patients with neuropathic pain experience spontaneous tingling sensations , hyperalgesia , and tactile allodynia , symptoms that deteriorate their quality of life .
the pathogenesis of neuropathic pain is believed to involve central sensitization of spinal dorsal horn neurons following central or peripheral nervous system damage .
glia interactions play a crucial role in the development of central sensitization . various cytokines or chemokines
interferon - gamma ( ifn ) is a pro - inflammatory cytokine and a major modulator of central and peripheral immune responses that plays roles in a variety of chronic pain conditions .
for example , patients with persistent pain due to viral infections or multiple sclerosis have persistently high ifn levels in the spinal cord as an inflammatory response .
this cytokine is produced primarily by t - cells and natural killer cells   and stimulates the janus activated kinase ( jak)-signal transducer and activator of transcription ( stat ) signaling pathway . upon peripheral nerve injury
, ifn is upregulated in the rat dorsal horn as t - cells that infiltrate the spinal cord , and it has been implicated as one of the causative agents of neuropathic pain .
interestingly , intrathecal injections of ifn induce pain - related behaviors in wildtype rats and mice but not ifn receptor knock - out mice .
however , little is known about the roles of ifn in the dorsal horn of the spinal cord .
the substantia gelatinosa ( sg , lamina ii ) of the spinal cord dorsal horn has been implicated in nociceptive transmission because both finely myelinated ( a ) and unmyelinated ( c ) primary nociceptive afferent fibers are heavily projected to this spinal region . to elucidate how ifn affects nociceptive responses in the spinal dorsal horn
, we analyzed the effects of ifn on postsynaptic action by using whole - cell patch - clamp recordings from sg neurons .
five to six - week - old male sprague dawley rats ( 180210  g ; obtained from slc , japan ) were used .
the protocol was approved by the ethics committee on animal experiments , wakayama medical university , and was in accordance with the uk animals ( scientific procedures ) act of 1986 and associated guidelines .
animals were housed in plastic cages at room temperature on a 12-h light / dark cycle ( light on between 8:00 a.m. and 8:00 p.m. ) with ad libitum access to food and water .
briefly , the adult rats were anesthetized with urethane ( 1.2  g / kg , intraperitoneal ) , and th10-l4 laminectomy was performed .
then , we excised the spinal cord and submerged it in preoxygenated krebs solution at 13. immediately after the removal of the spinal cord , the rats were killed by exsanguination under urethane .
the dura , arachnoid , and pia mater were removed , and the spinal segment was sliced to 650-m thickness by using a microslicer ( dtk-1000 , dousaka from kyoto , japan ) .
the slices were continuously perfused with krebs solution ( 10  ml / min ) and saturated with 95% o2 and 5% co2 .
the perfusion solution was heated to 36    1 by using a temperature controller .
the krebs solution contained the following ( in mm ) : 117 nacl , 3.6 kcl , 1.2 nah2po4 , 2.5 cacl2 , 1.2 mgcl2 , 25 nahco3 , and 11 glucose , at ph 7.4 .
blind whole - cell patch - clamp recordings were made from lamina ii ( sg ) neurons in voltage - clamp mode .
the patch - pipette had a resistance of 510   , and the composition of the patch - pipette internal solution was as follows ( in mm ) : 135 potassium gluconate , 5 kcl , 0.5 cacl2 , 2 mgcl2 , 5 egta , 5 hepes , and 5 atp  mg .
membrane currents were amplified by using an amplifier ( axopatch 200b from molecular devices , sunnyvale , ca , usa ) in voltage - clamp mode .
holding potential was set to 70  mv for recording exogenous -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid ( ampa ) current and to 50  mv for n - methyl - d - aspartate ( nmda ) current .
data were digitized using an analog - to - digital converter ( digidata 1440a from molecular devices ) , stored , and then analyzed using a personal computer with the pclamp 10 data acquisition program ( molecular devices ) .
the drugs used in this study were ifn ( peprotech , rocky hill , nj , usa ) ; ifn antagonist ( vwr international , radnor , pa , usa ) ; monocyte chemoattractant protein 1 ( mcp-1 ) ( r&d , minneapolis , mn , usa ) ; and , ampa , nmda , minocycline hydrochloride , teijin compound 1 hydrochloride , gdp--s , and tofacitinib citrate ( sigma  aldrich , st .
