Advances in Head and Neck Cancer Pain.

Head and neck cancer (HNC) affects over 890,000 people annually worldwide and has a mortality rate of 50%. Aside from poor survival, HNC pain impairs eating, drinking, and talking in patients, severely reducing quality of life. Different pain phenotype in patients (allodynia, hyperalgesia, and spontaneous pain) results from a combination of anatomical, histopathological, and molecular differences between cancers. Poor pathologic features (e.g., perineural invasion, lymph node metastasis) are associated with increased pain. The use of syngeneic/immunocompetent animal models, as well as a new mouse model of perineural invasion, provides novel insights into the pathobiology of HNC pain. Glial and immune modulation of the tumor microenvironment affect not only cancer progression but also pain signaling. For example, Schwann cells promote cancer cell proliferation, migration, and secretion of nociceptive mediators, whereas neutrophils are implicated in sex differences in pain in animal models of HNC. Emerging evidence supports the existence of a functional loop of cross-activation between the tumor microenvironment and peripheral nerves, mediated by a molecular exchange of bioactive contents (pronociceptive and protumorigenic) via paracrine and autocrine signaling. Brain-derived neurotrophic factor, tumor necrosis factor α, legumain, cathepsin S, and A disintegrin and metalloprotease 17 expressed in the HNC microenvironment have recently been shown to promote HNC pain, further highlighting the importance of proinflammatory cytokines, neurotrophic factors, and proteases in mediating HNC-associated pain. Pronociceptive mediators, together with nerve injury, cause nociceptor hypersensitivity. Oncogenic, pronociceptive mediators packaged in cancer cell-derived exosomes also induce nociception in mice. In addition to increased production of pronociceptive mediators, HNC is accompanied by a dampened endogenous antinociception system (e.g., downregulation of resolvins and µ-opioid receptor expression). Resolvin treatment or gene delivery of µ-opioid receptors provides pain relief in preclinical HNC models. Collectively, recent studies suggest that pain and HNC progression share converging mechanisms that can be targeted for cancer treatment and pain management.

Polyester Nanoparticle Encapsulation Mitigates Paclitaxel-Induced Peripheral Neuropathy.

Chemotherapy utilizing cytotoxic drugs, such as paclitaxel (PTX), is still a commonly used therapeutic approach to treat both localized and metastasized cancers. Unlike traditional regimens in which PTX is administered at the maximum tolerated dose, alternative regimens like metronomic dosing are beneficial by administering PTX more frequently and in much lower doses exploiting antiangiogenic and immunomodulatory effects. However, PTX-induced peripheral neuropathy and lack of patient compliant dosage forms of PTX are major roadblocks for the successful implementation of metronomic regimens. Because of the success of polyester nanoparticle drug delivery, we explored the potential of nanoparticle-encapsulated paclitaxel (nPTX) in alleviating peripheral neuropathy using a rat model. Rats were injected intraperitoneally with 2 mg/kg body weight of PTX or nPTX on four alternate days, and neuropathic pain and neuronal damage were characterized using behavioral assessments, histology, and immunohistochemistry. The reduction in tactile and nociceptive pressure thresholds was significantly less in nPTX-treated rats than in PTX-treated rats over a 16-day study period. Histological analysis showed that the degree of dorsal root ganglion (DRG) degeneration and reduction in motor neurons in the spinal cord was significantly lower in the nPTX group than the PTX group. Further, immunofluorescence data reveals that nPTX-treated rats had an increased density of a neuronal marker, ß-tubulin-III, reduced TUNEL positive cells, and increased high molecular weight neurofilament in the spinal cord, DRG, and sciatic nerves compared with PTX-treated rats. Therefore, this work has important implications in improving risk-benefit profile of PTX, paving the way for metronomic regimens.

Electroacupuncture reduces the expression of proinflammatory cytokines in inflamed skin tissues through activation of cannabinoid CB2 receptors.

