTP was potentiated by nerve injury. This TBS-driven synaptic plasticity required α2δ-1 and its interaction with NMDARs. Furthermore, TBS of sensory nerves induced persistent pain, which was maintained by α2δ-1-bound NMDARs. Thus, TBS-induced LTP at primary afferent-dorsal horn neuron synapses is an appropriate cellular model for studying mechanisms of chronic pain.

HDAC2 in Primary Sensory Neurons Constitutively Restrains Chronic Pain by Repressing α2δ-1 Expression and Associated NMDA Receptor Activity.

α2δ-1 (encoded by the Cacna2d1 gene) is a newly discovered NMDA receptor-interacting protein and is the therapeutic target of gabapentinoids (e.g., gabapentin and pregabalin) frequently used for treating patients with neuropathic pain. Nerve injury causes sustained α2δ-1 upregulation in the dorsal root ganglion (DRG), which promotes NMDA receptor synaptic trafficking and activation in the spinal dorsal horn, a hallmark of chronic neuropathic pain. However, little is known about how nerve injury initiates and maintains the high expression level of α2δ-1 to sustain chronic pain. Here, we show that nerve injury caused histone hyperacetylation and diminished enrichment of histone deacetylase-2 (HDAC2), but not HDAC3, at the Cacna2d1 promoter in the DRG. Strikingly, Hdac2 knockdown or conditional knockout in DRG neurons in male and female mice consistently induced long-lasting mechanical pain hypersensitivity, which was readily reversed by blocking NMDA receptors, inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-NMDA receptor interaction at the spinal cord level. Hdac2 deletion in DRG neurons increased histone acetylation levels at the Cacna2d1 promoter, upregulated α2δ-1 in the DRG, and potentiated α2δ-1-dependent NMDA receptor activity at primary afferent central terminals in the spinal dorsal horn. Correspondingly, Hdac2 knockdown-induced pain hypersensitivity was blunted in Cacna2d1 knockout mice. Thus, our findings reveal that HDAC2 functions as a pivotal transcriptional repressor of neuropathic pain via constitutively suppressing α2δ-1 expression and ensuing presynaptic NMDA receptor activity in the spinal cord. HDAC2 enrichment levels at the Cacna2d1 promoter in DRG neurons constitute a unique epigenetic mechanism that governs acute-to-chronic pain transition.SIGNIFICANCE STATEMENT Excess α2δ-1 proteins produced after nerve injury directly interact with glutamate NMDA receptors to potentiate synaptic NMDA receptor activity in the spinal cord, a prominent mechanism of nerve pain. Because α2δ-1 upregulation after nerve injury is long lasting, gabapentinoids relieve pain symptoms only temporarily. Our study demonstrates for the first time the unexpected role of intrinsic HDAC2 activity at the α2δ-1 gene promoter in limiting α2δ-1 gene transcription, NMDA receptor-dependent synaptic plasticity, and chronic pain development after nerve injury. These findings challenge the prevailing view about the role of general HDAC activity in promoting chronic pain. Restoring the repressive HDAC2 function and/or reducing histone acetylation at the α2δ-1 gene promoter in primary sensory neurons could lead to long-lasting relief of nerve pain.

α2δ-1 protein drives opioid-induced conditioned reward and synaptic NMDA receptor hyperactivity in the nucleus accumbens.