ifn antagonist , mcp-1 , ampa , nmda , and minocycline hydrochloride were dissolved in distilled water as 1,000    stock solutions , and teijin compound 1 hydrochloride and tofacitinib citrate were first dissolved in 5% dmso as 1,000    stock solutions .
the 1,000    stock solutions of ifn were prepared in distilled water containing 1% bovine serum albumin ( bsa ) ( sigma  aldrich ) .
all perfusion drugs were stored at 20 and diluted to the final concentration in krebs solution just before use , and then were perfused via a three - way stopcock without any change in the rate or temperature .
the time necessary for the solution to flow from the stopcock to the spinal cord section surface was about 1  min .
the tubing of our perfusion system was coated with aquasil siliconizing fluid ( thermo fisher scientific , waltham , ma , usa ) to reduce the loss of ifn through nonspecific interactions .
all numerical data are expressed as mean    standard error ( se ) .
paired student s t tests were used for statistical comparison , and the criterion for statistical significance was p  <  0.05 ; n refers to the number of neurons studied .
briefly , the adult rats were anesthetized with urethane ( 1.2  g / kg , intraperitoneal ) , and th10-l4 laminectomy was performed .
then , we excised the spinal cord and submerged it in preoxygenated krebs solution at 13. immediately after the removal of the spinal cord , the rats were killed by exsanguination under urethane .
the dura , arachnoid , and pia mater were removed , and the spinal segment was sliced to 650-m thickness by using a microslicer ( dtk-1000 , dousaka from kyoto , japan ) .
the slices were continuously perfused with krebs solution ( 10  ml / min ) and saturated with 95% o2 and 5% co2 .
the perfusion solution was heated to 36    1 by using a temperature controller .
the krebs solution contained the following ( in mm ) : 117 nacl , 3.6 kcl , 1.2 nah2po4 , 2.5 cacl2 , 1.2 mgcl2 , 25 nahco3 , and 11 glucose , at ph 7.4 .
blind whole - cell patch - clamp recordings were made from lamina ii ( sg ) neurons in voltage - clamp mode .
the patch - pipette had a resistance of 510   , and the composition of the patch - pipette internal solution was as follows ( in mm ) : 135 potassium gluconate , 5 kcl , 0.5 cacl2 , 2 mgcl2 , 5 egta , 5 hepes , and 5 atp  mg .
membrane currents were amplified by using an amplifier ( axopatch 200b from molecular devices , sunnyvale , ca , usa ) in voltage - clamp mode .
holding potential was set to 70  mv for recording exogenous -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid ( ampa ) current and to 50  mv for n - methyl - d - aspartate ( nmda ) current .
data were digitized using an analog - to - digital converter ( digidata 1440a from molecular devices ) , stored , and then analyzed using a personal computer with the pclamp 10 data acquisition program ( molecular devices ) .
the drugs used in this study were ifn ( peprotech , rocky hill , nj , usa ) ; ifn antagonist ( vwr international , radnor , pa , usa ) ; monocyte chemoattractant protein 1 ( mcp-1 ) ( r&d , minneapolis , mn , usa ) ; and , ampa , nmda , minocycline hydrochloride , teijin compound 1 hydrochloride , gdp--s , and tofacitinib citrate ( sigma  aldrich , st .
ifn antagonist , mcp-1 , ampa , nmda , and minocycline hydrochloride were dissolved in distilled water as 1,000    stock solutions , and teijin compound 1 hydrochloride and tofacitinib citrate were first dissolved in 5% dmso as 1,000    stock solutions .
the 1,000    stock solutions of ifn were prepared in distilled water containing 1% bovine serum albumin ( bsa ) ( sigma  aldrich ) .
all perfusion drugs were stored at 20 and diluted to the final concentration in krebs solution just before use , and then were perfused via a three - way stopcock without any change in the rate or temperature .
the time necessary for the solution to flow from the stopcock to the spinal cord section surface was about 1  min .
the tubing of our perfusion system was coated with aquasil siliconizing fluid ( thermo fisher scientific , waltham , ma , usa ) to reduce the loss of ifn through nonspecific interactions .
all numerical data are expressed as mean    standard error ( se ) .
paired student s t tests were used for statistical comparison , and the criterion for statistical significance was p  <  0.05 ; n refers to the number of neurons studied .
only sg neurons , with resting membrane potentials lower than 50  mv in the current - clamp mode , were used for recording .
