Chemotherapy for pain: reversing inflammatory and neuropathic pain with the anticancer agent mithramycin A.

ABSTRACT: The persistence of inflammatory and neuropathic pain is poorly understood. We investigated a novel therapeutic paradigm by targeting gene networks that sustain or reverse persistent pain states. Our prior observations found that Sp1-like transcription factors drive the expression of TRPV1, a pain receptor, that is blocked in vitro by mithramycin A (MTM), an inhibitor of Sp1-like factors. Here, we investigate the ability of MTM to reverse in vivo models of inflammatory and chemotherapy-induced peripheral neuropathy (CIPN) pain and explore MTM's underlying mechanisms. Mithramycin reversed inflammatory heat hyperalgesia induced by complete Freund adjuvant and cisplatin-induced heat and mechanical hypersensitivity. In addition, MTM reversed both short-term and long-term (1 month) oxaliplatin-induced mechanical and cold hypersensitivity, without the rescue of intraepidermal nerve fiber loss. Mithramycin reversed oxaliplatin-induced cold hypersensitivity and oxaliplatin-induced TRPM8 overexpression in dorsal root ganglion (DRG). Evidence across multiple transcriptomic profiling approaches suggest that MTM reverses inflammatory and neuropathic pain through broad transcriptional and alternative splicing regulatory actions. Mithramycin-dependent changes in gene expression following oxaliplatin treatment were largely opposite to and rarely overlapped with changes in gene expression induced by oxaliplatin alone. Notably, RNAseq analysis revealed MTM rescue of oxaliplatin-induced dysregulation of mitochondrial electron transport chain genes that correlated with in vivo reversal of excess reactive oxygen species in DRG neurons. This finding suggests that the mechanism(s) driving persistent pain states such as CIPN are not fixed but are sustained by ongoing modifiable transcription-dependent processes.

N-Oleoyl dopamine induces IL-10 via central nervous system TRPV1 and improves endotoxemia and sepsis outcomes.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) participates in thermosensation and inflammatory pain, but its immunomodulatory mechanisms remain enigmatic. N-Oleoyl dopamine (OLDA), an endovanilloid and endocannabinoid, is a TRPV1 agonist that is produced in the central nervous system and the peripheral nervous system. We studied the anti-inflammatory effects and TRPV1-dependent mechanisms of OLDA in models of inflammation and sepsis. METHODS: Mice were challenged intratracheally or intravenously with LPS, or intratracheally with S. aureus to induce pneumonia and sepsis, and then were treated intravenously with OLDA. Endpoints included plasma cytokines, leukocyte activation marker expression, mouse sepsis scores, lung histopathology, and bacterial counts. The role of TRPV1 in the effects of OLDA was determined using Trpv1-/- mice, and mice with TRPV1 knockdown pan-neuronally, in peripheral nervous system neurons, or in myeloid cells. Circulating monocytes/macrophages were depleted using clodronate to determine their role in the anti-inflammatory effects of OLDA in endotoxemic mice. Levels of exogenous OLDA, and of endovanilloids and endocannabinoids, at baseline and in endotoxemic mice, were determined by LC-MS/MS. RESULTS: OLDA administration caused an early anti-inflammatory response in endotoxemic and septic mice with high serum levels of IL-10 and decreased levels of pro-inflammatory cytokines. OLDA also reduced lung injury and improved mouse sepsis scores. Blood and lung bacterial counts were comparable between OLDA- and carrier-treated mice with S. aureus pneumonia. OLDA's effects were reversed in mice with pan-neuronal TRPV1 knockdown, but not with TRPV1 knockdown in peripheral nervous system neurons or myeloid cells. Depletion of monocytes/macrophages reversed the IL-10 upregulation by OLDA in endotoxemic mice. Brain and blood levels of endovanilloids and endocannabinoids were increased in endotoxemic mice. CONCLUSIONS: OLDA has strong anti-inflammatory actions in mice with endotoxemia or S. aureus pneumonia. Prior studies focused on the role of peripheral nervous system TRPV1 in modulating inflammation and pneumonia. Our results suggest that TRPV1-expressing central nervous system neurons also regulate inflammatory responses to endotoxemia and infection. Our study reveals a neuro-immune reflex that during acute inflammation is engaged proximally by OLDA acting on neuronal TRPV1, and through a multicellular network that requires circulating monocytes/macrophages, leads to the systemic production of IL-10.

