Limits...
Activation of toll like receptor 4 attenuates GABA synthesis and postsynaptic GABA receptor activities in the spinal dorsal horn via releasing interleukin-1 beta.

Yan X, Jiang E, Weng HR - J Neuroinflammation (2015)

Bottom Line: We found that activation of TLR4 by lipopolysaccharide (LPS) reduces GABAergic synaptic activities through both presynaptic and postsynaptic mechanisms.GABA synthesis at the presynaptic site is reduced upon activation of TLR4.The suppression of glial glutamate transporter activities leads to a deficiency of glutamine supply, which results in an attenuation of the glutamate-glutamine cycle-dependent GABA synthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical and Biomedical Sciences, The University of Georgia College of Pharmacy, 240 West Green Street, Athens, GA, 30602, USA. xisheng@uga.edu.

ABSTRACT
Toll like receptor 4 (TLR4) is an innate immune pattern recognition receptor, expressed predominantly on microglia in the CNS. Activation of spinal TLR4 plays a critical role in the genesis of pathological pain induced by nerve injury, bone cancer, and tissue inflammation. Currently, it remains unknown how synaptic activities in the spinal dorsal horn are regulated by TLR4 receptors. Through recording GABAergic currents in neurons and glial glutamate transporter currents in astrocytes in rodent spinal slices, we determined whether and how TLR4 modulates GABAergic synaptic activities in the superficial spinal dorsal horn. We found that activation of TLR4 by lipopolysaccharide (LPS) reduces GABAergic synaptic activities through both presynaptic and postsynaptic mechanisms. Specifically, LPS causes the release of IL-1β from microglia. IL-1β in turn suppresses GABA receptor activities at the postsynaptic site through activating protein kinase C (PKC) in neurons. GABA synthesis at the presynaptic site is reduced upon activation of TLR4. Glial glutamate transporter activities are suppressed by IL-1β and PKC activation induced by LPS. The suppression of glial glutamate transporter activities leads to a deficiency of glutamine supply, which results in an attenuation of the glutamate-glutamine cycle-dependent GABA synthesis. These findings shed light on understanding synaptic plasticity induced by activation of TLR4 under neuroinflammation and identify GABA receptors, glial glutamate transporters, IL-1β and PKC as therapeutic targets to abrogate abnormal neuronal activities following activation of TLR4 in pathological pain conditions.

No MeSH data available.


Related in: MedlinePlus

Lipopolysaccharide (LPS) suppresses GABAergic inhibitory postsynaptic currents (IPSCs) through activating microglia. GABAergic miniature IPSCs (mIPSCs) recordings obtained from superficial spinal dorsal horn neurons before, during and after washout of LPS (1 μg/ml) in the absence (A) and presence (B) of minocycline (100 μM) are shown. (C) shows the mean (+SE) GABAergic mIPSC amplitude and frequency before (baseline), during and after washout of LPS. The mean amplitude and frequency in 2-minute bins during LPS perfusion are displayed. (D) shows the mean (+SE) GABAergic mIPSC amplitude and frequency before and during perfusion of LPS in the presence of minocycline. Number of neurons included in each group for the analysis is shown in each bar. *P < 0.05; **P < 0.01; ***P < 0.001; NS: no statistical significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4302431&req=5

Fig1: Lipopolysaccharide (LPS) suppresses GABAergic inhibitory postsynaptic currents (IPSCs) through activating microglia. GABAergic miniature IPSCs (mIPSCs) recordings obtained from superficial spinal dorsal horn neurons before, during and after washout of LPS (1 μg/ml) in the absence (A) and presence (B) of minocycline (100 μM) are shown. (C) shows the mean (+SE) GABAergic mIPSC amplitude and frequency before (baseline), during and after washout of LPS. The mean amplitude and frequency in 2-minute bins during LPS perfusion are displayed. (D) shows the mean (+SE) GABAergic mIPSC amplitude and frequency before and during perfusion of LPS in the presence of minocycline. Number of neurons included in each group for the analysis is shown in each bar. *P < 0.05; **P < 0.01; ***P < 0.001; NS: no statistical significance.