perfusion of exogenous ampa ( 10  m ) for 30  s onto the spinal cord slice induces slow inward currents at a holding potential of 70  mv , and nmda ( 50  m ) does so at 50  mv .
this discrepancy is because nmda receptors are blocked by mg when the cell membrane is held at 70  mv .
ampa superfusion repeatedly produces an inward current with the same peak amplitude in an sg neuron ( 101.3    0.6% , n  =  5 , p  =  0.09 ) , observed for nmda ( 97.9    3.1% ,
we first examined the effects of ifn on inward currents induced by ampa and nmda when maintaining the voltage at 70 and 50  mv , respectively .
bath application of ifn ( 30  nm , 2.5  min ) did not affect the ampa - induced current ( 100.0  3.7% , n  =  11 , p  =  0.87 , mean amplitude of control : 51.5  pa ; figure 1(a),(c ) ) .
on the other hand , nmda - induced current was significantly increased by ifn ( 147.3    10.7% , n  =  14 , p  =  0.0002 , mean amplitude of control : 16.8  pa ; figure 1(b),(d ) ) .
this ifn-induced facilitation of nmda current was inhibited by an ifn receptor - selective antagonist ( 300  nm , 5.5  min ) ( 90.0    3.6% , n  =  10 , p  =  0.053 ; figure 2 ) .
these results suggest that ifn binds to own receptor and enhances the activity of nmda but not ampa receptors in the dorsal horn of the spinal cord . 

figure 1.ampa- and nmda - induced currents under bath application of ifn in adult rat sg neurons .
( a ) recordings of ampa - induced inward currents at a holding potential of 70  mv .
perfusion of ifn ( 30  nm , 2.5  min ) did not result in any significant change in ampa - induced currents .
( b ) effects of ifn on nmda - induced inward currents at a holding potential of 50  mv .
nmda - induced current was significantly increased by ifn ( 30  nm , 2.5  min ) .
( c ) the peak amplitude of the ampa - induced currents after ifn treatment was 100.0    3.7% of baseline .
( d ) the peak amplitude of the nmda - induced currents after ifn treatment was 147.3    10.7% of baseline . in figure 1(b ) and subsequent figures , nmda responses were recorded in voltage - clamp mode ( vh  =  50  mv ) . n.s . : not significant . * 
ampa : -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid ; ifn : interferon - gamma ; nmda : n - methyl - d - aspartate . 

( a ) perfusion of the ifn receptor - selective antagonist ( 300  nm , 5.5  min ) inhibited the ifn-induced facilitation of nmda current .
( b ) bar graphs show the peak amplitude of nmda - induced current compared to baseline when the ifn receptor - selective antagonist and ifn are perfused simultaneously .
therefore , ifn likely binds to ifn receptors and activates nmda receptors in sg neurons .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate . ampa- and
( a ) recordings of ampa - induced inward currents at a holding potential of 70  mv .
perfusion of ifn ( 30  nm , 2.5  min ) did not result in any significant change in ampa - induced currents .
( b ) effects of ifn on nmda - induced inward currents at a holding potential of 50  mv .
nmda - induced current was significantly increased by ifn ( 30  nm , 2.5  min ) .
( c ) the peak amplitude of the ampa - induced currents after ifn treatment was 100.0    3.7% of baseline .
( d ) the peak amplitude of the nmda - induced currents after ifn treatment was 147.3    10.7% of baseline . in figure 1(b ) and subsequent figures , nmda responses were recorded in voltage - clamp mode ( vh  =  50  mv ) . n.s . : not significant . *  p  <  0.05 .
ampa : -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid ; ifn : interferon - gamma ; nmda : n - methyl - d - aspartate .
( a ) perfusion of the ifn receptor - selective antagonist ( 300  nm , 5.5  min ) inhibited the ifn-induced facilitation of nmda current .
( b ) bar graphs show the peak amplitude of nmda - induced current compared to baseline when the ifn receptor - selective antagonist and ifn are perfused simultaneously .
therefore , ifn likely binds to ifn receptors and activates nmda receptors in sg neurons .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate . because both neurons and microglia are reported as sites of ifn receptors , we investigated
as shown in figure 3 , the ifn-induced facilitation of nmda currents was inhibited by minocycline ( 20  m , 5.5  min ) , an inhibitor of microglia activation ( 90.3    6.4% , n  =  12 , p  =  0.12 ) . in addition , we recorded the ifn-induced facilitation of nmda current during inhibition of jak - stat signaling .
we added the jak inhibitor tofacitinib ( 1  mm ) to the pipette solution . despite blocking jak - stat signaling , ifn-induced facilitation of nmda currents
was not affected even after 30  min ( 98.9    6.0% , n  =  7 , p  =  0.99 ; figure 4 ) .
therefore , we considered that ifn activates the microglial ifn receptor and enhances the activity of nmda receptors in the dorsal horn neuron . 

