Subramanian S. Parvathy

Gait analysis of C57BL/6 mice with complete Freund's adjuvant-induced arthritis using the CatWalk system

BackgroundThe CatWalk system, a video based automated gait analysis system developed to evaluate footfall and gait changes in rodents, has been used for studying rodent models of arthritis, mainly the rat model. However, it has not been used to study static and dynamic gait parameters in mice with Complete Freund’s adjuvant (CFA). CFA is used extensively to induce arthritis in rodents including mice.MethodsThe CatWalk system was used to study gait of freely moving mice with CFA-induced monoarthritis and evaluate pharmacological pain relief in this model of arthritis. CFA (20 μl) was injected intra-articularly into the right hind (RH) limb ankle joint through the Achilles tendon of C57BL/6 mice.ResultsMice had less regularity in their walking patterns after CFA inoculation compared to baseline walking patterns, which was significant at 2 days post inoculation (dpi). The mice also showed changes in static parameters (paw pressure (light intensity) and print area) as well as dynamic parameters (stance phase duration, swing phase duration and speed, and duty cycle). The ratio of the RH limb (ipslateral) to the left hind (LH) limb (contralateral) for paw pressure, print area, stance phase duration, duty cycle (stance phase duration/sum of stance and swing phase duration), and swing speed were significantly reduced compared to baseline ratios at 1–6 and/or 7 dpi. On the other hand, RH/LH limb ratio of the swing phase duration increased at 3 dpi compared to baseline values. Treatment with indomethacin (10 mg/kg) improved or restored the gait parameters of CFA inoculated mice i.e. similar to baseline values or LH limb.ConclusionsThese data show that the CatWalk system can be used to assess static and dynamic gait changes and pharmacological pain relief in freely moving mice with CFA-induced monoarthritis.

Dopamine and adenosine mediate substance P‐induced depression of evoked IPSCs in the rat nucleus accumbens in vitro

The major projection cells of the nucleus accumbens (NAc) are under a strong inhibitory influence from GABAergic afferents and depend on afferent excitation to produce their output. We have earlier reported that substance P (SP), a peptide which is colocalized with GABA in these neurons, depresses excitatory synaptic transmission in this nucleus (Kombian, S.B., Ananthalakshmi, K.V.V., Parvathy, S.S. & Matowe, W.C. (2003) J. Neurophysiol., 89, 728–738). In order to better understand the role of this peptide in the synaptic physiology of the NAc, it is important to determine its effects on inhibitory synaptic responses. Using whole‐cell recording in rat forebrain slices, we show here that SP also depresses evoked inhibitory postsynaptic currents (IPSCs) in the NAc via intermediate neuromodulators. SP caused a partially reversible, dose‐dependent decrease in evoked IPSC amplitude. This effect was present without measurable changes in the holding current, input resistance of recorded cells or decay rate (τ) of IPSCs. It was mimicked by a neurokinin‐1 (NK1) receptor‐selective agonist, [Sar9, Met (O2)11]‐SP, and blocked by an NK1 receptor‐selective antagonist, L 732 138. The SP‐induced IPSC depression was prevented by SCH23390, a dopamine D1‐like receptor antagonist and by 8‐cyclopentyltheophylline, an adenosine A1 receptor blocker. Furthermore, the SP effect was also markedly attenuated by exogenous adenosine, dipyridamole, rolipram and barium. These data show that SP, acting on NK1 receptors, depresses inhibitory synaptic transmission indirectly by enhancing extracellular dopamine and adenosine levels. SP therefore acts in the NAc to modulate both excitatory and inhibitory afferent inputs using the same mechanism(s).

Cholecystokinin activates CCKB receptors to excite cells and depress EPSCs in the rat rostral nucleus accumbens in vitro