Glutamate NMDA receptors (NMDARs) in the nucleus accumbens (NAc) are critically involved in drug dependence and reward. α2δ-1 is a newly discovered NMDAR-interacting protein that promotes synaptic trafficking of NMDARs independently of its conventional role as a calcium channel subunit. However, it remains unclear how repeated opioid exposure affects synaptic NMDAR activity and α2δ-1-NMDAR interaction in the NAc. In this study, whole-cell patch-clamp recordings showed that repeated treatment with morphine in mice markedly increased the NMDAR-mediated frequency of miniature excitatory postsynaptic currents (mEPSCs) and amplitude of puff NMDAR currents in medium spiny neurons in the NAc core region. Morphine treatment significantly increased the physical interaction of α2δ-1 with GluN1 and their synaptic trafficking in the NAc. In Cacna2d1 knockout mice, repeated treatment with morphine failed to increase the frequency of mEPSCs and amplitude of puff NMDAR currents in the NAc core. Furthermore, inhibition of α2δ-1 with gabapentin or disruption of the α2δ-1-NMDAR interaction with the α2δ-1 C terminus-interfering peptide blocked the morphine-elevated frequency of mEPSCs and amplitude of puff NMDAR currents in the NAc core. Correspondingly, systemically administered gabapentin, Cacna2d1 ablation, or microinjection of the α2δ-1 C terminus-interfering peptide into the NAc core attenuated morphine-induced conditioned place preference and locomotor sensitization. Our study reveals that repeated opioid exposure strengthens presynaptic and postsynaptic NMDAR activity in the NAc via α2δ-1. The α2δ-1-bound NMDARs in the NAc have a key function in the rewarding effect of opioids and could be targeted for treating opioid use disorder and addiction.

α2δ-1 Upregulation in Primary Sensory Neurons Promotes NMDA Receptor-Mediated Glutamatergic Input in Resiniferatoxin-Induced Neuropathy.

Systemic treatment with resiniferatoxin (RTX) induces small-fiber sensory neuropathy by damaging TRPV1-expressing primary sensory neurons and causes distinct thermal sensory impairment and tactile allodynia, which resemble the unique clinical features of postherpetic neuralgia. However, the synaptic plasticity associated with RTX-induced tactile allodynia remains unknown. In this study, we found that RTX-induced neuropathy is associated with α2δ-1 upregulation in the dorsal root ganglion (DRG) and increased physical interaction between α2δ-1 and GluN1 in the spinal cord synaptosomes. RNAscope in situ hybridization showed that RTX treatment significantly increased α2δ-1 expression in DRG neurons labeled with calcitonin gene-related peptide, isolectin B4, NF200, and tyrosine hydroxylase. Electrophysiological recordings revealed that RTX treatment augmented the frequency of miniature excitatory postsynaptic currents (mEPSCs) and the amplitude of evoked EPSCs in spinal dorsal horn neurons, and these effects were reversed by blocking NMDA receptors with AP-5. Inhibiting α2δ-1 with gabapentin, genetically ablating α2δ-1, or targeting α2δ-1-bound NMDA receptors with α2δ-1Tat peptide largely normalized the baseline frequency of mEPSCs and the amplitude of evoked EPSCs potentiated by RTX treatment. Furthermore, systemic treatment with memantine or gabapentin and intrathecal injection of AP-5 or Tat-fused α2δ-1 C terminus peptide reversed allodynia in RTX-treated rats and mice. In addition, RTX-induced tactile allodynia was attenuated in α2δ-1 knock-out mice and in mice in which GluN1 was conditionally knocked out in DRG neurons. Collectively, our findings indicate that α2δ-1-bound NMDA receptors at presynaptic terminals of sprouting myelinated afferent nerves contribute to RTX-induced potentiation of nociceptive input to the spinal cord and tactile allodynia.SIGNIFICANCE STATEMENT Postherpetic neuralgia (PHN), associated with shingles, is a distinct form of neuropathic pain commonly seen in elderly and immunocompromised patients. The synaptic plasticity underlying touch-induced pain hypersensitivity in PHN remains unclear. Using a nonviral animal model of PHN, we found that glutamatergic input from primary sensory nerves to the spinal cord is increased via tonic activation of glutamate NMDA receptors. Also, we showed that α2δ-1 (encoded by Cacna2d1), originally considered a calcium channel subunit, serves as an auxiliary protein that promotes activation of presynaptic NMDA receptors and pain hypersensitivity. This new information advances our understanding of the molecular mechanism underlying PHN and suggests new strategies for treating this painful condition.

Dynamic increases in AMPA receptor phosphorylation in the rat hippocampus in response to amphetamine.