( a ) minocycline ( 20  m , 5.5  min ) , an inhibitor of microglia activation , inhibited the ifn-induced facilitation of the nmda current .
( b ) bar graphs show the peak amplitude of the nmda - induced currents compared with baseline when minocycline and ifn were perfused simultaneously .
therefore , ifn likely binds to microglial ifn receptors and enhances the activity of nmda receptors in dorsal horn neurons .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate . 

( a ) tofacitinib ( 1  mm ) addition to the pipette solution did not affect the ifn-induced nmda current .
in this experimental system , the control corresponds to the ifn-induced nmda currents recorded immediately after patch clamping .
( b ) bar graphs show the average rate of nmda current enhancement by ifn at the start of patch - clamp recording and 30  min later .
the peak amplitude of the ifn-induced nmda currents did not change compared with the control recording .
this confirmed that ifn enhances neuronal nmda - induced inward current via the microglial ifn receptor .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate ; n.s . :
( a ) minocycline ( 20  m , 5.5  min ) , an inhibitor of microglia activation , inhibited the ifn-induced facilitation of the nmda current .
( b ) bar graphs show the peak amplitude of the nmda - induced currents compared with baseline when minocycline and ifn were perfused simultaneously .
therefore , ifn likely binds to microglial ifn receptors and enhances the activity of nmda receptors in dorsal horn neurons .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate .
( a ) tofacitinib ( 1  mm ) addition to the pipette solution did not affect the ifn-induced nmda current . in this experimental system
( b ) bar graphs show the average rate of nmda current enhancement by ifn at the start of patch - clamp recording and 30  min later .
the peak amplitude of the ifn-induced nmda currents did not change compared with the control recording .
this confirmed that ifn enhances neuronal nmda - induced inward current via the microglial ifn receptor .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate ; n.s . : not significant . as
jak - stat signaling was not the intraneuronal signal transducer underlying the effect of ifn on nmda currents , we examined another possible signaling pathway .
first , we added 1  mm gdp--s , a non - hydrolysable analog of gdp that competitively inhibits g proteins , to the pipette solution .
nmda - induced inward currents were significantly suppressed when ifn and nmda were applied 30  min after patch clamping with pipettes containing gdp--s ( 78.7  5.1% , n  =  6 , p  =  0.023 ; figure 5 ) .
these findings suggested that the ifn-induced increase of nmda currents involves the activation of g protein - coupled receptors such as chemokine receptors .
specifically , the chemokine mcp-1 , also known as chemokine ligand 2 , ccl2 and its receptor c - c chemokine receptor type 2 ( ccr2 ) are reportedly involved in the generation of neuropathic pain and are related to ifn. we then investigated the participation of ccl2/ccr2 in this effect of ifn on nmda currents .
ifn-induced enhancement of nmda currents was blocked by a selective antagonist of ccr2 , teijin compound 1 hydrochloride ( 10  m , 5.5  min ) ( 95.0    5.3% , n  =  6 , p  =  0.43 ; figure 6 ) .
in addition , bath - applied ccl2 ( 100  ng / ml ) for 2.5  min enhanced nmda - induced inward current ( 128.3    6.4% , n 
, it is possible that microglia activated by ifn release ccl2 , which binds to ccr2 and enhances neuronal nmda - induced current . 

( a ) in a potassium gluconate pipette solution containing gdp--s ( 1  mm ) , the ifn-induced nmda current after 30  min of recording was significantly lower than the control recording of ifn-induced nmda currents recorded at the start of patch clamping .
( b ) bar graphs show the average rate of ifn-induced enhancement of nmda current in the same neuron at the beginning of the recording and after 30  min .
the value of the right bar is 78.7    5.1% of the left bar .
this finding suggested that the ifn-induced enhancement of nmda currents involves the activation of g protein - coupled receptors . * p  <  0.05 .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate . 

figure 6.the enhancement of nmda - induced inward current by ifn was mediated by ccr2 .
