Hiroshi Ikeda

Long‐term potentiation of neuronal excitation by neuron–glia interactions in the rat spinal dorsal horn

By imaging neuronal excitation in rat spinal cord slices with a voltage‐sensitive dye, we examined the role of glial cells in the P2X receptor agonist αβ‐methylene ATP (αβmeATP)‐triggered long‐term potentiation (LTP) in the dorsal horn. Bath application of αβmeATP potentiated neuronal excitation in the superficial dorsal horn. The potentiation was inhibited in the presence of the P2X receptor antagonists TNP‐ATP, PPADS and A‐317491, and was not induced in slices taken from rats neonatally treated with capsaicin. These results suggest that αβmeATP acts on P2X receptors, possibly P2X3 and/or P2X2/3, in capsaicin‐sensitive primary afferent terminals. Furthermore, the potentiation was inhibited by treatment with the glial metabolism inhibitor monofluoroacetic acid. Results obtained with the p38 mitogen‐activated protein kinase (p38 MAPK) inhibitor SB203580, tumour necrosis factor‐α (TNF‐α) and interleukin (IL)‐6, and antibodies to TNF‐α and IL‐6, as well as by double immunolabelling of activated p38 MAPK with markers of astrocytes and microglia, demonstrated that αβmeATP activated p38 MAPK in astrocytes, and that the presence of proinflammatory cytokines and p38 MAPK activation were necessary for the induction of αβmeATP‐triggered LTP. These findings indicate that glial cells contribute to the αβmeATP‐induced LTP, which might be part of a cellular mechanism for the induction of persistent pain.

Glial Nitric Oxide-Mediated Long-Term Presynaptic Facilitation Revealed by Optical Imaging in Rat Spinal Dorsal Horn

We investigated a presynaptic form of long-term potentiation (LTP) in horizontal slices of the rat spinal cord by visualizing presynaptic and postsynaptic excitation with a voltage-sensitive dye. To record presynaptic excitation, we stained primary afferent fibers anterogradely from the dorsal root. A single-pulse test stimulation of C fiber-activating strength to the dorsal root elicited action potential (AP)-like or compound AP-like optical signals throughout the superficial dorsal horn. After conditioning (240 pulses at 2 Hz for 2 min), the presynaptic excitation was augmented. Furthermore, new excitation was elicited in the areas that were silent before conditioning. For postsynaptic recording, projection neurons in spinal lamina I were stained retrogradely from the periaqueductal gray in the brain stem. The test stimulation elicited AP-like or EPSP-like optical signals in the stained neurons. After conditioning, the EPSP-like responses were augmented, and previously silent neurons were converted to active ones. Results obtained with a nitric oxide (NO) donor, NO synthase inhibitors, metabotropic glutamate receptor (mGluR) agonist and mGluR1 antagonist, and a glial metabolism inhibitor suggest that after conditioning, presynaptic excitation is facilitated by NO released from glial cells via the activation of mGluR1. The results also indicate the possible presence of additional presynaptic and postsynaptic mechanism(s) for the LTP induction. Activity-dependent LTP of nociceptive afferent synaptic transmission in the spinal cord is believed to underlie central sensitization after inflammation or nerve injury. This glial NO-mediated control of presynaptic excitation may contribute to the induction at least in part.

Contribution of microglia and astrocytes to the central sensitization, inflammatory and neuropathic pain in the juvenile rat

BackgroundThe development of pain after peripheral nerve and tissue injury involves not only neuronal pathways but also immune cells and glia. Central sensitization is thought to be a mechanism for such persistent pain, and ATP involves in the process. We examined the contribution of glia to neuronal excitation in the juvenile rat spinal dorsal horn which is subjected to neuropathic and inflammatory pain.ResultsIn rats subjected to neuropathic pain, immunoreactivity for the microglial marker OX42 was markedly increased. In contrast, in rats subjected to inflammatory pain, immunoreactivity for the astrocyte marker glial fibrillary acidic protein was increased slightly. Optically-recorded neuronal excitation induced by single-pulse stimulation to the dorsal root was augmented in rats subjected to neuropathic and inflammatory pain compared to control rats. The bath application of a glial inhibitor minocycline and a p38 mitogen-activated protein kinase inhibitor SB203580 inhibited the neuronal excitation in rats subjected to neuropathic pain. A specific P2X1,2,3,4 antagonist TNP-ATP largely inhibited the neuronal excitation only in rats subjected to neuropathic pain rats. In contrast, an astroglial toxin L-alpha-aminoadipate, a gap junction blocker carbenoxolone and c-Jun N-terminal kinase inhibitor SP600125 inhibited the neuronal excitation only in rats subjected to inflammatory pain. A greater number of cells in spinal cord slices from rats subjected to neuropathic pain showed Ca2+ signaling in response to puff application of ATP. This Ca2+ signaling was inhibited by minocycline and TNP-ATP.ConclusionsThese results directly support the notion that microglia is more involved in neuropathic pain and astrocyte in inflammatory pain.

Optical responses evoked by single-pulse stimulation to the dorsal root in the rat spinal dorsal horn in slice.