The peptide cholecystokinin (CCK) is abundant in the rat nucleus accumbens (NAc). Although it is colocalized with dopamine (DA) in afferent terminals in this region, neurochemical and behavioural reports are equally divided as to whether CCK enhances or diminishes DAs actions in this nucleus. To better understand the role of this peptide in the physiology of the NAc, we examined the effects of CCK on excitatory synaptic transmission and tested whether these are dependent on DA and/or other neuromodulators. Using whole‐cell recording in rat forebrain slices containing the NAc, we show that sulphated CCK octapeptide (CCK‐8S), the endogenously active neuropeptide, consistently depolarized cells and depressed evoked excitatory postsynaptic currents (EPSCs) in the rostral NAc. It caused a reversible, dose‐dependent decrease in evoked EPSC amplitude that was accompanied by an increase in the decay constant of the EPSC but with no apparent change in paired pulse ratio. It was mimicked by unsulphated CCK‐8 (CCK‐8US), a CCKB receptor‐selective agonist, and blocked by LY225910, a CCKB receptor‐selective antagonist. Both CCK‐8S and CCK‐8US induced an inward current with a reversal potential around −90 mV that was accompanied by an increase in input resistance and action potential firing. The CCK‐8S‐induced EPSC depression was slightly reduced in the presence of SCH23390 but not in the presence of sulpiride or 8‐cyclopentyltheophylline. By contrast, it was completely blocked by CGP55845, a potent GABAB receptor‐selective antagonist. These results indicate that CCK excites NAc cells directly while depressing evoked EPSCs indirectly, mainly through the release of GABA.

Matrix Metalloproteinase Inhibitor COL-3 Prevents the Development of Paclitaxel-Induced Hyperalgesia in Mice

Objective: To study the potential of chemically modified tetracycline-3 (COL-3), a potent matrix metalloproteinase (MMP) inhibitor, to protect against the development of paclitaxel-induced painful neuropathy and its immunomodulatory effects. Materials and Methods: The reaction latency to thermal stimuli (hot plate test) of female BALB/c mice was recorded before and after treatment with paclitaxel (2 mg/kg i.p.), paclitaxel plus COL-3 (4, 20 or 40 mg/kg p.o.) or their vehicles for 5 consecutive days. Gene transcripts of CD11b (marker for microglia), 5 cytokines (IFN-γ, IL-1β, IL-6, IL-10 and TNF-α) and 3 chemokines (CCL2, CXCL10 and CX3CL1) were quantified by real-time PCR in the brains, spinal cords and spleens of mice sacrificed on day 7 after treatment. Results: Treatment with paclitaxel reduced the reaction latency time to thermal stimuli (thermal hyperalgesia) for 4 weeks, with maximum effect on days 7 and 10. The coadministration of paclitaxel with COL-3 40 mg/kg, but not lower doses, prevented the development of paclitaxel-induced thermal hyperalgesia. Treatment with paclitaxel alone or coadministration with COL-3 increased CD11b transcript levels in the brain but not in the spinal cord. Treatment with paclitaxel reduced IL-6 transcript levels in the spinal cord but did not alter the transcript levels of other cytokines or chemokines in the brain, spinal cord or spleen. The coadministration of COL-3 with paclitaxel significantly increased the transcript levels of IL-6 in the spleen and decreased CX3CL1 transcripts in the brain in comparison to treatment with paclitaxel alone. Conclusion: Our results indicate that the MMP inhibitor COL-3 protected against paclitaxel-induced thermal hyperalgesia and, thus, could be useful in the prevention of chemotherapy-induced painful neuropathy.

Cholecystokinin inhibits evoked inhibitory postsynaptic currents in the rat nucleus accumbens indirectly through γ-aminobutyric acid and γ-aminobutyric acid type B receptors

We recently reported that cholecystokinin (CCK) excited nucleus accumbens (NAc) cells and depressed excitatory synaptic transmission indirectly through γ‐aminobutyric acid (GABA), acting on presynaptic GABAB receptors (Kombian et al. [ 2004 ] J. Physiol. 555:71–84). The present study tested the hypothesis that CCK modulates inhibitory synaptic transmission in the NAc. Using in vitro forebrain slices containing the NAc and whole‐cell patch recording, we examined the effects of CCK on evoked inhibitory postsynaptic currents (IPSCs) recorded at a holding potential of −80 mV throughout CCK‐8S caused a reversible inward current accompanied by a concentration‐dependent decrease in evoked IPSC amplitude. Maximum IPSC depression was ∼25% at 10 μM, with an estimated EC50 of 0.1 μM. At 1 μM, CCK‐8S induced an inward current of 28.3 ± 4.8 pA (n = 6) accompanied by an IPSC depression of −18.8% ± 1.6% (n = 6). This CCK‐induced IPSC depression was blocked by pretreatment with proglumide (100 μM; −3.7% ± 6.9%; n = 4) and by LY225910 (100 nM), a selective CCKB receptor antagonist (4.4% ± 2.6%; n = 4). It was not blocked by SCH23390 (10 μM; −23.5% ± 1.3%; P < 0.05; n = 7) or sulpiride (10 μM; −21.8% ± 5.1%; P < 0.05; n = 4), dopamine receptor antagonists. By contrast, it was blocked by CGP55845 (1 μM; −0.4% ± 3.4%; n = 5) a potent GABAB receptor antagonist, and by forskolin (50 μM; 9.9% ± 5.2%; n = 4), an adenylyl cyclase activator, and H‐89 (1 μM; 6.9% ± 3.9%; n = 4), a protein kinase A (PKA) inhibitor. These results indicate that CCK acts on CCKB receptors to increase extracellular levels of GABA, which then acts on GABAB receptors to decrease IPSC amplitude.