Increasing evidence supports the critical role of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors in psychostimulant action. These receptors are regulated via a phosphorylation-dependent mechanism in their trafficking, distribution, and function. The hippocampus is a brain structure important for learning and memory and is emerging as a critical site for processing psychostimulant effects. To determine whether the hippocampal pool of AMPA receptors is regulated by stimulants, we investigated and characterized the impact of amphetamine (AMPH) on phosphorylation of AMPA receptors in the adult rat hippocampus in vivo. We found that AMPH markedly increased phosphorylation of AMPA receptor GluA1 subunits at serine 845 (S845) in the hippocampus. The effect of AMPH was dose dependent. A single dose of AMPH induced a rapid and transient increase in S845 phosphorylation. Among different hippocampal subfields, AMPH primarily elevated S845 phosphorylation in the Cornu Ammonis area 1 and dentate gyrus. In contrast to S845, serine 831 phosphorylation of GluA1 and serine 880 phosphorylation of GluA2 were not altered by AMPH. In addition, surface expression of hippocampal GluA1 was up-regulated, while the amount of intracellular GluA1 fraction was concurrently reduced in response to AMPH. GluA2 protein levels in either the surface or intracellular pool were insensitive to AMPH. These data demonstrate that the AMPA receptor in the hippocampus is sensitive to dopamine stimulation. Acute AMPH administration induces dose-, time-, site-, and subunit-dependent phosphorylation of AMPA receptors and facilitates surface trafficking of GluA1 AMPA receptors in hippocampal neurons in vivo. Acute injection of amphetamine increased phosphorylation of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 subunits at a protein kinase A (PKA)-sensitive site (S845) in the rat hippocampus. This increase was dose- and time-dependent and correlated with an increase in surface GluA1 expression. Thus, amphetamine can upregulate GluA1 phosphorylation and surface trafficking of GluA1 in hippocampal neurons in vivo.

Regulation of synaptic MAPK/ERK phosphorylation in the rat striatum and medial prefrontal cortex by dopamine and muscarinic acetylcholine receptors.

Dopamine and acetylcholine are two principal transmitters in the striatum and are usually balanced to modulate local neural activity and to maintain striatal homeostasis. This study investigates the role of dopamine and muscarinic acetylcholine receptors in the regulation of a central signaling protein, i.e., the mitogen-activated protein kinase (MAPK). We focus on the synaptic pool of MAPKs because of the fact that these kinases reside in peripheral synaptic structures in addition to their somatic locations. We show that a systemic injection of dopamine D1 receptor (D1R) agonist SKF81297 enhances phosphorylation of extracellular signal-regulated kinases (ERKs), a prototypic subclass of MAPKs, in the adult rat striatum. Similar results were observed in another dopamine-responsive region, the medial prefrontal cortex (mPFC). The dopamine D2 receptor agonist quinpirole had no such effects. Pretreatment with a positive allosteric modulator (PAM) of muscarinic acetylcholine M4 receptors (M4Rs), VU0152100, attenuated the D1R agonist-stimulated ERK phosphorylation in the two regions, whereas the PAM itself did not alter basal ERK phosphorylation. All drug treatments had no effect on phosphorylation of c-Jun N-terminal kinases (JNKs), another MAPK subclass, in the striatum and mPFC. These results demonstrate that dopamine and acetylcholine are integrated to control synaptic ERK but not JNK activation in striatal and mPFC neurons in vivo. Activation of M4Rs exerts an inhibitory effect on the D1R-mediated upregulation of synaptic ERK phosphorylation.

Phosphorylation and feedback regulation of metabotropic glutamate receptor 1 by calcium/calmodulin-dependent protein kinase II.

