Brigham Young University, Provo, UT, 84602
1
Interleukin 10 increases dopamine neuron activity in the ventral tegmental area and increases dopamine release in the nucleus accumbens via reduction of GABA inhibition A. Payne, S. Williams, B. Williams, S. Stapley, J. Hiatt, R. Leavitt, C. Kanyuck, K. Peterson, R. Jensen, B. Sorhus, M. Woodbury, J. T. Yorgason and S. C. Steffensen Brigham Young University, Provo, UT, 84602 This work was supported by PHS NIH grant AA020919 to SCS Figure 2. Localizing VTA GABA neurons. (A) VTA GABA neurons were localized in the horizontal slice to an area in the midbrain between the MT and midline. (B) Expression of GAD GFP enabled their visualization via fluorescence microscopy. Substantia nigra was typically characterized by diffuse GAD labeling, likely due to the abundance of GABA terminals. (C) Under high power VTA GABA neurons could be positively identified via fluorescence. (D) Their visualization under fluorescence facilitated their patching under IRDIC. SNr Dent MT SNc 3V mt -4.7 mm HPC SNr PAG ml * B C D Electrophysiology: Mice (P28-45) were anesthetized with isoflurane and decapitated. Brains were sectioned in high Mg2+, low Ca2+, ice-cold cutting solution consisted of (in mM) 3 KCl, 1.25 NaH2PO4, 25 NaH2CO3, 12 MgSO4, 10 Glucose, 0.2 CaCl2, and 220 Sucrose. VTA targeted horizontal slices (210 µm thick) were then immediately placed into 36 degree, normal ACSF, bubbled with 95% O2 / 5% CO2 consisting of (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 25 NaHCO3, 11 glucose, 1.3 MgCl2, 2 CaCl2, pH 7.4, osmolarity 315 ± 3 mOsm, and allowed to incubate for at least 30 minutes before studies began. Electrodes were pulled from borosilicate glass capillaries and then filled with ACSF. Positive pressure was applied to the electrode when approaching the neuron. By applying suction to the electrode, a seal (10 MΩ –1GΩ) was created between the cell membrane and the recording pipette. Neurons were voltage-clamped at 0 mV throughout the experiment. • Dopamine (DA) transmission is a key player in the rewarding aspects of ethanol as well as ethanol dependence. • The current dogma is that DA transmission is increased during ethanol via the inhibition of ventral tegmental area (VTA) GABA neurons and that the excitation of VTA GABA neurons during withdrawal results in decreased DA transmission. • Much of the recent evidence indicates that neuroimmune interactions may mediate this process. • Microglia, the major neuroimmune effector in the brain, may be a key mediator in this process by releasing cytokines following activation. Figure 3: Acute ethanol effects on IL-10 Levels. We evaluated the effect of ethanol on cytokine concentrations in the VTA and NAc, and found that low dose ethanol (1.0 g/kg) decreased interleukin (IL)-10 levels, but high dose ethanol (4.0 g/kg) increased IL-10 levels. Effects of IL-6 and BDNF on VTA Neuron Firing Rate Effects of IL-10 on VTA DA FR and oIPSCs Figure 5: IL-6 had no effect on VTA GABA or DA neuron firing rate. A,B show ratemeters for representative neurons. C No change in firing rate of GABA (101.17± 11.23, n=5) or DA (114.71 ± 13.36, n=4) neurons. Figure 1. Theoretical Framework for proposed studies: In the ethanol Naive condition, VTA DA neurons and a population of GABA neurons project to the nucleus accumbens (NAc). These GABA neurons inhibit DA neurons and subsequent DA release into the NAc. In the ethanol Intoxicated condition, we propose that ethanol enhances cytokine release from microglia in the VTA leading to inhibition of VTA GABA neurons and subsequent disinhibition of DA neurons and enhanced DA release. We hypothesize that select cytokines will affect the excitability of VTA GABA neurons and DA release. Key: Darker shading indicates excitation while lighter shading indicates inhibition relative to the Naïve condition. D2Rs (blue squares); GABA(A)Rs (green squares). Optogenetic experiments: VGAT-ChR2-EYFP mice were used for stimulation of all GABAergic neurons and terminals. In this case, we recorded oIPSCs from dopamine neurons in the VTA. BDNF Protein Determination : Tissue punches containing the VTA were excised from 1 mm thick brain slices and immediately placed on dry ice. Samples were stored at -20 degrees Celsius until used in the protein assay. An enzyme-linked immunosorbent assay (ELISA) kit from Biosensis was used to determine mature BDNF concentrations in the tissue. Cytokine determination: Tissue punches containing the VTA or Nac were excised from 1 mm thick brain slices and immediately place on dry ice. Tissue was