Quantitative analysis of synaptic release at the photoreceptor synapse.

Exocytosis from the rod photoreceptor is stimulated by submicromolar Ca(2+) and exhibits an unusually shallow dependence on presynaptic Ca(2+). To provide a quantitative description of the photoreceptor Ca(2+) sensor for exocytosis, we tested a family of conventional and allosteric computational models describing the final Ca(2+)-binding steps leading to exocytosis. Simulations were fit to two measures of release, evoked by flash-photolysis of caged Ca(2+): exocytotic capacitance changes from individual rods and postsynaptic currents of second-order neurons. The best simulations supported the occupancy of only two Ca(2+) binding sites on the rod Ca(2+) sensor rather than the typical four or five. For most models, the on-rates for Ca(2+) binding and maximal fusion rate were comparable to those of other neurons. However, the off-rates for Ca(2+) unbinding were unexpectedly slow. In addition to contributing to the high-affinity of the photoreceptor Ca(2+) sensor, slow Ca(2+) unbinding may support the fusion of vesicles located at a distance from Ca(2+) channels. In addition, partial sensor occupancy due to slow unbinding may contribute to the linearization of the first synapse in vision.

Role of the synaptic ribbon in transmitting the cone light response.

Cone photoreceptors distinguish small changes in light intensity while operating over a wide dynamic range. The cone synapse encodes intensity by modulating tonic neurotransmitter release, but precise encoding is limited by the quantal nature of synaptic vesicle exocytosis. Cones possess synaptic ribbons, structures that are thought to accelerate the delivery of vesicles for tonic release. Here we show that the synaptic ribbon actually constrains vesicle delivery, resulting in a maintained state of synaptic depression in darkness. Electron microscopy of cones from the lizard Anolis segrei revealed that depression is caused by the depletion of vesicles on the ribbon, indicating that resupply, not fusion, is the rate-limiting step that controls release. Responses from postsynaptic retinal neurons from the salamander Ambystoma tigrinum showed that the ribbon behaves like a capacitor, charging with vesicles in light and discharging in a phasic burst at light offset. Phasic release extends the operating range of the cone synapse to more accurately encode changes in light intensity, accentuating features that are salient to photopic vision.

Calcium-induced calcium release in rod photoreceptor terminals boosts synaptic transmission during maintained depolarization.

We examined the contribution of calcium-induced calcium release (CICR) to synaptic transmission from rod photoreceptor terminals. Whole-cell recording and confocal calcium imaging experiments were conducted on rods with intact synaptic terminals in a retinal slice preparation from salamander. Low concentrations of ryanodine stimulated calcium increases in rod terminals, consistent with the presence of ryanodine receptors. Application of strong depolarizing steps (-70 to -10 mV) exceeding 200 ms or longer in duration evoked a wave of calcium that spread across the synaptic terminals of voltage-clamped rods. This secondary calcium increase was blocked by high concentrations of ryanodine, indicating it was due to CICR. Ryanodine (50 microm) had no significant effect on rod calcium current (I(ca)) although it slightly diminished rod light-evoked voltage responses. Bath application of 50 microm ryanodine strongly inhibited light-evoked currents in horizontal cells. Whether applied extracellularly or delivered into the rod cell through the patch pipette, ryanodine (50 microm) also inhibited excitatory post-synaptic currents (EPSCs) evoked in horizontal cells by depolarizing steps applied to rods. Ryanodine caused a preferential reduction in the later portions of EPSCs evoked by depolarizing steps of 200 ms or longer. These results indicate that CICR enhances calcium increases in rod terminals evoked by sustained depolarization, which in turn acts to boost synaptic exocytosis from rods.

