Transcriptomic and proteomic profiling of the anterior cingulate cortex in neuropathic pain model rats.

Background: Neuropathic pain (NP) takes a heavy toll on individual life quality, yet gaps in its molecular characterization persist and effective therapy is lacking. This study aimed to provide comprehensive knowledge by combining transcriptomic and proteomic data of molecular correlates of NP in the anterior cingulate cortex (ACC), a cortical hub responsible for affective pain processing. Methods: The NP model was established by spared nerve injury (SNI) in Sprague-Dawley rats. RNA sequencing and proteomic data from the ACC tissue isolated from sham and SNI rats 2 weeks after surgery were integrated to compare their gene and protein expression profiles. Bioinformatic analyses were performed to figure out the functions and signaling pathways of the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) enriched in. Results: Transcriptomic analysis identified a total of 788 DEGs (with 49 genes upregulated) after SNI surgery, while proteomic analysis found 222 DEPs (with 89 proteins upregulated). While Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of the DEGs suggested that most of the altered genes were involved in synaptic transmission and plasticity, bioinformatics analysis of the DEPs revealed novel critical pathways associated with autophagy, mitophagy, and peroxisome. Notably, we noticed functionally important NP-related changes in the protein that occurred in the absence of corresponding changes at the level of transcription. Venn diagram analysis of the transcriptomic and proteomic data identified 10 overlapping targets, among which only three genes (XK-related protein 4, NIPA-like domain-containing 3, and homeodomain-interacting protein kinase 3) showed concordance in the directions of change and strong correlations between mRNA and protein levels. Conclusion: The present study identified novel pathways in the ACC in addition to confirming previously reported mechanisms for NP etiology, and provided novel mechanistic insights for future research on NP treatment. These findings also imply that mRNA profiling alone fails to provide a complete landscape of molecular pain in the ACC. Therefore, explorations of changes at the level of protein are necessary to understand NP processes that are not transcriptionally modulated.

Inhibitory effects of endomorphin-2 on excitatory synaptic transmission and the neuronal excitability of sacral parasympathetic preganglionic neurons in young rats.

The function of the urinary bladder is partly controlled by parasympathetic preganglionic neurons (PPNs) of the sacral parasympathetic nucleus (SPN). Our recent work demonstrated that endomorphin-2 (EM-2)-immunoreactive (IR) terminals form synapses with µ-opioid receptor (MOR)-expressing PPNs in the rat SPN. Here, we examined the effects of EM-2 on excitatory synaptic transmission and the neuronal excitability of the PPNs in young rats (24-30 days old) using a whole-cell patch-clamp approach. PPNs were identified by retrograde labeling with the fluorescent tracer tetramethylrhodamine-dextran (TMR). EM-2 (3 µM) markedly decreased both the amplitude and the frequency of the spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) of PPNs. EM-2 not only decreased the resting membrane potentials (RMPs) in 61.1% of the examined PPNs with half-maximal response at the concentration of 0.282 µM, but also increased the rheobase current and reduced the repetitive action potential firing of PPNs. Analysis of the current-voltage relationship revealed that the EM-2-induced current was reversed at -95 ± 2.5 mV and was suppressed by perfusion of the potassium channel blockers 4-aminopyridine (4-AP) or BaCl2 or by the addition of guanosine 5'-[ß-thio]diphosphate trilithium salt (GDP-ß-S) to the pipette solution, suggesting the involvement of the G-protein-coupled inwardly rectifying potassium (GIRK) channel. The above EM-2-invoked inhibitory effects were abolished by the MOR selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), indicating that the effects of EM-2 on PPNs were mediated by MOR via pre- and/or post-synaptic mechanisms. EM-2 activated pre- and post-synaptic MORs, inhibiting excitatory neurotransmitter release from the presynaptic terminals and decreasing the excitability of PPNs due to hyperpolarization of their membrane potentials, respectively. These inhibitory effects of EM-2 on PPNs at the spinal cord level may explain the mechanism of action of morphine treatment and morphine-induced bladder dysfunction in the clinic.

Activation of mu-opioid receptors in the ventrolateral orbital cortex inhibits the GABAergic miniature inhibitory postsynaptic currents in rats.

Previous studies have indicated that mu-opioid receptors in the ventrolateral orbital cortex (VLO) are involved in antinociception in tail flick tests and GABAergic neurons or terminals express mu-opioid receptors in the VLO. The current study examined the effect of selective mu-opioid receptor agonist DAMGO on the GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in the VLO in rats using the whole-cell patch clamp. The results demonstrated that 5 µM DAMGO application into the rat VLO slices significantly reduced the GABAergic mIPSCs frequency, without any effect on its amplitude, and this effect of DAMGO was reversed by pretreatment with selective mu-opioid receptor antagonist 1 µM CTOP. Importantly, application of CTOP alone into the VLO slices did not produce any effect on the frequency and amplitude of GABAergic mIPSCs. These results indicate a presynaptic effect of mu-opioid receptor activation on the GABAergic neurons in the VLO. The current data suggests that a presynaptic inhibition of the GABA release may contribute to the mu-opioid receptor mediated effects in the VLO and provides novel electrophysiological evidence for the underlying mechanisms of mu-opioid receptors in the VLO.