Mentions: To study the impact of TLR4 activation on GABAergic synaptic activities, GABAergic mIPSCs were first recorded from neurons located in spinal lamina II in rats in the presence of 1 μM TTX before and after bath perfusion of the well-known TLR4 agonist LPS (1 μg/ml) [26]. As shown in Figure 1A, LPS significantly reduced mIPSC amplitudes and frequencies at the same time. These effects appeared at 4 minutes from the beginning of LPS perfusion and plateaued between 6 to 8 minutes. At 10 minutes after the perfusion, LPS significantly reduced the mean mIPSC amplitudes by 15.35 ± 1.45% (n = 15, P < 0.001) and mean mIPSC frequencies by 29.53 ± 2.91% (n = 15, P < 0.001). Similarly, when neurons in the same area in mice were recorded, LPS (1 μg/ml) significantly reduced the mean mIPSC amplitudes by 14.69 ± 3.28% (n = 7, P < 0.01) and frequencies by 27.11 ± 2.36% (n = 7, P < 0.001). It is generally believed that TLR4 is predominantly expressed in microglia [1,2,27]. If this is the case, the effects induced by LPS on mIPSCs should be abolished when activation of microglia is blocked. Previous studies have shown that minocycline inhibits the production of immune activators by macrophages, microglia [28,29], and monocytes [30]. More specifically, minocycline has been shown to specifically suppress microglial activation in the spinal dorsal horn at the concentration of 100 μM [31]. We then perfused minocycline (100 μM) into the recording for 10 minutes prior to addition of LPS into the bath. In the presence of minocycline, LPS no longer altered the amplitudes or frequencies of GABAergic mIPSCs recorded from rats (Figure 1B) and mice (amplitudes: n = 7, P = 0.82; frequencies: n = 7, P = 0.55, data not shown). Previous studies show that intraperitoneal injection of minocycline (40 mg/kg) suppresses phosphorylation of extracellular signal regulating kinases (ERK) in spinal dorsal horn neurons induced by intraperitoneal injection of acetic acid [32]. To confirm that the minocycline treatment under our experimental conditions did not alter the GABAergic synaptic activities, we examined the effects of minocycline on GABAergic mIPSCs. We found that perfusion of minocycline at 100 μM for 20 minutes did not significantly alter the amplitudes (P = 0.62) or frequencies (P = 0.52) of mIPSCs recorded from neurons in mice (n = 5). These data indicate that LPS suppresses GABAergic synaptic activities through activation of microglia. Several mechanisms may underlie the LPS-induced attenuation of GABAergic synaptic activities, which at least include (a) a reduced release probability of GABA at presynaptic terminals; (b) a decreased function of GABA receptors at postsynaptic neurons; and (c) the decreased synthesis of GABA. We conducted the following experiments to determine which of these mechanisms contributes to the changes of GABAergic synaptic activities induced by LPS.Figure 1


Activation of toll like receptor 4 attenuates GABA synthesis and postsynaptic GABA receptor activities in the spinal dorsal horn via releasing interleukin-1 beta.

Yan X, Jiang E, Weng HR - J Neuroinflammation (2015)

Lipopolysaccharide (LPS) suppresses GABAergic inhibitory postsynaptic currents (IPSCs) through activating microglia. GABAergic miniature IPSCs (mIPSCs) recordings obtained from superficial spinal dorsal horn neurons before, during and after washout of LPS (1 μg/ml) in the absence (A) and presence (B) of minocycline (100 μM) are shown. (C) shows the mean (+SE) GABAergic mIPSC amplitude and frequency before (baseline), during and after washout of LPS. The mean amplitude and frequency in 2-minute bins during LPS perfusion are displayed. (D) shows the mean (+SE) GABAergic mIPSC amplitude and frequency before and during perfusion of LPS in the presence of minocycline. Number of neurons included in each group for the analysis is shown in each bar. *P < 0.05; **P < 0.01; ***P < 0.001; NS: no statistical significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4302431&req=5