The metabotropic glutamate receptor 1 (mGluR1) is a Gα(q)-protein-coupled receptor and is distributed in broad regions of the mammalian brain. As a key element in excitatory synaptic transmission, the receptor regulates a wide range of cellular and synaptic activities. In addition to regulating its targets, the receptor itself is believed to be actively regulated by intracellular signals, although underlying mechanisms are essentially unknown. Here we found that a synapse-enriched protein kinase, Ca²âº/calmodulin-dependent protein kinase IIα (CaMKIIα), directly binds to the intracellular C terminus (CT) of mGluR1a. This binding is augmented by Ca²âº in vitro. The direct interaction promotes CaMKIIα to phosphorylate mGluR1a at a specific threonine site (T871). In rat striatal neurons, the mGluR1 agonist triggers the receptor-associated phosphoinositide signaling pathway to induce Ca²âº-dependent recruitment of CaMKIIα to mGluR1a-CT. This enables the kinase to inhibit the response of the receptor to subsequent agonist exposure. Our data identify an agonist-induced and Ca²âº-dependent protein-protein interaction between a synaptic kinase and mGluR1, which constitutes a feedback loop facilitating desensitization of mGluR1a.

Activity-dependent regulation of release probability at excitatory hippocampal synapses: a crucial role of fragile X mental retardation protein in neurotransmission.

Transcriptional silencing of the Fmr1 gene encoding fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS), the most common form of inherited intellectual disability and the leading genetic cause of autism. FMRP has been suggested to play important roles in regulating neurotransmission and short-term synaptic plasticity at excitatory hippocampal and cortical synapses. However, the origins and mechanisms of these FMRP actions remain incompletely understood, and the role of FMRP in regulating synaptic release probability and presynaptic function remains debated. Here we used variance-mean analysis and peak-scaled nonstationary variance analysis to examine changes in both presynaptic and postsynaptic parameters during repetitive activity at excitatory CA3-CA1 hippocampal synapses in a mouse model of FXS. Our analyses revealed that loss of FMRP did not affect the basal release probability or basal synaptic transmission, but caused an abnormally elevated release probability specifically during repetitive activity. These abnormalities were not accompanied by changes in excitatory postsynaptic current kinetics, quantal size or postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor conductance. Our results thus indicate that FMRP regulates neurotransmission at excitatory hippocampal synapses specifically during repetitive activity via modulation of release probability in a presynaptic manner. Our study suggests that FMRP function in regulating neurotransmitter release is an activity-dependent phenomenon that may contribute to the pathophysiology of FXS.

Phosphorylation and regulation of glutamate receptors by CaMKII.

Ca²âº/calmodulin-dependent protein kinase II (CaMKII) is the most abundant kinase within excitatory synapses in the mammalian brain. It interacts with and phosphorylates a large number of synaptic proteins, including major ionotropic glutamate receptors (iGluRs) and group I metabotropic glutamate receptors (mGluRs), to constitutively and/or activity-dependently regulate trafficking, subsynaptic localization, and function of the receptors. Among iGluRs, the N-methyl-D-aspartate receptor (NMDAR) is a direct target of CaMKII. By directly binding to an intracellular C-terminal (CT) region of NMDAR GluN2B subunits, CaMKII phosphorylates a serine residue (S1303) in the GluN2B CT. CaMKII also phosphorylates a serine site (S831) in the CT of α-amino-3-hydroxy-5- methylisoxazole-4-propionic acid receptors. This phosphorylation enhances channel conductance and is critical for synaptic plasticity. In addition to iGluRs, CaMKII binds to the proximal CT region of mGluR1a, which enables the kinase to phosphorylate threonine 871. Agonist stimulation of mGluR1a triggers a CaMKII-mediated negative feedback to facilitate endocytosis and desensitization of the receptor. CaMKII also binds to the mGluR5 CT. This binding seems to anchor and accumulate inactive CaMKII at synaptic sites. Active CaMKII dissociates from mGluR5 and may then bind to adjacent GluN2B to mediate the mGluR5-NMDAR coupling. Together, glutamate receptors serve as direct substrates of CaMKII. By phosphorylating these receptors, CaMKII plays a central role in controlling the number and activity of the modified receptors and determining the strength of excitatory synaptic transmission.

