Cellular, molecular, and epigenetic mechanisms in non-associative conditioning: implications for pain and memory

Abstract

Sensitization is a form of non-associative conditioning in which amplification of behavioral responses can occur following presentation of an aversive or noxious stimulus. Understanding the cellular and molecular underpinnings of sensitization has been an overarching theme spanning the field of learning and memory as well as that of pain research. In this review we examine how sensitization, both in the context of learning as well as pain processing, shares evolutionarily conserved behavioral, cellular/synaptic, and epigenetic mechanisms across phyla. First, we characterize the behavioral phenomenon of sensitization both in invertebrates and vertebrates. Particular emphasis is placed on long-term sensitization (LTS) of withdrawal reflexes in Aplysia following aversive stimulation or injury, although additional invertebrate models are also covered. In the context of vertebrates, sensitization of mammalian hyperarousal in a model of post-traumatic stress disorder (PTSD), as well as mammalian models of inflammatory and neuropathic pain is characterized. Second, we investigate the cellular and synaptic mechanisms underlying these behaviors. We focus our discussion on serotonin-mediated long-term facilitation (LTF) and axotomy-mediated long-term hyperexcitability (LTH) in reduced Aplysia systems, as well as mammalian spinal plasticity mechanisms of central sensitization. Third, we explore recent evidence implicating epigenetic mechanisms in learning- and pain-related sensitization. This review illustrates the fundamental and functional overlay of the learning and memory field with the pain field which argues for homologous persistent plasticity mechanisms in response to sensitizing stimuli or injury across phyla.

Keywords: 4EBP; 5-HT; 5-aza; 5-azacytidine; Aplysia; BDNF; C/EBP; CAMKII; CBS; CCAAT enhancer binding protein; CCI; CFA; COX-2; CPB; CPEB; CRE; CREB; CREB-binding protein; Ca(2+); Central sensitization; DHPG; DNA methyltransferase; DNA methyltransferase inhibitor; DNMT; DNMTi; DRG; EIF4E; EIF4E binding protein; ERK; Epigenetic; FCS; FK 506 binding protein; FKBP5; FMRFa; HAT; HDAC; HDACi; Histone; IL-6; IP3; ITF; LE; LG; LTD; LTF; LTH; LTP; LTS; Left E cluster; MAPK; MeCP2; Methylation; NF-κB; NO; NRM; NRSE; NRSF; PARP; PGE2; PKA; PKC; PKG; PKM; PSNL; PTM; PTSD; Pi-RNAs; Piwi-interacting RNAs; Pre-Met-ARg-Phe-NH(2); S-adenosyl-methionine; S6 kinase; S6K; SAHA; SAM; SGK1; SGWR; SNI; SNL; STF; SULT1A1; Sensitization; TOR; TSA; TrkB; UCH; VC; ZIP; brain derived neurotrophic factor; cAMP; cAMP response element; cAMP response element binding protein; cAMP-dependent protein kinase; cGMP; calcium; calmodulin-dependent protein kinase II; chronic constriction injury; complete Freund’s adjuvant; cyclic adenosine monophosphate; cyclic guanosine monophosphate; cyclooxygenase-2; cystathionine-β-synthase; cytoplasmic polyadenylation element binding protein; dihydroxyphenylglycine; dorsal root ganglion; eEF2; eukaryotic elongation factor 2; eukaryotic translation initiation factor 4E; extracellular receptor kinase; fast conducting system; histone acetyltransferase; histone acetyltransferase E1A binding protein p300; histone deacetylase; histone deacetylase inhibitor; inositol 1,4,5-trisphosphate; interleukin 6; intermediate-term facilitation; lateral giant neuron; long-term depression; long-term facilitation; long-term hyperexcitability; long-term potentiation; long-term sensitization; methyl-CpG-binding protein 2; mitogen activated protein kinase; neuron-restrictive silence factor; neuron-restrictive silencer element; nitric oxide; nuclear factor kappa-light-chain-enhancer of activated B cells; nucleus raphe magnus; p300; partial sciatic nerve ligation; poly-(ADP-ribose) polymerase; post-translational modifications; post-traumatic stress disorder; prostaglandin E2; protein kinase C; protein kinase G; protein kinase M; serotonin; serum- and glucocorticoid-inducible kinase; short-term facilitation; siphon-gill withdrawal reflex; spared nerve injury; spinal nerve ligation; suberoylanilide hydroxamic acid; sulfotransferase family 1A, phenol-preferring, member 1; target of rapamycin; trichostatin A; tyrosine kinase receptor B; ubiquitin C-terminal hydrolase; ventrocaudal clusters; zeta inhibitory peptide.