Fig1: Lipopolysaccharide (LPS) suppresses GABAergic inhibitory postsynaptic currents (IPSCs) through activating microglia. GABAergic miniature IPSCs (mIPSCs) recordings obtained from superficial spinal dorsal horn neurons before, during and after washout of LPS (1 μg/ml) in the absence (A) and presence (B) of minocycline (100 μM) are shown. (C) shows the mean (+SE) GABAergic mIPSC amplitude and frequency before (baseline), during and after washout of LPS. The mean amplitude and frequency in 2-minute bins during LPS perfusion are displayed. (D) shows the mean (+SE) GABAergic mIPSC amplitude and frequency before and during perfusion of LPS in the presence of minocycline. Number of neurons included in each group for the analysis is shown in each bar. *P < 0.05; **P < 0.01; ***P < 0.001; NS: no statistical significance.
Mentions: To study the impact of TLR4 activation on GABAergic synaptic activities, GABAergic mIPSCs were first recorded from neurons located in spinal lamina II in rats in the presence of 1 μM TTX before and after bath perfusion of the well-known TLR4 agonist LPS (1 μg/ml) [26]. As shown in Figure 1A, LPS significantly reduced mIPSC amplitudes and frequencies at the same time. These effects appeared at 4 minutes from the beginning of LPS perfusion and plateaued between 6 to 8 minutes. At 10 minutes after the perfusion, LPS significantly reduced the mean mIPSC amplitudes by 15.35 ± 1.45% (n = 15, P < 0.001) and mean mIPSC frequencies by 29.53 ± 2.91% (n = 15, P < 0.001). Similarly, when neurons in the same area in mice were recorded, LPS (1 μg/ml) significantly reduced the mean mIPSC amplitudes by 14.69 ± 3.28% (n = 7, P < 0.01) and frequencies by 27.11 ± 2.36% (n = 7, P < 0.001). It is generally believed that TLR4 is predominantly expressed in microglia [1,2,27]. If this is the case, the effects induced by LPS on mIPSCs should be abolished when activation of microglia is blocked. Previous studies have shown that minocycline inhibits the production of immune activators by macrophages, microglia [28,29], and monocytes [30]. More specifically, minocycline has been shown to specifically suppress microglial activation in the spinal dorsal horn at the concentration of 100 μM [31]. We then perfused minocycline (100 μM) into the recording for 10 minutes prior to addition of LPS into the bath. In the presence of minocycline, LPS no longer altered the amplitudes or frequencies of GABAergic mIPSCs recorded from rats (Figure 1B) and mice (amplitudes: n = 7, P = 0.82; frequencies: n = 7, P = 0.55, data not shown). Previous studies show that intraperitoneal injection of minocycline (40 mg/kg) suppresses phosphorylation of extracellular signal regulating kinases (ERK) in spinal dorsal horn neurons induced by intraperitoneal injection of acetic acid [32]. To confirm that the minocycline treatment under our experimental conditions did not alter the GABAergic synaptic activities, we examined the effects of minocycline on GABAergic mIPSCs. We found that perfusion of minocycline at 100 μM for 20 minutes did not significantly alter the amplitudes (P = 0.62) or frequencies (P = 0.52) of mIPSCs recorded from neurons in mice (n = 5). These data indicate that LPS suppresses GABAergic synaptic activities through activation of microglia. Several mechanisms may underlie the LPS-induced attenuation of GABAergic synaptic activities, which at least include (a) a reduced release probability of GABA at presynaptic terminals; (b) a decreased function of GABA receptors at postsynaptic neurons; and (c) the decreased synthesis of GABA. We conducted the following experiments to determine which of these mechanisms contributes to the changes of GABAergic synaptic activities induced by LPS.Figure 1

Bottom Line: We found that activation of TLR4 by lipopolysaccharide (LPS) reduces GABAergic synaptic activities through both presynaptic and postsynaptic mechanisms.GABA synthesis at the presynaptic site is reduced upon activation of TLR4.The suppression of glial glutamate transporter activities leads to a deficiency of glutamine supply, which results in an attenuation of the glutamate-glutamine cycle-dependent GABA synthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical and Biomedical Sciences, The University of Georgia College of Pharmacy, 240 West Green Street, Athens, GA, 30602, USA. xisheng@uga.edu.

ABSTRACT
Toll like receptor 4 (TLR4) is an innate immune pattern recognition receptor, expressed predominantly on microglia in the CNS. Activation of spinal TLR4 plays a critical role in the genesis of pathological pain induced by nerve injury, bone cancer, and tissue inflammation. Currently, it remains unknown how synaptic activities in the spinal dorsal horn are regulated by TLR4 receptors. Through recording GABAergic currents in neurons and glial glutamate transporter currents in astrocytes in rodent spinal slices, we determined whether and how TLR4 modulates GABAergic synaptic activities in the superficial spinal dorsal horn. We found that activation of TLR4 by lipopolysaccharide (LPS) reduces GABAergic synaptic activities through both presynaptic and postsynaptic mechanisms. Specifically, LPS causes the release of IL-1β from microglia. IL-1β in turn suppresses GABA receptor activities at the postsynaptic site through activating protein kinase C (PKC) in neurons. GABA synthesis at the presynaptic site is reduced upon activation of TLR4. Glial glutamate transporter activities are suppressed by IL-1β and PKC activation induced by LPS. The suppression of glial glutamate transporter activities leads to a deficiency of glutamine supply, which results in an attenuation of the glutamate-glutamine cycle-dependent GABA synthesis. These findings shed light on understanding synaptic plasticity induced by activation of TLR4 under neuroinflammation and identify GABA receptors, glial glutamate transporters, IL-1β and PKC as therapeutic targets to abrogate abnormal neuronal activities following activation of TLR4 in pathological pain conditions.

No MeSH data available.


Related in: MedlinePlus