Endogenous cannabinoids and peripheral cannabinoid CB2 receptors (CB2Rs) are involved in the antinociceptive effect of electroacupuncture (EA) on inflammatory pain. However, it is not clear how CB2R activation contributes to the antinociceptive effect of EA. The major proinflammatory cytokines, such as tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6, are involved in inflammatory pain. Here we determined the effects of CB2R activation and EA on the expression level of IL-1ß, IL-6 and TNF-α in inflamed skin tissues. Inflammatory pain was induced by injection of complete Freund's adjuvant into the left hindpaw of rats. Thermal hyperalgesia was tested with a radiant heat stimulus, and mechanical allodynia was quantified using von Frey filaments. The mRNA and protein levels of IL-1ß, IL-6 and TNF-α in inflamed skin tissues were measured using real-time polymerase chain reaction and Western blot, respectively. Local injection of the selective CB2R agonist AM1241 or EA applied to GB30 and GB34 significantly reduced thermal hyperalgesia and mechanical allodynia induced by tissue inflammation. The specific CB2R antagonist AM630 significantly attenuated the antinociceptive effect of EA. Furthermore, EA or AM1241 treatment significantly decreased the mRNA and protein levels of IL-1ß, IL-6 and TNF-α in inflamed skin tissues. In addition, pretreatment with AM630 significantly reversed the inhibitory effect of EA on these cytokine levels in inflamed skin tissues. Our results suggest that EA reduces inflammatory pain and proinflammatory cytokines in inflamed skin tissues through activation of CB2Rs.

Effects of activation of group III metabotropic glutamate receptors on spinal synaptic transmission in a rat model of neuropathic pain.

Chronic neuropathic pain remains an unmet clinical problem because it is often resistant to conventional analgesics. Metabotropic glutamate receptors (mGluRs) are involved in nociceptive processing at the spinal level, but their functions in neuropathic pain are not fully known. In this study, we investigated the role of group III mGluRs in the control of spinal excitatory and inhibitory synaptic transmission in a rat model of neuropathic pain induced by L5/L6 spinal nerve ligation. Whole-cell recording of lamina II neurons was performed in spinal cord slices from control and nerve-ligated rats. The baseline amplitude of glutamatergic EPSCs evoked from primary afferents was significantly larger in nerve-injured rats than in control rats. However, the baseline frequency of GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) was much lower in nerve-injured rats than in control rats. The group III mGluR agonist l(+)-2-amino-4-phosphonbutyric acid (l-AP4) produced a greater inhibition of the amplitude of monosynaptic and polysynaptic evoked EPSCs in nerve-injured rats than in control rats. l-AP4 inhibited the frequency of miniature EPSCs in 66.7% of neurons in control rats but its inhibitory effect was observed in all neurons tested in nerve-injured rats. Furthermore, l-AP4 similarly inhibited the frequency of GABAergic and glycinergic IPSCs in control and nerve-injured rats. Our study suggests that spinal nerve injury augments glutamatergic input from primary afferents but decreases GABAergic and glycinergic input to spinal dorsal horn neurons. Activation of group III mGluRs attenuates glutamatergic input from primary afferents in nerve-injured rats, which could explain the antinociceptive effect of group III mGluR agonists on neuropathic pain.

Stimulation of alpha(1)-adrenoceptors reduces glutamatergic synaptic input from primary afferents through GABA(A) receptors and T-type Ca(2+) channels.

Activation of the descending noradrenergic system inhibits nociceptive transmission in the spinal cord. Although both alpha(1)- and alpha(2)-adrenoceptors in the spinal cord are involved in the modulation of nociceptive transmission, it is not clear how alpha(1)-adrenoceptors regulate excitatory and inhibitory synaptic transmission at the spinal level. In this study, inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs, respectively) were recorded from lamina II neurons in rat spinal cord slices. The specific alpha(1)-adrenoceptor agonist phenylephrine significantly increased the frequency of GABAergic spontaneous IPSCs in a concentration dependent manner, and this effect was abolished by the alpha(1)-adrenoceptor antagonist 2-(2,6-dimethoxyphenoxy)ethylaminomethyl-1,4-benzodioxane (WB4101). Phenylephrine also significantly reduced the amplitude of monosynaptic and polysynaptic EPSCs evoked from primary afferents. The inhibitory effect of phenylephrine on evoked monosynaptic glutamatergic EPSCs was largely blocked by the GABA(A) receptor antagonist picrotoxin and, to a lesser extent, by the GABA(B) receptor antagonist CGP55845. Furthermore, blocking T-type Ca(2+) channels with amiloride or mibefradil diminished the inhibitory effect produced by phenylephrine or the GABA(A) receptor agonist muscimol on monosynaptic EPSCs evoked from primary afferents. Collectively, these findings suggest that activation of alpha(1)-adrenoceptors in the spinal cord increases synaptic GABA release, which attenuates glutamatergic input from primary afferents mainly through GABA(A) receptors and T-type Ca(2+) channels. This mechanism of presynaptic inhibition in the spinal cord may be involved in the regulation of nociception by the descending noradrenergic system.