Emetine treatment masks initial LTP without affecting long-term stability.

Applying emetine, a protein synthesis inhibitor, at 20-40µM for 90-120 min prior to LTP induction in hippocampal slices from young rats (2-3 weeks) and washing it out afterwards revealed a slowly developing potentiation that reached maximum after 20-30 min, distinct from the LTP observed under normal conditions. Nevertheless, the later phase of this potentiation was similar to standard LTP as judged by experiments lasting up to 8h after induction. Emetine preapplication for 3h without subsequent washout resulted in a substantial decay of evoked responses. By comparison between test and control pathways, LTP could still be assessed in these experiments for up to 4-6h after induction and was found not to differ from normal, except for the slow onset. The NMDA-R blocker AP5 fully blocked LTP; however, with emetine pretreatment there was an initial depression of responses with a gradual recovery during 20-30 min. This depression involved not only the field EPSP but also the presynaptic fiber volley. However, when using the protein synthesis inhibitors cycloheximide and anisomycin there was essentially no such depression. In conclusion, the present results support the idea that preexisting proteins are sufficient for inducing stable LTP. Moreover, emetine but not anisomycin or cycloheximide impairs presynaptic action potentials, leading to an apparent slow onset of LTP. The emetine-dependent effect could be due to a characteristic blocking spectrum of the drug, preferred targeting of presynaptic compartments or effects unrelated to protein synthesis.

Activation of serotonin 1A receptors in ventrolateral orbital cortex depresses persistent nociception: a presynaptic inhibition mechanism.

The present study examined the effect of serotonin 1A (5-HT(1A)) receptor activation in the ventrolateral orbital cortex (VLO) upon formalin-evoked flinching behavior and spinal Fos expression, and further determined whether activation of 5-HT(1A) receptors affected the spontaneous GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in rat VLO slice by pharmacologically separated neurons to understand the possible mechanism underlying this effect. Microinjection of the 5-HT(1A) receptors agonist 8-OH-DPAT (8-hydro-2-(di-n-propylamino) tetralin) into the VLO depressed the formalin-evoked nociceptive behavior flinching response and the Fos expression in the lumbar spinal cord dorsal, which was antagonized by pre-treatment with 5-HT(1A) receptors antagonist NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide). Furthermore, application of 8-OH-DPAT into VLO slice inhibited GABAergic mIPSC frequency in a dose-dependent manner without effects on amplitude of the GABAergic mIPSCs, this effect was blocked by NAN-190. These results provide evidence for the involvement of 5-HT(1A) receptors in VLO in the modulation of persistent inflammatory nociception, and suggest that a presynaptic inhibition of the GABA release may contribute to the 5-HT(1A) receptor-mediated descending antinociception.

Bidirectional synaptic plasticity in response to single or paired pulse activation of NMDA receptors.

It is still incompletely known how NMDA receptors (NMDA-R) regulate bidirectional synaptic plasticity. We examined this issue by an experimental protocol in which paired pulse stimulation (PPS) with 50ms interstimulus interval and basal frequency of 0.1Hz was applied to CA1 area of rat hippocampal slices during low Mg(2+) perfusion. Under blockade of NMDA-Rs by AP5, PPS for 12-60min led to only a minor depression. In contrast, when PPS was applied in the absence of AP5, there was a prominent short-term potentiation (STP), mainly of AMPA-R mediated responses, with peak at 1min and lasting 10-15min. The STP was followed by a slowly developing long-term depression (LTD). Applying AP5 during the STP, converted it to a stable increase relative to the control pathway. Following peak STP, plasticity was controlled in a composite manner. Whereas the initial decay was counteracted by NMDA-R activation, the following LTD was dependent on such activation. Our data suggest that synaptic changes do not only depend on the instantaneous, NMDA-dependent Ca(2+) concentration in the dendritic spine, but are also influenced by prior induction events. In addition to NMDA-R driven processes, passive relaxation contributes to the synaptic plasticity and in some cases outbalances the active control.

Persistent LTP without triggered protein synthesis.