homogenized and then cytokine levels were measured using a cytometric bead array (BD biosciences). Figure 7: IL-10 increases DA neuron firing rate via decreases GABA inhibition. (A) Ratemeter of firing rate for a representative GABA neuron. (B) Ratemeter of firing rate for a representative DA neuron. (C) IL-10 (20 pg/ml) had no effect on the firing rate of VTA GABA neurons. (D) IL-10 (20 pg/ml) increased the firing rate of VTA DA neurons. (E) Optical stimulation in VGAT::ChR2 mice induces robust GABA(A) receptor-mediated IPSCs in DA neurons which were inhibited by IL- 10. (F) IL-10 (20 pg/ml) reduced the amplitude of oIPSC on VTA DA neurons. • We show a change in cytokine expression in the VTA following an acute ethanol injection (1.0 g/kg and 4.0 g/kg), but no change in BDNF expression following an acute ethanol injection (2.5 g/kg) • The pro-inflammatory cytokine, IL-6, had no effect on VTA neuron firing rate. • BDNF also had no effect on VTA neuron firing rate. • The anti-inflammatory cytokine, IL-10, caused an increase in VTA dopamine neuron firing rate. This increase in firing appears to be due to an decrease in inhibitory GABA transmission. • IL-10 also caused an increase in DA release in vivo, but not ex vivo. • These findings suggest that IL-10 may mediate the rewarding and/or addictive properties of ethanol. INTRODUCTION METHODS RESULTS RESULTS SUMMARY AND CONCLUSIONS ACKNOWLEDGMENTS Effects of Acute Ethanol on IL-10 and BDNF Concentrations Effects of IL-10 on DA Release Figure 8: IL-10 increases DA release in vivo, but not ex vivo in the Nucleus Accumbens. (A) Superfusion of IL-10 had no effect on DA release with a paired-pulse stimulation protocol ex vivo. (B) An intracerebroventricular injection of IL-10 caused an increase in DA release in the Nucleus Accumbens. A B Figure 6: BDNF had no effect on VTA GABA neuron firing rate. (A) Superfusion of BDNF (100ng/ml) and ANA-12 (10uM) had a slight effect on this representative GABA neuron. (B) No significant difference in firing rate with BDNF or ANA-12, a TrkB receptor antagonist. Figure 4: BDNF expression in the VTA and NAc. A single injection of ethanol (2.5g/kg) did not cause a change in BDNF expression in the VTA or NAc Cytokines? E F
Transcript of Brigham Young University, Provo, UT, 84602
Interleukin 10 increases dopamine neuron activity in the ventral tegmental area and increases dopamine release in the nucleus accumbens via reduction of GABA inhibition
A. Payne, S. Williams, B. Williams, S. Stapley, J. Hiatt, R. Leavitt, C. Kanyuck, K. Peterson, R. Jensen, B. Sorhus, M. Woodbury, J. T. Yorgason and S. C. Steffensen
Brigham Young University, Provo, UT, 84602
This work was supported by PHS NIH grant AA020919 to SCS
Figure 2. Localizing VTA GABA neurons. (A) VTA GABA neurons werelocalized in the horizontal slice to an area in the midbrain between the MTand midline. (B) Expression of GAD GFP enabled their visualization viafluorescence microscopy. Substantia nigra was typically characterized bydiffuse GAD labeling, likely due to the abundance of GABA terminals. (C)Under high power VTA GABA neurons could be positively identified viafluorescence. (D) Their visualization under fluorescence facilitated theirpatching under IRDIC.
SNr
Dent
MT SNc
3V
mt
-4.7 mm
HPCSNr
PAG
ml
*
B C
D
Electrophysiology: Mice (P28-45) were anesthetized with isoflurane and decapitated. Brains weresectioned in high Mg2+, low Ca2+, ice-cold cutting solution consisted of (in mM) 3 KCl, 1.25NaH2PO4, 25 NaH2CO3, 12 MgSO4, 10 Glucose, 0.2 CaCl2, and 220 Sucrose. VTA targetedhorizontal slices (210 µm thick) were then immediately placed into 36 degree, normal ACSF, bubbledwith 95% O2 / 5% CO2 consisting of (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 25 NaHCO3, 11glucose, 1.3 MgCl2, 2 CaCl2, pH 7.4, osmolarity 315 ± 3 mOsm, and allowed to incubate for at least30 minutes before studies began.
Electrodes were pulled from borosilicate glass capillaries and then filled with ACSF. Positivepressure was applied to the electrode when approaching the neuron. By applying suction to theelectrode, a seal (10 MΩ – 1 GΩ) was created between the cell membrane and the recording pipette.Neurons were voltage-clamped at 0 mV throughout the experiment.
• Dopamine (DA) transmission is a key player in the rewarding aspectsof ethanol as well as ethanol dependence.
• The current dogma is that DA transmission is increased duringethanol via the inhibition of ventral tegmental area (VTA) GABAneurons and that the excitation of VTA GABA neurons duringwithdrawal results in decreased DA transmission.