Signaling mechanisms mediating muscarinic enhancement of GABAergic synaptic transmission in the spinal cord.

Activation of muscarinic acetylcholine receptors (mAChRs) inhibits spinal nociceptive transmission by potentiation of GABAergic tone through M(2), M(3), and M(4) subtypes. To study the signaling mechanisms involved in this unique mAChR action, GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of lamina II neurons were recorded using whole-cell patch clamp techniques in rat spinal cord slices. The mAChR agonist oxotremorine-M caused a profound increase in the frequency of GABAergic sIPSCs, which was abolished in the Ca(2+)-free solution. Inhibition of voltage-gated Ca(2+) channels with Cd(2+) and Ni(2+) largely reduced the effect of oxotremorine-M on sIPSCs. Blocking nonselective cation channels (NSCCs) with SKF96365 or 2-APB also largely attenuated the effect of oxotremorine-M. However, the KCNQ channel blocker XE991 and the adenylyl cyclase inhibitor MDL12330A had no significant effect on oxotremorine-M-induced increases in sIPSCs. Furthermore, the phosphoinositide-3-kinase (PI3K) inhibitor wortmannin or LY294002 significantly reduced the potentiating effect of oxotremorine-M on sIPSCs. In the spinal cord in which the M(3) subtype was specifically knocked down by intrathecal small interfering RNA (siRNA) treatment, SKF96365 and wortmannin still significantly attenuated the effect of oxotremorine-M. In contrast, SKF96365 and wortmannin both failed to alter the effect of oxotremorine-M on sIPSCs when the M(2)/M(4) mAChRs were blocked. Therefore, our study provides new evidence that activation of mAChRs increases synaptic GABA release through Ca(2+) influx and voltage-gated Ca(2+) channels. The PI3K-NSCC signaling cascade is primarily involved in the excitation of GABAergic interneurons by the M(2)/M(4) mAChRs in the spinal dorsal horn.

Distinct inhibition of voltage-activated Ca2+ channels by delta-opioid agonists in dorsal root ganglion neurons devoid of functional T-type Ca2+ currents.

Both mu- and delta-opioid agonists selectively inhibit nociception but have little effect on other sensory modalities. Voltage-activated Ca(2+) channels in the primary sensory neurons are important for the regulation of nociceptive transmission. In this study, we determined the effect of delta-opioid agonists on voltage-activated Ca(2+) channel currents (I(Ca)) in small-diameter rat dorsal root ganglion (DRG) neurons that do and do not bind isolectin B(4) (IB(4)). The delta-opioid agonists [d-Pen(2),d-Pen(5)]-enkephalin (DPDPE) and deltorphin II produced a greater inhibition of high voltage-activated I(Ca) in IB(4)-negative than IB(4)-positive neurons. Furthermore, DPDPE produced a greater inhibition of N-, P/Q-, and L-type I(Ca) in IB(4)-negative than IB(4)-positive neurons. However, DPDPE had no significant effect on the R-type I(Ca) in either type of cells. We were surprised to find that DPDPE failed to inhibit either the T-type or high voltage-activated I(Ca) in all the DRG neurons with T-type I(Ca). Double immunofluorescence labeling showed that the majority of the delta-opioid receptor-immunoreactive DRG neurons had IB(4) labeling, while all DRG neurons immunoreactive to delta-opioid receptors exhibited Cav(3.2) immunoreactivity. Additionally, DPDPE significantly inhibited high voltage-activated I(Ca) in Tyrode's or N-methyl-d-glucamine solution but not in tetraethylammonium solution. This study provides new information that delta-opioid agonists have a distinct effect on voltage-activated Ca(2+) channels in different phenotypes of primary sensory neurons. High voltage-activated Ca(2+) channels are more sensitive to inhibition by delta-opioid agonists in IB(4)-negative than IB(4)-positive neurons, and this opioid effect is restricted to DRG neurons devoid of functional T-type Ca(2+) currents.

Kv1.1/1.2 channels are downstream effectors of nitric oxide on synaptic GABA release to preautonomic neurons in the paraventricular nucleus.