Coadministration of indomethacin and minocycline attenuates established paclitaxel-induced neuropathic thermal hyperalgesia: Involvement of cannabinoid CB1 receptors

Taxanes such as paclitaxel, which are chemotherapeutic drugs, cause dose-dependent painful neuropathy in some patients. We investigated whether coadministration of minocycline and indomethacin produces antinociceptive effects in mice with paclitaxel-induced neuropathic thermal hyperalgesia and if the cannabinoid system is involved. Previously, we reported that coadministration of these two drugs results in antinociception against inflammatory pain at doses where either drug alone lack significant activity. In the current study, we observed that treatment of female mice with indomethacin or minocycline alone did not affect established paclitaxel-induced thermal hyperalgesia, whereas coadministration of the two drugs attenuated it. In male mice indomethacin had some antihyperalgesic activity, whilst minocycline did not. Coadministration of the two drugs had supraadditive antihyperalgesic activity in male mice. Administration of a cannabinoid CB1 receptor antagonist AM 251 blocked the antihyperalgesic effects of the combination of minocycline and indomethacin in both male and female mice. In conclusion our results indicate that coadministration of minocycline and indomethacin abrogates established paclitaxel-induced neuropathic thermal hyperalgesia in mice, and the potentiation of the antinociceptive effects of this combination involves the cannabinoid system.

Preventative and therapeutic effects of a GABA transporter 1 inhibitor administered systemically in a mouse model of paclitaxel-induced neuropathic pain

Background There is a dearth of drugs to manage a dose-limiting painful peripheral neuropathy induced by paclitaxel in some patients during the treatment of cancer. Gamma-aminobutyric acid transporter-1 (GAT-1) whose expression is increased in the brain and spinal cord during paclitaxel-induced neuropathic pain (PINP) might be a potential therapeutic target for managing PINP. Thus, our aim was to evaluate if systemic administration of a GAT-1 inhibitor ameliorates PINP. Methods The reaction latency to thermal stimuli (hot plate test; at 55 °C) and cold stimuli (cold plate test; at 4 °C) of female BALB/c mice was recorded before and after intraperitoneal treatment with paclitaxel, its vehicle, and/or a selective GAT-1 inhibitor NO-711. The effects of NO-711 on motor coordination were evaluated using the rotarod test at a constant speed of 4 rpm or accelerating mode from 4 rpm to 40 rpm over 5 min. Results The coadministration of paclitaxel with NO-711 3 mg/kg prevented the development of paclitaxel-induced thermal hyperalgesia and cold allodynia at day 7 after drug treatment. NO-711 at 3 mg/kg produced antihyperalgesic activity up to 1 h and antiallodynic activity up to 2 h in mice with established paclitaxel-induced thermal hyperalgesia and cold allodynia. No motor deficits were observed with NO-711 at a dose of 3 mg/kg, whereas a higher dose 5 mg/kg caused motor impairment and reduced mean time spent on the rotarod at a constant speed of 4 rpm. However, at a rotarod accelerating mode from 4 rpm to 40 rpm over 5 min, NO-711 3 mg/kg caused motor impairment up to 1 h, but had recovered by 2 h. Conclusions These results show that systemic administration of the GAT-1 inhibitor NO-711 has preventative and therapeutic activity against paclitaxel-induced thermal hyperalgesia and cold allodynia. NO-711’s antiallodynic effects, but not antihyperalgesic effects, were independent of its motor impairment/sedation properties. Thus, low doses of GAT-1 inhibitors could be useful for the prevention and treatment of PINP with proper dose titration to reduce motor impairment/sedation side effects.