Protein synthesis is believed to be involved in stabilizing synaptic plasticity. Effects lasting longer than about 2-3h are considered to require synthesis of new proteins, implying a functional separation between early (E) and late (L) components. However, the issue of constitutive vs. new protein synthesis is still unclear, especially in young animals. Here, we examined the effects of two protein synthesis inhibitors, anisomycin and emetine, on long-term-potentiation (LTP) in CA1 area of hippocampal slices from 12- to 20-day-old rats. Either drug was applied from -30 min to +30 min with respect to LTP induction, a time window previously reported to be critical. However, the LTP remained stable under the entire recording period of 4h (anisomycin), or 8h (emetine). Proper preparation of emetine solution was evidenced by the fact that, in separate experiments, prolonged treatment with emetine gradually blocked baseline responses. Although no corresponding effect was observed with anisomycin, the drug was judged to be potent by its ability to inhibit yeast growth. The ability of anisomycin to inhibit protein synthesis was further confirmed by radiolabeling experiments assessing the degree of leucine incorporation. Our data suggest that LTP up to at least 8h is not dependent on triggered protein synthesis but can be attained by utilizing proteins already available at induction time.

Microinjection of morphine into thalamic nucleus submedius depresses bee venom-induced inflammatory pain in the rat.

Previous studies have provided evidence of the existence of a pain modulatory feedback pathway consisting of thalamic nucleus submedius (Sm)-ventrolateral orbital cortex-periaqueductal grey pathway, which is activated during acute pain and leads to depression of transmission of nociceptive information in the spinal dorsal horn. The aim of this study was to test the hypothesis that morphine microinjection into the Sm decreased spontaneous pain and bilateral thermal hyperalgesia, as well as ipsilateral mechanical allodynia, induced by subcutaneous injections of bee venom into the rat hind paw. Morphine (1.0, 2.5 or 5.0 microg in 0.5 microL) injected into the Sm, contralateral to the bee venom-injected paw, depressed spontaneous nociceptive behaviour in a dose-dependent manner. Furthermore, morphine significantly decreased bilateral thermal hyperalgesia and ipsilateral mechanical allodynia 2 h after bee venom injection. These morphine-induced effects were antagonized by 1.0 microg naloxone (an opioid antagonist) microinjected into the Sm 5 min before morphine administration. The results provided further support for the important role of the Sm and Sm-opioid receptors in inhibiting nociceptive behaviour and indicated for the first time that Sm opioid receptors were also effective in inhibiting the hypersensitivity provoked by bee venom-induced inflammation.

Mu-opioid receptor in the nucleus submedius: involvement in opioid-induced inhibition of mirror-image allodynia in a rat model of neuropathic pain.

The current study investigated the roles of various subtypes of opioid receptors expressed in the thalamic nucleus submedius (Sm) in inhibition of mirror-image allodynia induced by L5/L6 spinal nerve ligation in rats. Morphine was microinjected into the Sm, which produced a dose-dependent inhibition of mirror-image allodynia; this effect was antagonized by pretreatment with non-selective opioid receptor antagonist naloxone. Microinjections of endomorphin-1 (mu-receptor agonist), or [D-Ala(2), D-Leu(5)]-enkephalin (DADLE, delta-/mu-receptor agonist), also inhibited mirror-image allodynia, and these effects were blocked by the selective mu-receptor antagonist, beta-funaltrexamine hydrochloride. The DADLE-induced inhibition, however, was not influenced by the delta-receptor antagonist naltrindole. The kappa-receptor agonist, spiradoline mesylate salt, failed to alter the mirror-image allodynia. These results suggest that Sm opioid receptor signaling is involved in inhibition of mirror-image allodynia; this effect is mediated by mu- (but not delta- and kappa-) opioid receptors in the rat model of neuropathic pain.

Analysis of high-frequency stimulation-evoked synaptic plasticity in mouse hippocampal CA1 region.

Extracellular recordings of field excitatory postsynaptic potential (fEPSP) is one of the most common ways for studies of synaptic plasticity, such as long-term potentiation (LTP) and paired-pulse plasticity (PPP). The measurement of the changes in the different components of fEPSP waveform, such as the initial slope, initial area, peak amplitude and whole area, were commonly used as criteria for the judgement of potentiation or depression of synaptic plasticity. However, the differences in the conclusions drawn from measuring different components of fEPSP waveform at the same recording have still been largely ignored. Here we compared high-frequency stimulation (HFS)-evoked synaptic plasticity, both LTP and PPP, by measuring different components of fEPSP waveform, including the initial slope, initial area, peak amplitude, whole area and time course. The results not only indicated the acceleration of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor kinetics underlies LTP in hippocampal CA1 region of mice, but also showed that different measurements of fEPSP waveform at the same recording result in different magnitudes of LTP and different forms of PPP in hippocampal CA1 region of mice. After HFS, the paired-pulse ratio was slightly decreased by measurement of the initial area, but obviously increased by measurement of the initial slope of the pair fEPSPs. These results might draw apparently contradictory conclusions. Therefore, careful and complete analysis of the data from different parts of fEPSP waveforms is important for reflection of the faithful changes in synaptic plasticity.