The paraventricular nucleus (PVN) of the hypothalamus is important for the neural regulation of cardiovascular function. Nitric oxide (NO) increases synaptic GABA release to presympathetic PVN neurons through the cyclic guanosine monophosphate (cGMP)/protein kinase G signaling pathway. However, the downstream signaling mechanisms underlying the effect of NO on synaptic GABA release remain unclear. In this study, whole-cell voltage-clamp recordings were performed on retrograde-labeled spinally projecting PVN neurons in rat brain slices. Bath application of the NO precursor l-arginine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly increased the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in labeled PVN neurons. A specific antagonist of cyclic ADP ribose, 8-bromo-cyclic ADP ribose (8-Br-cADPR), had no significant effect on l-arginine-induced potentiation of mIPSCs. Surprisingly, blocking of voltage-gated potassium channels (Kv) with 4-aminopyridine or alpha-dendrotoxin eliminated the effect of l-arginine on mIPSCs in all labeled PVN neurons tested. The membrane permeable cGMP analog mimicked the effect of l-arginine on mIPSCs, and this effect was blocked by alpha-dendrotoxin. Furthermore, the specific Kv channel blocker for Kv1.1 (dendrotoxin-K) or Kv1.2 (tityustoxin-Kalpha) abolished the effect of l-arginine on mIPSCs in all neurons tested. SNAP failed to inhibit the firing activity of labeled PVN neurons in the presence of dendrotoxin-K, Kalpha. Additionally, the immunoreactivity of Kv1.1 and Kv1.2 subunits was colocalized extensively with synaptophysin in the PVN. These findings suggest that NO increases GABAergic input to PVN presympathetic neurons through a downstream mechanism involving the Kv1.1 and Kv1.2 channels at the nerve terminals.

Resistance to morphine analgesic tolerance in rats with deleted transient receptor potential vanilloid type 1-expressing sensory neurons.

Deletion of transient receptor potential vanilloid type 1 (TRPV1)-expressing afferent neurons reduces presynaptic mu opioid receptors but paradoxically potentiates the analgesic efficacy of mu opioid agonists. In this study, we determined if removal of TRPV1-expressing afferent neurons by resiniferatoxin (RTX), an ultrapotent capsaicin analog, influences the development of opioid analgesic tolerance. Morphine tolerance was induced by daily intrathecal injections of 10 microg of morphine for 14 consecutive days or by daily i.p. injections of 10 mg/kg of morphine for 10 days. In vehicle-treated rats, the effect of intrathecal or systemic morphine on the mechanical withdrawal threshold was gradually diminished within 7 days. However, the analgesic effect of intrathecal and systemic morphine was sustained in RTX-treated rats at the time the morphine effect was lost in the vehicle group. Furthermore, the mu opioid receptor-G protein coupling in the spinal cord was significantly decreased ( approximately 22%) in vehicle-treated morphine tolerant rats, but was not significantly altered in RTX-treated rats receiving the same treatment with morphine. Additionally, there was a large reduction in protein kinase Cgamma-immunoreactive afferent terminals in the spinal dorsal horn of RTX-treated rats. These findings suggest that loss of TRPV1-expressing sensory neurons attenuates the development of morphine analgesic tolerance possibly by reducing mu opioid receptor desensitization through protein kinase Cgamma in the spinal cord. These data also suggest that the function of presynaptic mu opioid receptors on TRPV1-expressing sensory neurons is particularly sensitive to down-regulation by mu opioid agonists during opioid tolerance development.

Regulation of synaptic input to hypothalamic presympathetic neurons by GABA(B) receptors.

The hypothalamic paraventricular (PVN) neurons projecting to the spinal cord and brainstem play an important role in the control of homeostasis and the sympathetic nervous system. Although GABA(B) receptors are present in the PVN, their function in the control of synaptic inputs to PVN presympathetic neurons is not clear. Using retrograde tracing and whole-cell patch-clamp recordings in rat brain slices, we determined the role of presynaptic GABA(B) receptors in regulation of glutamatergic and GABAergic inputs to spinally projecting PVN neurons. The GABA(B) receptor agonist baclofen (1-50 microM) dose-dependently decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs). The effect of baclofen on sEPSCs and sIPSCs was completely blocked by 10 microM CGP52432, a selective GABA(B) receptor antagonist. Baclofen also significantly reduced the frequency of both miniature excitatory and miniature inhibitory postsynaptic currents (mEPSCs and mIPSCs). Furthermore, uncoupling pertussis toxin-sensitive G(i/o) proteins with N-ethylmaleimide abolished baclofen-induced inhibition of mEPSCs and mIPSCs. However, the inhibitory effect of baclofen on the frequency of mIPSCs and mEPSCs persisted in the presence of either Cd2+, a voltage-gated Ca2+ channel blocker, or 4-aminopyridine, a blocker of voltage-gated K+ channels. Our results suggest that activation of presynaptic GABA(B) receptors inhibits synaptic GABA and glutamate release to PVN presympathetic neurons. This presynaptic action of GABA(B) receptors is mediated by the N-ethylmaleimide-sensitive G(i/o) proteins, but independent of voltage-gated Ca2+ and K+ channels.