Differential regulation of CaMKIIα interactions with mGluR5 and NMDA receptors by Ca(2+) in neurons.

Two glutamate receptors, metabotropic glutamate receptor 5 (mGluR5), and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca(2+) /calmodulin-dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse-enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state-dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca(2+) /calmodulin binding and activation) loses its affinity for the receptor. Ca(2+) also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα-binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca(2+) -dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα-sensitive site. Together, the long intracellular C-terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5-dependent Ca(2+) transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to cross-talk between the two receptors. We show that activation of mGluR5 with a selective agonist triggers intracellular Ca(2+) release in striatal neurons. Released Ca(2+) dissociates preformed CaMKIIα from mGluR5 and meanwhile promotes active CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα-sensitive site. This agonist-induced cascade seems to mediate crosstalk between mGluR5 and NMDA receptors in neurons.

Lipocalin 2 is a selective modulator of peroxisome proliferator-activated receptor-gamma activation and function in lipid homeostasis and energy expenditure.

We have previously identified lipocalin 2 (Lcn2) as a cytokine playing a critical role in the regulation of body fat mass, lipid metabolism, and insulin resistance. Lcn2 deficiency reduces PPARγ gene expression in adipocytes. In this study, we investigated the role of Lcn2 in PPARγ activation and function via assessing the insulin sensitization and fatty acid (FA) homeostasis of PPARγ agonist in high-fat diet (HFD)-induced obesity in Lcn2(-/-) mice. We found that rosiglitazone (Rosi) significantly improved insulin sensitivity in Lcn2(-/-) mice as effectively as in wild-type (WT) mice; unfed-state levels of blood glucose, free FAs, and triglycerides (TGs) were significantly reduced after a 25-d treatment of Rosi in Lcn2(-/-) mice. However, Rosi action on fat deposition and FA homeostasis was altered; Rosi-induced body weight and subcutaneous fat gain and liver lipid accumulation were markedly lessened in Lcn2(-/-) mice. The results of in vivo metabolic labeling showed that Rosi markedly reduced de novo lipogenesis in adipose tissue of Lcn2(-/-) mice. In brown adipose tissue (BAT), the expression of the genes functioning in TG hydrolysis and mitochondrial oxidation was up-regulated more in Lcn2(-/-) than in WT mice. Most strikingly, Rosi stimulated significantly higher levels of uncoupling protein-1 expression in BAT, and completely rescued cold intolerance in Lcn2(-/-) mice. We demonstrate that Lcn2 is a critical selective modulator of PPARγ activation and function in lipid homeostasis and energy expenditure.

δ-Opioid receptors in primary sensory neurons tonically restrain nociceptive input in chronic pain but do not enhance morphine analgesic tolerance.

δ-Opioid receptors (DORs, encoded by the Oprd1 gene) are expressed throughout the peripheral and central nervous system, and DOR stimulation reduces nociception. Previous studies suggest that DORs promote the development of analgesic tolerance of µ-opioid receptor (MOR) agonists. It is uncertain whether DORs expressed in primary sensory neurons are involved in regulating chronic pain and MOR agonist-induced tolerance. In this study, we generated Oprd1 conditional knockout (Oprd1-cKO) mice by crossing Advillin-Cre mice with Oprd1-floxed mice. DOR expression in the dorsal root ganglion was diminished in Oprd1-cKO mice. Systemic or intrathecal injection of the DOR agonist SNC-80 produced analgesia in wild-type (WT), but not Oprd1-cKO, mice. In contrast, intracerebroventricular injection of SNC-80 produced a similar analgesic effect in WT and Oprd1-cKO mice. However, morphine-induced analgesia, hyperalgesia, or analgesic tolerance did not differ between WT and Oprd1-cKO mice. Compared with WT mice, Oprd1-cKO mice showed increased mechanical and heat hypersensitivity after nerve injury or tissue inflammation. Furthermore, blocking DORs with naltrindole increased nociceptive sensitivity induced by nerve injury or tissue inflammation in WT, but not Oprd1-cKO, mice. In addition, naltrindole potentiated glutamatergic input from primary afferents to spinal dorsal horn neurons increased by nerve injury or CFA in WT mice; this effect was absent in Oprd1-cKO mice. Our findings indicate that DORs in primary sensory neurons are critically involved in the analgesic effect of DOR agonists but not morphine-induced analgesic tolerance. Presynaptic DORs at primary afferent central terminals constitutively inhibit inflammatory and neuropathic pain by restraining glutamatergic input to spinal dorsal horn neurons.