Neuronal excitation evoked after dorsal-root (DR) stimulation in the spinal dorsal horn (DH) of rats was visualized with a high-resolution optical-imaging method, and the propagation mechanism was studied. Transverse slices of the spinal cord were obtained from 2-4 week-old rats and stained with the voltage-sensitive dye RH-482. Single-pulse stimulation to the primary-afferent A fibers in the DR attached to the slice evoked a weak, brief (<10 ms) excitatory optical response in the laminae I and III-V. When the stimulus intensity and duration were increased to activate both A and C fibers, an additional, much greater, and longer-lasting (>100 ms) excitatory response was generated in the laminae I-III, most intensely in the lamina II. A treatment with excitatory amino acid (EAA) antagonists, dl-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2, 3-dione, significantly reduced the amplitude and duration of the response in the lamina II. The optical response in the antagonists-containing solution was quite similar to that recorded in a Ca2+-free solution that blocked afferent synaptic transmission. The late component (>10 ms) was, however, slightly greater than that in the Ca2+-free solution. Treatment with the ATP-receptor antagonist, suramin, had a minimal effect on the response in the presence of EAA antagonists. These results suggested that the propagation of the DR-stimulus-elicited excitation was contributed largely by EAA receptors, but also by other receptors to a much lesser extent.

Synaptic plasticity in the spinal dorsal horn.

Long-term potentiation (LTP) at synapses in the spinal dorsal horn is thought to be a cellular mechanism for abnormal pain sensitivity. In this article, we review LTP in spinal projection neurons and presynaptic mechanisms of LTP in the spinal dorsal horn revealed by patch-clamp recording and optical imaging with voltage-sensitive dye.

Clinicopathologic profile, immunophenotype, and genotype of CD274 (PD-L1)-positive colorectal carcinomas

The CD274 (PD-L1)/PDCD1 (PD-1) pathway is crucial for the modulation of immune responses and self-tolerance. Aberrantly expressed CD274 allows tumor cells to evade host immune system and is considered to be a mechanism of adaptive immune resistance. Inhibition of the CD274/PDCD1 immune checkpoint offers a promising new therapeutic strategy. Although CD274-expressing tumor cells have been identified in different types of tumors including colorectal cancer, clinicopathologic profile of these CD274-positive tumors has not been extensively studied. In this study, 454 primary colorectal carcinomas were analyzed histologically and immunohistochemically for CD274, mismatch repair (MMR) proteins, intestinal differentiation marker (CDX2), and stem cell markers (ALCAM, ALDH1A1, and SALL4). CD274-positive colorectal carcinomas (54/454 (12%)) usually (83%) involved the right or transverse colon with poorly differentiated and solid/medullary histology. On the basis of multivariate logistic regression analysis, CD274 positivity was significantly associated with poorly differentiated histotype (OR: 3.32; 95% CI: 1.46–7.51; P=0.004), MMR deficiency (OR: 10.0; 95% CI: 4.66–21.5; P<0.001), and ‘stem-like’ immunophenotype defined by the loss or weak expression of CDX2 and ALCAM-positivity (OR: 5.51; 95% CI: 1.66–18.3; P=0.005). Mutation analysis of 66 arbitrary selected colorectal carcinomas revealed that CD274-positive tumors usually (88%) carried the BRAF V600E mutation. Thus, colorectal carcinomas defined by CD274 positivity displayed features associated with tumors arising via the serrated neoplasia pathway. Moreover, colorectal carcinomas characterized by lack of CDX2 and prominent expression of ALCAM frequently (71%) showed CD274 positivity. This might suggest association of CD274 expression with ‘stem-like’ phenotype. Further evaluation of a larger cohort or experimental analyses would be needed to confirm this notion.

Slow intrinsic optical signals in the rat spinal dorsal horn in slice.

TETANIC stimulation of high-threshold primary afferent fibers in the dorsal root was found to elicit intrinsic optical signals (IOSs) in transverse slices of 11 to 20-day-old rat spinal cords. The IOS, lasting for 30 s or longer, was most prominent in the lamina II of the dorsal horn. Treatment with a Na+-K+-2Cl− co-transport blocker, furosemide, abolished the IOS, suggesting that the origin of the IOS is the cellular swelling due to an activity-dependent rise in extracellular K+. Substance P antagonist spantide, glutamate antagonists 2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, and the μ-opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin suppressed IOSs. Thus, IOSs represent at least in part the slow excitatory response that is known to be generated in dorsal horn neurons after tetanic activation of unmyelinated afferent fibers.

Astrocytes are involved in long-term facilitation of neuronal excitation in the anterior cingulate cortex of mice with inflammatory pain.