Signaling mechanisms of down-regulation of voltage-activated Ca2+ channels by transient receptor potential vanilloid type 1 stimulation with olvanil in primary sensory neurons.

Olvanil ((N-vanillyl)-9-oleamide), a non-pungent transient receptor potential vanilloid type 1 agonist, desensitizes nociceptors and alleviates pain. But its molecular targets and signaling mechanisms are little known. Calcium influx through voltage-activated Ca(2+) channels plays an important role in neurotransmitter release and synaptic transmission. Here we determined the effect of olvanil on voltage-activated Ca(2+) channel currents and the signaling pathways in primary sensory neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Olvanil (1 microM) elicited a delayed but sustained inward current, and caused a profound inhibition (approximately 60%) of N-, P/Q-, L-, and R-type voltage-activated Ca(2+) channel current. Pretreatment with a specific transient receptor potential vanilloid type 1 antagonist or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the inhibitory effect of olvanil on voltage-activated Ca(2+) channel current. Calmodulin antagonists (ophiobolin-A and calmodulin inhibitory peptide) largely blocked the effect of olvanil and capsaicin on voltage-activated Ca(2+) channel current. Furthermore, calcineurin (protein phosphatase 2B) inhibitors (deltamethrin and FK-506) eliminated the effect of olvanil on voltage-activated Ca(2+) channel current. Notably, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin antagonists, and calcineurin inhibitors each alone significantly increased the amplitude of voltage-activated Ca(2+) channel current. In addition, double immunofluorescence labeling revealed that olvanil induced a rapid internalization of Ca(V)2.2 immunoreactivity from the membrane surface of dorsal root ganglion neurons. Collectively, this study suggests that stimulation of non-pungent transient receptor potential vanilloid type 1 inhibits voltage-activated Ca(2+) channels through a biochemical pathway involving intracellular Ca(2+)-calmodulin and calcineurin in nociceptive neurons. This new information is important for our understanding of the signaling mechanisms of desensitization of nociceptors by transient receptor potential vanilloid type 1 analogues and the feedback regulation of intracellular Ca(2+) and voltage-activated Ca(2+) channels in nociceptive sensory neurons.

Activation of muscarinic receptors inhibits spinal dorsal horn projection neurons: role of GABAB receptors.

Spinally administered muscarinic receptor agonists or acetylcholinesterase inhibitors produce efficacious analgesia. However, the mechanisms of the antinociceptive actions of muscarinic agonists in the spinal cord are not fully known. Previous in vitro studies have shown that muscarinic agonists increase GABA release and reduce the glutamatergic synaptic input to lamina II interneurons through GABAB receptors in the spinal cord. In the present study, we studied the effect of muscarinic agents on dorsal horn projection neurons and the role of spinal GABAB receptors in their action. Single-unit activity of ascending dorsal horn neurons was recorded in the lumbar spinal cord of anesthetized rats. The responses of dorsal horn neurons to graded mechanical stimuli were determined before and after topical spinal application of muscarine and neostigmine. We found that topical application of 0.1-5 microM muscarine or 0.5-5 microM neostigmine significantly suppressed the evoked response of dorsal horn neurons in a concentration-dependent manner. The inhibitory effect of muscarine or neostigmine on dorsal horn neurons was completely abolished in the presence of 1 microM atropine and by intrathecal pretreatment with 1 microg pertussis toxin to inactivate inhibitory G proteins. Furthermore, the inhibitory effect of both muscarine and neostigmine on the evoked response of dorsal horn neurons was significantly attenuated in the presence of 1 microM CGP55845, a GABAB receptor antagonist. Collectively, these data suggest that muscarinic agents inhibit dorsal horn projection neurons through muscarinic receptors coupled to pertussis toxin-sensitive Gi/o proteins. The inhibitory action of muscarinic agonists on these dorsal horn neurons is mediated in part by spinal GABAB receptors.

Role of primary afferent nerves in allodynia caused by diabetic neuropathy in rats.