Paired-pulse depression at photoreceptor synapses.

Synaptic depression produced by repetitive stimulation is likely to be particularly important in shaping responses of second-order retinal neurons at the tonically active photoreceptor synapse. We analyzed the time course and mechanisms of synaptic depression at rod and cone synapses using paired-pulse protocols involving two complementary measurements of exocytosis: (1) paired whole-cell recordings of the postsynaptic current (PSC) in second-order retinal neurons and (2) capacitance measurements of vesicular membrane fusion in rods and cones. PSCs in ON bipolar, OFF bipolar, and horizontal cells evoked by stimulation of either rods or cones recovered from paired-pulse depression (PPD) at rates similar to the recovery of exocytotic capacitance changes in rods and cones. Correlation between presynaptic and postsynaptic measures of recovery from PPD suggests that 80-90% of the depression at these synapses is presynaptic in origin. Consistent with a predominantly presynaptic mechanism, inhibiting desensitization of postsynaptic glutamate receptors had little effect on PPD. The depression of exocytotic capacitance changes exceeded depression of the presynaptic calcium current, suggesting that it is primarily caused by a depletion of synaptic vesicles. In support of this idea, limiting Ca2+ influx by using weaker depolarizing stimuli promoted faster recovery from PPD. Although cones exhibit much faster exocytotic kinetics than rods, exocytotic capacitance changes recovered from PPD at similar rates in both cell types. Thus, depression of release is not likely to contribute to differences in the kinetics of transmission from rods and cones.

Kinetics of exocytosis is faster in cones than in rods.

Cone-driven responses of second-order retinal neurons are considerably faster than rod-driven responses. We examined whether differences in the kinetics of synaptic transmitter release from rods and cones may contribute to differences in postsynaptic response kinetics. Exocytosis from rods and cones was triggered by membrane depolarization and monitored in two ways: (1) by measuring EPSCs evoked in second-order neurons by depolarizing steps applied to presynaptic rods or cones during simultaneous paired whole-cell recordings or (2) by direct measurements of exocytotic increases in membrane capacitance. The kinetics of release was assessed by varying the length of the depolarizing test step. Both measures of release revealed two kinetic components to the increase in exocytosis as a function of the duration of a step depolarization. In addition to slow sustained components in both cell types, the initial fast component of exocytosis had a time constant of <5 ms in cones, >10-fold faster than that of rods. Rod/cone differences in the kinetics of release were substantiated by a linear correlation between depolarization-evoked capacitance increases and EPSC charge transfer. Experiments on isolated rods indicate that the slower kinetics of exocytosis from rods was not a result of rod-rod coupling. The initial rapid release of vesicles from cones can shape the postsynaptic response and may contribute to the faster responses of cone-driven cells observed at light offset.

A highly Ca2+-sensitive pool of vesicles contributes to linearity at the rod photoreceptor ribbon synapse.

Studies of the properties of synaptic transmission have been carried out at only a few synapses. We analyzed exocytosis from rod photoreceptors with a combination of physiological and ultrastructural techniques. As at other ribbon synapses, we found that rods exhibited rapid kinetics of release, and the number of vesicles in the releasable pool is comparable to the number of vesicles tethered at ribbon-style active zones. However, unlike other previously studied neurons, we identified a highly Ca(2+)-sensitive pool of releasable vesicles with a relatively shallow relationship between the rate of exocytosis and [Ca(2+)](i) that is nearly linear over a presumed physiological range of intraterminal [Ca(2+)]. The low-order [Ca(2+)] dependence of release promotes a linear relationship between Ca(2+) entry and exocytosis that permits rods to relay information about small changes in illumination with high fidelity at the first synapse in vision.

Reciprocal interactions between calcium and chloride in rod photoreceptors.