Summary Astrocytes are involved in long‐term facilitation of neuronal excitation in the anterior cingulate cortex of mice with inflammatory pain. Hiroshi Ikeda, Keiichi Mochizuki, Kazuyuki Murase. Activated astrocytes in the anterior cingulate cortex play a crucial role in the long‐term potentiation of and the development of negative emotions in conditions of inflammatory pain. Abstract Neuronal plasticity in the pain‐processing pathway is thought to be a mechanism underlying pain hypersensitivity and negative emotions occurring during a pain state. Recent evidence suggests that the activation of astrocytes in the anterior cingulate cortex (ACC) contributes to the development of negative emotions during pain hypersensitivity after peripheral inflammation. However, it is unknown whether these activated astrocytes contribute to neuronal plasticity in the ACC. In this study, by using optical imaging with voltage‐ and Ca2+‐sensitive dyes, we examined the long‐term facilitation of neuronal excitation induced by high‐frequency conditioning stimulation (HFS) in ACC slices of control mice and mice with peripheral inflammation induced by the injection of complete Freund adjuvant (CFA) to the hind paw. Immunoreactivity of glial fibrillary acidic protein in laminae II–III of the ACC in the CFA‐injected mice was higher than in the control mice. Neuronal excitation in ACC slices from the CFA‐injected mice was gradually increased by HFS, and the magnitude of this long‐term facilitation was greater than in the control mice. The long‐term facilitation in the CFA‐injected mice was inhibited by the astroglial toxin, the N‐methyl‐d‐aspartate (NMDA) receptor antagonist and NMDA receptor glycine binding site antagonist. The increase of intracellular Ca2+ concentration in astrocytes during HFS was higher in the CFA‐injected mice than in the control mice and was inhibited by l‐&agr;‐aminoadipate (l‐&agr;‐AA). These results suggest that the activation of astrocytes in the ACC plays a crucial role in the development of negative emotions and LTP during pain hypersensitivity after peripheral inflammation.

Effects of corticotropin-releasing factor on plasticity of optically recorded neuronal activity in the substantia gelatinosa of rat spinal cord slices.

&NA; We examined the effects of corticotropin‐releasing factor (CRF) on plasticity of optically recorded neuronal activity in the substantia gelatinosa (lamina II) of 12–18‐day‐old rat spinal cord slices stained with a voltage‐sensitive dye. Single‐pulse test stimulation to the dorsal root that activated A and C fibres evoked prolonged (>100 ms) light‐absorption change in the lamina II. This response represents the gross membrane potential change of all elements along the slice depth. After conditioning high‐frequency stimulation of A‐fibre‐activating strength, test stimulus elicited less neuronal activity [−27±1% (7), (average±SE (n)), P<0.01 (*) at 45–60 min after conditioning]. When CRF (1 &mgr;M, 10 min) was applied during conditioning, the neuronal activity was facilitated rather than suppressed [+20±3% (5), P<0.05]. CRF alone exhibited insignificant effect [−5±1% (4), P=0.2]. In the presence of the inhibitory amino acid antagonists bicuculline (1 &mgr;M) and strychnine (0.3 &mgr;M) in the perfusate, in contrast, the conditioning facilitated it [+27±1% (12)*], and CRF treatment during conditioning inhibited the facilitation dose‐dependently [0.1 &mgr;M: +18±2% (5)*, 1 &mgr;M: +13±1% (7)*]. Although interneuronal actions might contribute, these results suggest that CRF may have dual effects on excitatory synaptic transmission within the lamina II depending upon cellular conditions: a conversion from the induction of long‐term depression to long‐term potentiation (LTP), and inhibition of LTP induction. Since the LTP is thought to be responsible at least in part for the persistent pain, CRF could regulate the induction.

Effect of halothane on neuronal excitation in the superficial dorsal horn of rat spinal cord slices: evidence for a presynaptic action

The action of the volatile anaesthetic halothane on optically recorded neuronal excitation in juvenile rat spinal cord slices was investigated. Prolonged neuronal excitation lasting ≈ 100 ms was evoked in the superficial dorsal horn after single‐pulse dorsal root stimulation that activated both A‐ and C‐fibres. Halothane depressed the neuronal excitation in a concentration‐dependent manner (IC50 0.21 mm, Imax 28%). In Ca2+‐free solution, dorsal root stimulation induced excitation with a short duration of several tens of milliseconds, in which the excitation of the postsynaptic component was largely eliminated. Under these conditions, halothane also depressed the excitation concentration‐dependently (IC50 0.46 mm, Imax 60%). Most of the suppression occurred within 5 min of halothane application, and the effect of halothane was fully reversible upon washout of the anaesthetic. Application of bicuculline and strychnine or picrotoxin, or reduction of extracellular Cl– concentration ([Cl–]o), had no effect on halothane inhibition. Applications of K+ channel blockers tetraethyl ammonium, 4‐aminopyridine, Cs+ or Ba2+ either had no effect or augmented the inhibitory effect of halothane. On the other hand, the degree of inhibition by halothane was found to be dependent on [K+]o; the higher [K+]o, the larger the depression. In addition, decreases in [Na+]o and [Mg2+]o reduced the excitation similar to that of halothane treatment, and the degree of halothane inhibition became larger with lower [Mg2+]o. These results lead to a hypothesis that halothane suppresses the excitation of presynaptic elements by inhibiting presynaptic Na+ channels by shifting the steady‐state inactivation curve in the hyperpolarizing direction.