Both myelinated and unmyelinated afferents are implicated in transmitting diabetic neuropathic pain. Although unmyelinated afferents are generally considered to play a significant role in diabetic neuropathic pain, pathological changes in diabetic neuropathy occur mostly in myelinated A-fibers. In the present study, we first examined the role of capsaicin-sensitive C-fibers in the development of allodynia induced by diabetic neuropathy. We then studied the functional changes of afferent nerves pertinent to diabetic neuropathic pain. Diabetes was induced in rats by i.p. streptozotocin. To deplete capsaicin-sensitive C-fibers, rats were treated with i.p. resiniferatoxin (300 microg/kg). Mechanical and thermal sensitivities were measured using von Frey filaments and a radiant heat stimulus. Single-unit activity of afferents was recorded from the tibial nerve. Tactile allodynia, but not thermal hyperalgesia, developed in diabetic rats. Resiniferatoxin treatment did not alter significantly the degree and time course of allodynia. Post-treatment with resiniferatoxin also failed to attenuate allodynia in diabetic rats. The electrophysiological recordings revealed ectopic discharges and a higher spontaneous activity mainly in Adelta- and Abeta-fiber afferents in diabetic rats regardless of resiniferatoxin treatment. Furthermore, these afferent fibers had a lower threshold for activation and augmented responses to mechanical stimuli. Thus, our study suggests that capsaicin-sensitive C-fiber afferents are not required in the development of allodynia in this rat model of diabetes. Our electrophysiological data provide substantial new evidence that the abnormal sensory input from Adelta- and Abeta-fiber afferents may play an important role in diabetic neuropathic pain.

Stereospecific effect of pregabalin on ectopic afferent discharges and neuropathic pain induced by sciatic nerve ligation in rats.

BACKGROUND: The new anticonvulsants, gabapentin and pregabalin, are effective in the treatment of neuropathic pain. The sites and mechanisms of their analgesic action are not fully known. The authors have previously demonstrated that systemic gabapentin suppresses ectopic afferent discharges recorded from injured sciatic nerves in rats. In the current study, they further examined the stereospecific effect of pregabalin on neuropathic pain and afferent ectopic discharges in a rodent model of neuropathic pain. METHODS: Tactile allodynia and thermal hyperalgesia were induced by partial ligation of the left sciatic nerve in rats. Single-unit activity of afferent ectopic discharges was recorded from the sciatic nerve proximal to the site of ligation. RESULTS: Intravenous injection of 10-30 mg/kg pregabalin dose-dependently attenuated tactile allodynia (n = 10) and thermal hyperalgesia (n = 8). The stereoisomer of pregabalin, R-3-isobutylgaba, had no analgesic effect in this dose range. Furthermore, intravenous injection of pregabalin, but not R-3-isobutylgaba, significantly inhibited the ectopic discharges from injured afferents in a dose-dependent manner (from 20.8 +/- 2.4 impulses/s during control to 2.3 +/- 0.7 impulses/s after treatment with 30 mg/kg pregabalin, n = 15). Pregabalin did not affect the conduction velocity of afferent fibers and the response of normal afferent nerves to mechanical stimulation. CONCLUSIONS: These data strongly suggest that the analgesic effect of pregabalin on neuropathic pain is likely mediated, at least in part, by its peripheral inhibitory action on the impulse generation of ectopic discharges caused by nerve injury.

Acetylcholine attenuates synaptic GABA release to supraoptic neurons through presynaptic nicotinic receptors.

Both inhibitory GABAergic and excitatory glutamatergic inputs to supraoptic nucleus (SON) neurons can influence the release of vasopressin and oxytocin. Acetylcholine is known to excite SON neurons and to increase vasopressin release. The functional significance of cholinergic receptors, located at the presynaptic nerve terminals, in the regulation of the excitability of SON neurons is not fully known. In this study, we determined the role of presynaptic cholinergic receptors in regulation of the inhibitory GABAergic inputs to the SON neurons. The magnocellular neurons in the rat hypothalamic slice were identified microscopically, and the spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were recorded using the whole-cell voltage-clamp technique. The mIPSCs were abolished by the GABA(A) receptor antagonist, bicuculline (10 microM). Acetylcholine (100 microM) significantly reduced the frequency of mIPSCs of SON neurons from 3.59+/-0.36 to 1.62+/-0.20 Hz (n=37), but did not alter the amplitude and the decay time constant of mIPSCs. Furthermore, the nicotinic receptor antagonist, mecamylamine (10 microM, n=13), eliminated the inhibitory effect of acetylcholine on mIPSCs of SON neurons. The muscarinic receptor antagonist, atropine (100 microM), did not alter significantly the effect of acetylcholine on mIPSCs in most of the 17 SON neurons studied. These results suggest that the excitatory effect of acetylcholine on the SON neurons is mediated, at least in part, by inhibition of presynaptic GABA release. Activation of presynaptic nicotinic receptors located in the GABAergic terminals plays a major role in the cholinergic regulation of the inhibitory GABAergic input to SON neurons.