Role of NMDA receptor subtypes in different forms of NMDA-dependent synaptic plasticity.

BACKGROUND: The involvement of different NMDA receptor (NMDAR) subunits has been implicated in several forms of synaptic plasticity. However, it is still controversial to what extent the involvement is specific, and little is known about the role of NMDAR subunits in certain "non-conventional" forms of plasticity. In this study we used subunit-specific blockers to test the roles of NR2A- and NR2B-containing NMDARs in a type of chemical long-term depression (LTD) induced by brief bath application of the NMDAR agonist NMDA to hippocampal slices from 12-18 days old rats. For comparison, we also examined other forms of plasticity, including a "slow LTD" induced by 0.1 Hz stimulation under low Mg2+ conditions as well as long-term potentiation (LTP). RESULTS: A blocker of NR2A-containing NMDARs, NVP-AAM077 (NVP), substantially reduced the two forms of studied depression whereas blockers of NR2B-containing NMDARs, Ro25-6981 (Ro) or Ifenprodil (Ife), had no significant effect on them. LTP appeared to be more sensitive as it was fully blocked by NVP and partially blocked by Ro or Ife. However, the blocking effects of NVP could be counteracted by general amplification of NMDA responses by lowering Mg2+ concentration in the perfusion solution. Applying NVP or Ro/Ife on isolated NMDA-EPSPs recorded in low Mg2+ solution reduced responses to about 70% and 20% of initial size, respectively, whereas coapplication of both blockers almost completely abolished the responses. Additionally, NMDA application caused depotentiation of a pathway with prior tetanus-induced LTP, and NVP but not Ro/Ife substantially prevented that depotentiation as well as the chemical LTD of the control pathway. A second tetanus on the LTP pathway induced repotentiation which was fully blocked by NVP but partially blocked by Ro/Ife. CONCLUSION: All of these results on hippocampal slices from young rats can be explained by a simple model, in which NR2A subunits dominate over NR2B subunits with respect to both plasticity and NMDAR-mediated responses. The model suggests that Ca2+ influx into the postsynaptic spine via different subtypes of NMDARs makes up a "final common pathway", controlling synaptic plasticity by its magnitude and temporal pattern regardless of the source.

Properties of paired-pulse firing thresholds and the relationship with paired-pulse plasticity in hippocampal CA3-CA1 synapses.

A large variability of paired-pulse plasticity (PPP) has been reported in CA3-CA1 synapses, and paired-pulse facilitation (PPF) has long been extensively used as a relative index of release probability (P(r)) from the presynaptic terminal. One of the most common ways of studying P(r) and PPP is to pass paired-pulse stimulation (PPS) through an electrode to fire an action potential (AP), which in turn opens Ca(2+) channels in the presynaptic boutons to evoke transmitter release. However, when the postsynaptic responses were elicited by electrical stimulations, the presynaptic APs were usually not monitored. The reliability of presynaptic activation, the difference of AP firing thresholds between the first and the second pulses of PPS, and its relationship with P(r) and PPP have been largely ignored. Here we show that the AP firing thresholds in the same CA3 pyramidal cells (axons) are obviously lower for the second pulses of the PPS than for the first pulses. When single (or small numbers of) presynaptic axons were stimulated by low-intensity PPS, a large variation in the postsynaptic response probability of the first pulses (P(res1)) and PPP was observed. Increasing stimulation intensities resulted in the conversion of a lower P(res1) and PPF to a higher P(res1) and paired-pulse depression. These results indicate that changes in the reliability of AP initiation by the first pulse of the PPS may account for the high variability of P(res1) and PPP observed in CA1 pyramidal cells of the hippocampus, therefore PPF may not be a reliable index of P(r) .

Contribution of AMPA and NMDA receptors to early and late phases of LTP in hippocampal slices.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor mediated responses were investigated in rat hippocampal slices under 4h of long-term potentiation (LTP) expression. A modified medium containing the NMDA receptor antagonist AP5 and low concentration of Mg(2+) was used to monitor isolated AMPA responses. NMDA components were determined from composite excitatory postsynaptic potentials (EPSPs) under brief (15-20 min) wash-out of AP5. LTP was induced in a medium with low concentration of AP5, resulting in an about two-fold larger increase of the AMPA component than of the NMDA component at both 1h and 4h after induction. Similar results were obtained if LTP was induced in "normal Mg(2+)" and the NMDA components were assessed at the end of experiment, from either composite or isolated NMDA EPSPs, with or without blockade of GABAergic inhibition. It is generally believed that LTP undergoes biochemical and/or structural conversions during the first few hours. Our study, however, shows constant expression of LTP, at least in terms of AMPA versus NMDA components, during this time. The data support the notion that LTP initiates as a predominant amplification of AMPA receptors and remains so for at least 4h.