Lipocalin 2 deficiency inhibits cell proliferation, autophagy, and mitochondrial biogenesis in mouse embryonic cells.

Lipocalin 2 (LCN2) has been recently implicated as a critical player in multiple cancer tumorigeneses. However, the molecular mechanisms for its tumorigenic role are poorly understood. Herein, we investigated the effects of LCN2 on cell proliferation, autophagy, and mitochondrial biogenesis in MEF cells. We observed that LCN2 deficiency significantly inhibited cell proliferation and autophagy in MEF cells. Furthermore, mitochondrial DNA content, mRNA expression levels of mitochondrial-encoded gene cytochrome oxidase 2 and PGC-1α were all markedly reduced in LCN2⁻/⁻ MEF cells. Additionally, when compared with wild-type MEF cells, LCN2⁻/⁻ MEF cells expressed significantly higher levels of IRS-1, and displayed more potent TNFα-stimulated NF-κB activation. These findings demonstrate that LCN2 is a critical regulator of cell proliferation, autophagy, and mitochondrial biogenesis.

Amphetamine activates non-receptor tyrosine kinase Fyn and stimulates ERK phosphorylation in the rat striatum in vivo.

The psychostimulant amphetamine (AMPH) has an impact on a variety of cellular activities in striatal neurons, although underlying signaling mechanisms are incompletely understood. The Src family kinase (SFK) is among key signaling molecules enriched in striatal neurons and is involved in the regulation of a set of discrete downstream targets. Given the likelihood that AMPH may regulate SFKs, we investigated and characterized the effect of AMPH on SFK phosphorylation and enzymatic activity in rat striatal neurons in vivo. We found that AMPH elevated SFK Y416 phosphorylation in striatal slices and the adult rat striatum. This elevation was concentration- and time-dependent and occurred in all subdivisions of the striatum, including the caudate putamen and nucleus accumbens (core and shell). The dopamine D1 receptor antagonist SCH23390 blocked the effect of AMPH. Between Fyn and Src, AMPH elevated phosphorylation of immunoprecipitated Fyn but not Src and increased Fyn kinase activity in the striatum. In parallel with SFKs, striatal ERK phosphorylation was increased by AMPH. This increase in ERK phosphorylation was reduced by the SFK inhibitor PP2. These results demonstrate that AMPH is able to activate SFKs (mainly Fyn) in striatal neurons via a D1 receptor-dependent mechanism. Activated SFKs participate in processing the concomitant ERK response to AMPH.

The Role of Extracellular Signal-Regulated Kinases (ERK) in the Regulation of mGlu5 Receptors in Neurons.

The metabotropic glutamate (mGlu) receptor 5 is a G protein-coupled receptor and is densely expressed in the mammalian brain. Like other glutamate receptors, mGlu5 receptors are tightly regulated by posttranslational modifications such as phosphorylation, although underlying mechanisms are incompletely investigated. In this study, we investigated the role of a prime kinase, extracellular signal-regulated kinase 1 (ERK1), in the phosphorylation and regulation of mGlu5 receptors in vitro and in striatal neurons. We found that recombinant ERK1 proteins directly bound to the C-terminal tail (CT) of mGlu5 receptors in vitro. Endogenous ERK1 also interacted with mGlu5 receptor proteins in adult rat striatal neurons in vivo. The kinase showed the ability to phosphorylate mGlu5 receptors. A serine residue in the distal region of mGlu5 CT was found to be a primary phosphorylation site sensitive to ERK1. In functional studies, we found that pharmacological inhibition of ERK with an inhibitor U0126 reduced the efficacy of mGlu5 receptors in stimulating production of cytoplasmic inositol-1,4,5-triphosphate, a major downstream conventional signaling event, in striatal neurons under normal conditions. These results identify mGlu5 as a new biochemical substrate of ERK1. The kinase can interact with and phosphorylate an intracellular domain of mGlu5 receptors in striatal neurons and thereby control its signaling efficacy.