Antiallodynic effect of intrathecal neostigmine is mediated by spinal nitric oxide in a rat model of diabetic neuropathic pain.

BACKGROUND: Intrathecal administration of acetylcholinesterase inhibitors produces antinociception in both animals and humans, but their effect on diabetic neuropathic pain has not been studied. In the current study, we determined the antiallodynic effect of intrathecal injection of an acetylcholinesterase inhibitor, neostigmine, in a rat model of diabetic neuropathic pain. In addition, since acetylcholine can increase release of nitric oxide in the spinal cord, we studied the role of spinal endogenous nitric oxide in the action of intrathecal neostigmine in diabetic neuropathic pain. METHODS: Rats were rendered diabetic with an intraperitoneal 50-mg/kg injection of streptozotocin. Intrathecal catheters were inserted, with tips in the lumbar intrathecal space. Mechanical allodynia was determined by application of von Frey filaments to the hind paw. We first determined the dose-dependent effect of intrathecal neostigmine on allodynia. The role of spinal nitric oxide in the action of intrathecal neostigmine was then examined through intrathecal treatments with a neuronal nitric oxide synthase inhibitor (TRIM), a nitric oxide scavenger (PTIO), L-arginine, or D-arginine. RESULTS: The diabetic rats developed a sustained tactile allodynia within 4 weeks after streptozotocin injection. Intrathecal injection of 0.1-0.5 microg neostigmine dose-dependently increased the withdrawal threshold in response to application of von Frey filaments. Intrathecal pretreatment with 30 microg TRIM or 30 microg PTIO abolished the antiallodynic effect of intrathecal neostigmine. Furthermore, the inhibitory effect of TRIM on the action of intrathecal neostigmine was reversed by intrathecal injection of 100 microg L-arginine but not D-arginine. CONCLUSIONS: Intrathecal neostigmine produces a profound analgesic effect in a rat model of diabetic neuropathic pain. Spinal endogenous nitric oxide contributes to the analgesic action of intrathecal neostigmine in this rat model of diabetic neuropathic pain.

Potentiation of glutamatergic synaptic input to supraoptic neurons by presynaptic nicotinic receptors.

The release of vasopressin and oxytocin from the supraoptic nucleus (SON) neurons is tonically regulated by excitatory glutamatergic and inhibitory GABAergic synaptic inputs. Acetylcholine is known to excite SON neurons and to elicit vasopressin release. Cholinergic receptors are located pre- and postsynaptically in the SON, but their functional significance in the regulation of SON neurons is not fully understood. In this study, we determined the role of presynaptic cholinergic receptors in regulation of the excitatory glutamatergic inputs to the SON neurons. The magnocellular neurons in the rat hypothalamic slices were identified microscopically, and the spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded using the whole cell voltage-clamp technique. The mEPSCs were abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). Acetylcholine (100 microM) significantly increased the frequency of mEPSCs of 38 SON neurons from 1.87 +/- 0.36 to 3.42 +/- 0.54 Hz but did not alter the amplitude (from 19.61 +/- 0.90 to 19.34 +/- 0.84 pA) and the decay time constant of mEPSCs. Furthermore, the nicotinic receptor antagonist mecamylamine (10 microM, n = 16), but not the muscarinic receptor antagonist atropine (100 microM, n = 12), abolished the excitatory effect of acetylcholine on the frequency of mEPSCs. These data provide new information that the excitatory effect of acetylcholine on the SON neurons is mediated, at least in part, by its effect on presynaptic glutamate release. Activation of presynaptic nicotinic, but not muscarinic, receptors located in the glutamatergic terminals increases the excitatory synaptic input to the SON neurons of the hypothalamus.

Allosteric adenosine modulation to reduce allodynia.