• Much of the recent evidence indicates that neuroimmune interactionsmay mediate this process.
• Microglia, the major neuroimmune effector in the brain, may be a keymediator in this process by releasing cytokines following activation.
Figure 3: Acute ethanol effects on IL-10 Levels. We evaluated the effect of ethanol on cytokine concentrations in the VTA and NAc, and found that low dose ethanol (1.0 g/kg) decreased interleukin (IL)-10 levels, but high dose ethanol (4.0 g/kg) increased IL-10 levels.
Effects of IL-6 and BDNF on VTA Neuron Firing Rate
Effects of IL-10 on VTA DA FR and oIPSCs
Figure 5: IL-6 had no effect on VTA GABA or DAneuron firing rate. A,B show ratemeters forrepresentative neurons. C No change in firing rateof GABA (101.17± 11.23, n=5) or DA (114.71 ±13.36, n=4) neurons.
Figure 1. Theoretical Framework for proposed studies: In the ethanolNaive condition, VTA DA neurons and a population of GABA neuronsproject to the nucleus accumbens (NAc). These GABA neurons inhibitDA neurons and subsequent DA release into the NAc. In the ethanolIntoxicated condition, we propose that ethanol enhances cytokinerelease from microglia in the VTA leading to inhibition of VTA GABAneurons and subsequent disinhibition of DA neurons and enhanced DArelease. We hypothesize that select cytokines will affect the excitabilityof VTA GABA neurons and DA release. Key: Darker shading indicatesexcitation while lighter shading indicates inhibition relative to the Naïvecondition. D2Rs (blue squares); GABA(A)Rs (green squares).
Optogenetic experiments: VGAT-ChR2-EYFP mice were usedfor stimulation of all GABAergic neurons and terminals. In thiscase, we recorded oIPSCs from dopamine neurons in the VTA.
BDNF Protein Determination : Tissue punches containing theVTA were excised from 1 mm thick brain slices and immediatelyplaced on dry ice. Samples were stored at -20 degrees Celsiusuntil used in the protein assay. An enzyme-linkedimmunosorbent assay (ELISA) kit from Biosensis was used todetermine mature BDNF concentrations in the tissue.
Cytokine determination: Tissue punches containing the VTA orNac were excised from 1 mm thick brain slices and immediatelyplace on dry ice. Tissue was homogenized and then cytokinelevels were measured using a cytometric bead array (BDbiosciences).
Figure 7: IL-10 increases DA neuronfiring rate via decreases GABAinhibition. (A) Ratemeter of firing rate for arepresentative GABA neuron. (B)Ratemeter of firing rate for a representativeDA neuron. (C) IL-10 (20 pg/ml) had noeffect on the firing rate of VTA GABAneurons. (D) IL-10 (20 pg/ml) increased thefiring rate of VTA DA neurons. (E) Opticalstimulation in VGAT::ChR2 mice inducesrobust GABA(A) receptor-mediated IPSCsin DA neurons which were inhibited by IL-10. (F) IL-10 (20 pg/ml) reduced theamplitude of oIPSC on VTA DA neurons.
• We show a change in cytokine expression in theVTA following an acute ethanol injection (1.0 g/kgand 4.0 g/kg), but no change in BDNF expressionfollowing an acute ethanol injection (2.5 g/kg)
• The pro-inflammatory cytokine, IL-6, had no effecton VTA neuron firing rate.
• BDNF also had no effect on VTA neuron firing rate.• The anti-inflammatory cytokine, IL-10, caused an
increase in VTA dopamine neuron firing rate. Thisincrease in firing appears to be due to an decreasein inhibitory GABA transmission.
• IL-10 also caused an increase in DA release in vivo,but not ex vivo.
• These findings suggest that IL-10 may mediate therewarding and/or addictive properties of ethanol.
INTRODUCTION
METHODS
RESULTS RESULTS
SUMMARY AND CONCLUSIONS
ACKNOWLEDGMENTS
Effects of Acute Ethanol on IL-10 and BDNF Concentrations Effects of IL-10 on DA Release
Figure 8: IL-10 increases DA release in vivo, but not ex vivo inthe Nucleus Accumbens. (A) Superfusion of IL-10 had no effecton DA release with a paired-pulse stimulation protocol ex vivo. (B)An intracerebroventricular injection of IL-10 caused an increase inDA release in the Nucleus Accumbens.
A
B
Figure 6: BDNF had no effect on VTA GABAneuron firing rate. (A) Superfusion of BDNF(100ng/ml) and ANA-12 (10uM) had a slight effecton this representative GABA neuron. (B) Nosignificant difference in firing rate with BDNF orANA-12, a TrkB receptor antagonist.
Figure 4: BDNF expression in the VTA and NAc. A single injection of ethanol (2.5g/kg) did not cause a change in BDNF expression in the VTA or NAc
Cytokines?
E F