Regulation of Phosphorylation of AMPA Glutamate Receptors by Muscarinic M4 Receptors in the Striatum In vivo.

The acetylcholine muscarinic 4 (M4) receptor is a principal muscarinic receptor subtype present in the striatum. Notably, Gαi/o-coupled M4 receptors and Gαs/Golf-coupled dopamine D1 receptors are coexpressed in striatonigral projection neurons and are thought to interact with each other to regulate neuronal excitability, although underlying molecular mechanisms are poorly understood. In this study, we investigated the role of M4 receptors in the regulation of phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the rat normal and dopamine-stimulated striatum in vivo. We found that a systemic injection of a M4 antagonist tropicamide increased AMPA receptor GluA1 subunit phosphorylation at a protein kinase A-dependent site (S845) in the striatum. The tropicamide-induced S845 phosphorylation was rapid, reversible, and dose-dependent and occurred in the two subdivisions of the striatum, i.e., the caudate putamen and nucleus accumbens. Coadministration of subthreshold doses of tropicamide and a D1 agonist SKF81297 induced a significant increase in S845 phosphorylation. Coadministered tropicamide and a dopamine psychostimulant amphetamine at their subthreshold doses also elevated S845 phosphorylation. Tropicamide alone or coinjected with SKF81297 or amphetamine had no effect on GluA1 phosphorylation at S831. Tropicamide did not affect GluA2 phosphorylation at S880. These results reveal a selective inhibitory linkage from M4 receptors to GluA1 in S845 phosphorylation in striatal neurons. Blockade of the M4-mediated inhibition significantly augments constitutive and dopamine-stimulated GluA1 S845 phosphorylation.

Pharmacological modulation of AMPA receptor phosphorylation by dopamine and muscarinic receptor agents in the rat medial prefrontal cortex.

Two key transmitters in the medial prefrontal cortex (mPFC), dopamine and acetylcholine, are believed to interact with each other to modulate local glutamatergic transmission, although molecular mechanisms underlying their crosstalk are poorly understood. Here we investigated effects of pharmacological manipulations of dopamine and muscarinic receptors on phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the adult rat mPFC in vivo. We found that an agonist selective for Gαs-coupled dopamine D1 receptors, SKF81297, increased AMPA receptor GluA1 subunit phosphorylation at a protein kinase A-sensitive site (S845), while SKF81297 had no effect on GluA1 phosphorylation at S831. An agonist for Gαi/o-coupled dopamine D2 receptors, quinpirole, also increased S845 but not S831 phosphorylation. When coinjected, the two agonists induced an additive increase in S845 phosphorylation. The D1 receptor antagonist SCH23390 blocked the SKF81297/quinpirole-stimulated S845 phosphorylation. The D2 antagonist eticlopride also partially blocked S845 responses to SKF81297/quinpirole. VU0152100, a positive allosteric modulator selective for Gαi/o-coupled muscarinic M4 receptors, reduced the S845 phosphorylation induced by SKF81297 and quinpirole injected alone or together. In contrast, coinjection of subthreshold doses of tropicamide, an M4 antagonist, and SKF81297 facilitated S845 phosphorylation. Additionally, coadministered SFK81297 and quinpirole increased the abundance of mPFC GluA1 at extrasynaptic sites. These data reveal that both D1 and D2 receptors upregulate GluA1 phosphorylation in mPFC neurons probably via a direct and indirect mechanism, respectively. The indirect mechanism involves M4 receptors which generally counteract the effect of dopamine on GluA1 phosphorylation.