( a ) bath application of the ccr2 antagonist teijin compound 1 hydrochloride ( 10  m , 5.5  min ) blocked the ifn-induced enhancement of nmda currents .
( b ) the peak amplitude of the nmda - induced currents after simultaneous application of teijin compound 1 hydrochloride and ifn was 95.0    5.3% that of baseline .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate ; n.s .
( a ) perfusion of spinal cord slices with ccl2 ( 100  ng / ml , 2.5  min ) increased nmda - induced inward currents .
( b ) the peak amplitude of the nmda - induced currents after simultaneous ccl2 application is 128.3    6.4% compared with baseline .
this suggests that ifn increases the nmda - induced inward current in sg neurons via ccl2/ccr2 signaling . *
mcp-1 : monocyte chemoattractant protein 1 ; nmda : n - methyl - d - aspartate .
( a ) in a potassium gluconate pipette solution containing gdp--s ( 1  mm ) , the ifn-induced nmda current after 30  min of recording was significantly lower than the control recording of ifn-induced nmda currents recorded at the start of patch clamping .
( b ) bar graphs show the average rate of ifn-induced enhancement of nmda current in the same neuron at the beginning of the recording and after 30  min .
the value of the right bar is 78.7    5.1% of the left bar .
this finding suggested that the ifn-induced enhancement of nmda currents involves the activation of g protein - coupled receptors . * p  <  0.05 .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate .
( a ) bath application of the ccr2 antagonist teijin compound 1 hydrochloride ( 10  m , 5.5  min ) blocked the ifn-induced enhancement of nmda currents .
( b ) the peak amplitude of the nmda - induced currents after simultaneous application of teijin compound 1 hydrochloride and ifn was 95.0    5.3% that of baseline .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate ; n.s .
( a ) perfusion of spinal cord slices with ccl2 ( 100  ng / ml , 2.5  min ) increased nmda - induced inward currents .
( b ) the peak amplitude of the nmda - induced currents after simultaneous ccl2 application is 128.3    6.4% compared with baseline .
this suggests that ifn increases the nmda - induced inward current in sg neurons via ccl2/ccr2 signaling . *
mcp-1 : monocyte chemoattractant protein 1 ; nmda : n - methyl - d - aspartate .
only sg neurons , with resting membrane potentials lower than 50  mv in the current - clamp mode , were used for recording .
perfusion of exogenous ampa ( 10  m ) for 30  s onto the spinal cord slice induces slow inward currents at a holding potential of 70  mv , and nmda ( 50  m ) does so at 50  mv .
this discrepancy is because nmda receptors are blocked by mg when the cell membrane is held at 70  mv .
ampa superfusion repeatedly produces an inward current with the same peak amplitude in an sg neuron ( 101.3    0.6% , n  =  5 , p  =  0.09 ) , observed for nmda ( 97.9    3.1% ,
we first examined the effects of ifn on inward currents induced by ampa and nmda when maintaining the voltage at 70 and 50  mv , respectively .
bath application of ifn ( 30  nm , 2.5  min ) did not affect the ampa - induced current ( 100.0  3.7% , n  =  11 , p  =  0.87 , mean amplitude of control : 51.5  pa ; figure 1(a),(c ) ) .
on the other hand , nmda - induced current was significantly increased by ifn ( 147.3    10.7% , n  =  14 , p  =  0.0002 , mean amplitude of control : 16.8  pa ; figure 1(b),(d ) ) .
this ifn-induced facilitation of nmda current was inhibited by an ifn receptor - selective antagonist ( 300  nm , 5.5  min ) ( 90.0    3.6% , n  =  10 , p  =  0.053 ; figure 2 ) .
these results suggest that ifn binds to own receptor and enhances the activity of nmda but not ampa receptors in the dorsal horn of the spinal cord . 

figure 1.ampa- and nmda - induced currents under bath application of ifn in adult rat sg neurons .
( a ) recordings of ampa - induced inward currents at a holding potential of 70  mv .
perfusion of ifn ( 30  nm , 2.5  min ) did not result in any significant change in ampa - induced currents .
( b ) effects of ifn on nmda - induced inward currents at a holding potential of 50  mv .
nmda - induced current was significantly increased by ifn ( 30  nm , 2.5  min ) .
( c ) the peak amplitude of the ampa - induced currents after ifn treatment was 100.0    3.7% of baseline .