BACKGROUND: Adenosine and adenosine agonists reduce hypersensitivity following inflammation and peripheral nerve injury models of chronic pain. Because inhibitors of adenosine reuptake or metabolism are also effective at reducing hypersensitivity, it is likely that there is a tonic release of spinal adenosine in these models. One approach to avoid adverse effects from direct agonists is to enhance the effect of the endogenous ligand by administering a positive allosteric modulator of its receptor. METHODS: Rats with mechanical hypersensitivity after spinal nerve ligation received intrathecal injections of adenosine, the allosteric adenosine receptor modulator T62, or their combination, or received systemic T62 alone or with intrathecal injection of a specific A1 adenosine antagonist. RESULTS: Both adenosine and T62 reduced hypersensitivity alone, with 50% maximal doses (ED50) of 14+/-5.9 and 3.7+/-0.8 microg, respectively. They interacted in an additive manner as determined by isobolography. T62 also reduced mechanical hypersensitivity after systemic administration (15 mg/kg), and this effect was blocked by intrathecal injection of 9 microg of the A1-specific adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. CONCLUSIONS: These results add to previous studies that suggest ongoing spinal release of adenosine, which is antiallodynic, in this animal model of neuropathic pain. Positive allosteric modulation of the adenosine receptor reduces hypersensitivity by a spinal mechanism involving A1 adenosine receptor stimulation. Although obvious adverse effects were not observed in this investigation, further study is required to determine the feasibility of the use of such modulators in the treatment of chronic pain associated with hyperalgesia and allodynia.

Spinal endogenous acetylcholine contributes to the analgesic effect of systemic morphine in rats.

BACKGROUND: Systemic morphine is known to cause increased release of acetyicholine in the spinal cord. Intrathecal injection of the cholinergic receptor agonists or acetyicholinesterase inhibitors produces antinociception in both animals and humans. In the present study, we explored the functional importance of spinal endogenous acetylcholine in the analgesic action produced by intravenous morphine. METHODS: Rats were implanted with intravenous and intrathecal catheters. The antinociceptive effect of morphine was determined by the paw-withdrawal latency in response to a radiant heat stimulus after intrathecal treatment with atropine (a muscarinic receptor antagonist), mecamylamine (a nicotinic receptor antagonist), or cholinergic neurotoxins (ethylcholine mustard aziridinium ion [AF64A] and hemicholinium-3). RESULTS: Intravenous injection of 2.5 mg/kg morphine increased significantly the paw-withdrawal latency. Intrathecal pretreatment with 30 microg atropine (n = 7) or 50 microg mecamylamine (n = 6) both attenuated significantly the antinociceptive effect of morphine. The inhibitory effect of atropine on the effect of morphine was greater than that of mecamylanilne. Furthermore, the antinociceptive effect of morphine was significantly reduced in rats pretreated with intrathecal AF64A (n = 7) or hemicholinium-3 (n = 6) to inhibit the high-affinity choline transporter and acetylcholine synthesis. We found that intrathecal AF64A reduced significantly the [3H]hemicholinium-3 binding sites but did not affect its affinity in the dorsal spinal cord. CONCLUSIONS: The data in the current study indicate that spinal endogenous acetylcholine plays an important role in mediating the analgesic effect of systemic morphine through both muscarinic and nicotinic receptors.

Differential roles for glutamate receptor subtypes within commissural NTS in cardiac-sympathetic reflex.

Ischemic stimulation of cardiac receptors evokes excitatory sympathetic reflexes. Although the nucleus of the solitary tract (NTS) is an important site for integration of visceral afferents, its involvement in the cardiac-renal sympathetic reflex remains to be fully defined. This study examined the role of glutamate receptor subtypes in the commissural NTS in the sympathetic responses to stimulation of cardiac receptors. Renal sympathetic nerve activity (RSNA) was recorded in anesthetized rats. Cardiac receptors were stimulated by epicardial application of bradykinin (BK; 10 microg/ml). Application of BK significantly increased the mean arterial pressure from 78.2 +/- 2.2 to 97.5 +/- 2.9 mmHg and augmented RSNA by 38.5 +/- 2.5% (P < 0.05). Bilateral microinjection of 10 pmol of 6-cyano-7-nitroquinoxaline-2,3-dione, a non-N-methyl-D-aspartate (NMDA) antagonist, into the commissural NTS eliminated the pressor and RSNA responses to BK application in 10 rats. However, microinjection of 2-amino-5-phosphonopentanoic acid (0.1 and 1 nmol, n = 8), an NMDA- receptor antagonist, or alpha-methyl-4-carboxyphenylglycine (0.1 and 1 nmol, n = 5), a glutamate metabotropic receptor antagonist, failed to attenuate significantly the pressor and RSNA responses to stimulation of cardiac receptors with BK. Thus this study suggests that non-NMDA, but not NMDA and glutamate metabotropic, receptors in the commissural NTS play an important role in the sympathoexcitatory reflex response to activation of cardiac receptors during myocardial ischemia.