This study used imaging and electrophysiological techniques in salamander retinal slices to correlate Ca2+ and Cl- levels in rods and thus test the hypothesis of a feedback interaction between Ca2+- and Ca2+-activated Cl- channels whereby Cl- efflux through Cl- channels can inhibit Ca2+ channels. Increasing [K+]o levels produced a concentration-dependent depolarization of rods accompanied by increases in [Ca2+]i measured with Fura-2. The voltage dependence of increases in [Ca2+]i was compared with the voltage dependence of the calcium current (ICa). [Cl-]i was measured with the dye, MEQ. Depolarization with high K+ to membrane potentials below -20 mV reduced [Cl-]i; larger depolarizations increased [Cl-]i. The Na/K/Cl cotransport inhibitor, bumetanide, shifted the apparent Cl- equilibrium potential (ECl) to more negative potentials, suggesting that this cotransporter helps establish a relatively depolarized ECl. MEQ fluorescence changes evoked by high K+ were inhibited by niflumic acid (0.1 mM), NPPB (2 microM), or replacement of Ca2+ with Ba2+, suggesting that depolarization-evoked Cl- changes result partly from stimulation of Ca2+-activated Cl- channels. Replacing >/=12 mM [Cl-]o with CH3SO4- produced a significant reduction in [Cl-]i. [Ca2+]i increases evoked by 20 or 50 mM K+ were also significantly inhibited by replacing >/=12 mM [Cl-]o with CH3SO4-. Thus modest depolarization can evoke increases in [Ca2+]i that lead to reductions in [Cl-]i, and conversely, reductions in [Cl-]i inhibit depolarization-evoked [Ca2+]i increases. These findings support the hypothesis that feedback interactions between Ca2+- and Ca2+-activated Cl- channels may contribute to the regulation of presynaptic Ca2+ currents involved in synaptic transmission from rod photoreceptors.

Activation of glutamate transporters in rods inhibits presynaptic calcium currents.

We found that L-glutamate (L-Glu) inhibits L-type Ca2+ currents (ICa) in rod photoreceptors. This inhibition was studied in isolated rods or rods in retinal slices from tiger salamander using perforated patch whole cell recordings and Cl(-)-imaging techniques. Application of L-Glu inhibited ICa by approximately 20% at 0.1 mM and approximately 35% at 1 mM. L-Glu also produced an inward current that reversed around ECl. The metabotropic glutamate receptor (mGluR) agonists t-ADA (Group I), DCG-IV (Group II), and L-AP4 (Group III) had no effect on ICa. However, the glutamate transport inhibitor, TBOA (0.1 mM), prevented L-Glu from inhibiting ICa. D-aspartate (D-Asp), a glutamate transporter substrate, also inhibited ICa with significantly more inhibition at 1 mM than 0.1 mM. Using Cl imaging, L-Glu (0.1-1 mM) and D-Asp (0.1-1 mM) were found to stimulate a Cl- efflux from terminals of isolated rods whereas the ionotropic glutamate receptor agonists NMDA, AMPA, and kainate and the mGluR agonist, 1S,3R-ACPD, did not. Glutamate-evoked Cl- effluxes were blocked by the glutamate transport inhibitors TBOA and DHKA. Cl- efflux inhibits Ca2+ channel activity in rod terminals (Thoreson et al. (2000), Visual Neuroscience 17, 197). Consistent with the possibility that glutamate-evoked Cl- efflux may play a role in the inhibition, reducing intraterminal Cl- prevented L-Glu from inhibiting ICa. In summary, the results indicate that activation of glutamate transporters inhibits ICa in rods possibly as a consequence of Cl- efflux. The neurotransmitter L-Glu released from rod terminals might thus provide a negative feedback signal to inhibit further L-Glu release.

Calcium-dependent inactivation and depletion of synaptic cleft calcium ions combine to regulate rod calcium currents under physiological conditions.