Figure 1. Molecular Mechanisms and Signal Transduction Cascades of Aplysia Long-term Facilitation (LTF)

Long-term facilitation in Aplysia sensorimotor neurons in response to serotonin (5-HT). 5-HT-induced Short-Term Facilitation (STF) of Aplysia sensorimotor synapse results in cAMP-dependent protein kinase (PKA)-mediated presynaptic covalent modifications of existing proteins thereby producing short-term enhancement of neuronal excitability. Intermediate-Term Facilitation (ITF) is associated with spontaneous release of presynaptic neurotransmitters, including glutamate, and trafficking of AMPA GluR1-type receptors to the postsynaptic membrane. ITF is marked by long-term hyperexcitability and postsynaptic modifications associated with increased levels of intracellular Ca²⁺ through NMDA-mediated influx and release from IP3-meditated intracellular stores. Release of an as yet, uncharacterized brain derived neurotrophic factor (BDNF)-like ligand activates presynaptic tyrosine kinase-like receptors (ApTrkl) that contribute to extracellular receptor kinase (ERK) activation. Long-Term Facilitation is marked by the growth of new synaptic connections and results in translocation of PKA and mitogen activated protein kinase (MAPK) to the nucleus where cAMP-response element-1 (CREB-1) is activated and CREB-2 is repressed. This results in the induction of several immediate early genes (e.g., ubiquitin C-terminal hydrolase (Ap-UCH) and CCAAT enhancer binding protein (C/EBP)).

Figure 2. Spinal Cord Dorsal Horn Central Sensitization

Mechanisms underlying Central Sensitization in dorsal horn spinal neurons. Nociceptive input from peripheral dorsal root ganglia (DRG) synapse in the dorsal horn of the spinal cord. DRG inputs release the neurotransmitter glutamate and the neurotrophic factor BDNF. Glutamate and BDNF bind to postsynpatic AMPA/NMDA ionotropic receptors and tyrosine kinase B (TrkB) receptors, respectively. Activation of AMPA and NMDA receptors results in increased intracellular Ca²⁺ thereby augmenting synaptic efficacy. Postsynaptic Ca²⁺ influx triggers protein kinase C (PKC), and Ca²⁺/calmodulin-dependent kinase II (CaMKII), both of which have the ability to phosphorylate AMPA receptor subunits. In addition, increased intracellular Ca²⁺ indirectly activates cAMP-dependent protein kinase (PKA) through increased calmodulin and cAMP. PKA and PKC phosphorylate NMDA receptors. PKA, CaMKII, and extracellular receptor kinase (ERK) contribute to recruitment of intracellular GluR1-type AMPA receptors to the membrane from intracellular vesicular stores, ultimately increasing calcium conductance and synaptic efficacy. ERK and PKA contribute to cAMP-response element (CREB)-mediated induction of gene transcription (e.g., c-FOS and cyclooxygenase-2 (COX-2)). Binding of BDNF to TrkB activates PKC- and ERK- mediated signaling cascades.

Figure 3. Epigenetic Mechanisms and Modifications

The DNA-protein complex of chromatin is achieved by packaging DNA into nucleosomes comprised of 147 nucleotide base pairs wrapped around octamers of histone proteins. Histone Modifications, including acetylation, methylation, and phosphorylation dictate chromatin conformation (euchromatin vs. heterochromatin); these modifications contribute to the accessibility of genes for transcription. Enzymatic machinery for histone post-translational modifications include histone acetyltransferases (HAT; acetylate histones), histone deacetylases (HDAC; remove histone acetyl groups), histone methyltransferases (HMT; methylate histones), histone demethylases (HDM; remove histone methyl groups), protein kinases (PK; phosphorylate histones), and protein phosphatases (PP; remove histone phosphate groups). DNA Methylation occurs when DNA methyltransferases (DNMTs) catalyze the addition of a methyl group to the 5’ position of the cytosine pyrimidine ring. In the absence of DNA methylation, transcription factors and RNA polymerase II (RNAP II) bind DNA, resulting in gene expression. Recent evidence suggests the potential for active DNA demethylation through members of the GADD45 and TET families. DNA methylation results in transcriptional repression via recruitment of proteins with methyl-binding domains such as methyl CpG binding protein 2 (MeCP2), that can further recruit corepressor complexes containing HDACs. TSS, transcription start site.