L-type Ca2+ currents (I(Ca)) in rod photoreceptors exhibit Ca2+-dependent inactivation. Perforated-patch whole-cell recordings were obtained from isolated rods of the tiger salamander using 1.8 mm Ca2+ in the bathing medium to determine the extent of Ca2+-dependent inactivation of I(Ca) with physiological [Ca2+] and endogenous buffering. I(Ca) was measured with voltage ramps applied before and after 5-s steps to -40, -30, -20, or -10 mV. Long depolarizing steps in isolated rods produced inactivation of I(Ca) ranging from 15% at -40 mV to > 80% at -10 mV. Because, in addition to Ca2+-dependent inactivation, depletion of synaptic cleft Ca2+ accompanying activation of I(Ca) can reduce presynaptic I(Ca) at calycal synapses, we investigated whether a similar mechanism worked at the invaginating rod synapse. Rods from retinal slices with intact synapses were compared with isolated rods in which synaptic cleft depletion is absent. I(Ca) was more strongly depressed by depolarization of rods in retinal slices, with ICa reduced by 47% following voltage steps to -40 mV. The depression of currents by depolarization was also greater for rods from retinal slices than isolated rods when Ca2+ was replaced with Ba2+ to reduce Ca2+-dependent inactivation. The stronger depolarization-evoked inhibition of I(Ca) in retinal slices compared to isolated rods probably reflects depletion of synaptic cleft Ca2+ arising from sustained Ca2+ influx. Inactivation of I(Ca) exhibited slow onset and recovery. These findings suggest that Ca2+-dependent inactivation and depletion of synaptic cleft Ca2+ may combine to regulate I(Ca) in response to light-evoked changes in rod membrane potential.

PACAP inhibits anoxia-induced changes in physiological responses in horizontal cells in the turtle retina.

Pituitary adenylate cyclase activating polypeptide (PACAP) has neurotrophic and neuroprotective effects against various cytotoxic agents in vitro, and ischemia in vivo. Anoxia tolerance is most highly developed in some species of turtles. Recently, we have demonstrated high levels of PACAP38 in the turtle brain, exceeding those in corresponding rat and human brain areas by 10- to 100-fold. The aim of the present study was to investigate with electrophysiological methods the protective effects of PACAP in anoxia-induced neuronal damage of turtle retinal horizontal cells. Adult turtles (Pseudemys scripta elegans) were used for the experiments. After decapitation, half of the isolated eyecup slices were placed into a non-oxygenated Ringer solution, the other half into 0.165 microM PACAP solution. Intracellular recordings were obtained from horizontal cells 18, 22, 42 and 46 h after removal of the eyes. The amplitudes of light responses with the exception of the 0-h measurement, were larger at all time-points in PACAP-incubated slices than in control retinal slices. After both 18 and 22 h, the response amplitudes of PACAP-treated cells exceeded those taken from control horizontal cells by 1.2-fold. At later times, this difference became larger than 2-fold. In summary, the present results provide evidence that PACAP has neuroprotective effects on the anoxic retinal cells in the turtle.

Electrophysiological evidence for push-pull interactions in the inner retina of turtle.

The responses of the inner retinal neurons of turtle to light spots of sizes were studied in an attempt to reveal characteristics that may reflect possible interactions of the neural circuits underlying the center and surround responses. For the ON-OFF cells, the responses were also analyzed to observe whether interference or augmentation of these responses occur. The intracellular recordings revealed several such interactions, observed either in the form of altered spike activity or as changes in the transiency of the light responses. The ON-responding amacrine cell presented in this study became more sustained, while for the ON-OFF amacrine cells larger light spots tended to make the responses more transient and both the ON and OFF components became more pronounced. The spiking activity of the OFF-type ganglion cell shifted in relation to the light stimulus and the number of spikes observed upon presentation of larger spots increased. We suggest that the surround circuits activated by increasing light spots may substantially influence and reorganize not only the overall center-surround balance, but also the center response of the cells. Although it cannot be excluded that intrinsic membrane properties also influence these processes to some extent, it is more likely that lateral inhibition and disinhibitory mechanisms play the leading role in this process.