( d ) the peak amplitude of the nmda - induced currents after ifn treatment was 147.3    10.7% of baseline . in figure 1(b ) and subsequent figures , nmda responses were recorded in voltage - clamp mode ( vh  =  50  mv ) . n.s . : not significant . * 
ampa : -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid ; ifn : interferon - gamma ; nmda : n - methyl - d - aspartate . 

( a ) perfusion of the ifn receptor - selective antagonist ( 300  nm , 5.5  min ) inhibited the ifn-induced facilitation of nmda current .
( b ) bar graphs show the peak amplitude of nmda - induced current compared to baseline when the ifn receptor - selective antagonist and ifn are perfused simultaneously .
therefore , ifn likely binds to ifn receptors and activates nmda receptors in sg neurons .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate . ampa- and
( a ) recordings of ampa - induced inward currents at a holding potential of 70  mv .
perfusion of ifn ( 30  nm , 2.5  min ) did not result in any significant change in ampa - induced currents .
( b ) effects of ifn on nmda - induced inward currents at a holding potential of 50  mv .
nmda - induced current was significantly increased by ifn ( 30  nm , 2.5  min ) .
( c ) the peak amplitude of the ampa - induced currents after ifn treatment was 100.0    3.7% of baseline .
( d ) the peak amplitude of the nmda - induced currents after ifn treatment was 147.3    10.7% of baseline . in figure 1(b ) and subsequent figures , nmda responses were recorded in voltage - clamp mode ( vh  =  50  mv ) . n.s . : not significant . *  p  <  0.05 .
ampa : -amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid ; ifn : interferon - gamma ; nmda : n - methyl - d - aspartate .
( a ) perfusion of the ifn receptor - selective antagonist ( 300  nm , 5.5  min ) inhibited the ifn-induced facilitation of nmda current .
( b ) bar graphs show the peak amplitude of nmda - induced current compared to baseline when the ifn receptor - selective antagonist and ifn are perfused simultaneously .
therefore , ifn likely binds to ifn receptors and activates nmda receptors in sg neurons .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate .
because both neurons and microglia are reported as sites of ifn receptors , we investigated which receptor is responsible for the ifn-induced potentiation of nmda - induced currents .
as shown in figure 3 , the ifn-induced facilitation of nmda currents was inhibited by minocycline ( 20  m , 5.5  min ) , an inhibitor of microglia activation ( 90.3    6.4% , n  =  12 , p  =  0.12 ) .
in addition , we recorded the ifn-induced facilitation of nmda current during inhibition of jak - stat signaling .
we added the jak inhibitor tofacitinib ( 1  mm ) to the pipette solution . despite blocking jak - stat signaling , ifn-induced facilitation of nmda currents
was not affected even after 30  min ( 98.9    6.0% , n  =  7 , p  =  0.99 ; figure 4 ) .
therefore , we considered that ifn activates the microglial ifn receptor and enhances the activity of nmda receptors in the dorsal horn neuron . 

( a ) minocycline ( 20  m , 5.5  min ) , an inhibitor of microglia activation , inhibited the ifn-induced facilitation of the nmda current .
( b ) bar graphs show the peak amplitude of the nmda - induced currents compared with baseline when minocycline and ifn were perfused simultaneously .
therefore , ifn likely binds to microglial ifn receptors and enhances the activity of nmda receptors in dorsal horn neurons .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate . 

( a ) tofacitinib ( 1  mm ) addition to the pipette solution did not affect the ifn-induced nmda current . in this experimental system
( b ) bar graphs show the average rate of nmda current enhancement by ifn at the start of patch - clamp recording and 30  min later .
the peak amplitude of the ifn-induced nmda currents did not change compared with the control recording .
this confirmed that ifn enhances neuronal nmda - induced inward current via the microglial ifn receptor .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate ; n.s . : not significant .
( a ) minocycline ( 20  m , 5.5  min ) , an inhibitor of microglia activation , inhibited the ifn-induced facilitation of the nmda current .
( b ) bar graphs show the peak amplitude of the nmda - induced currents compared with baseline when minocycline and ifn were perfused simultaneously .
therefore , ifn likely binds to microglial ifn receptors and enhances the activity of nmda receptors in dorsal horn neurons .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate .
( a ) tofacitinib ( 1  mm ) addition to the pipette solution did not affect the ifn-induced nmda current .
in this experimental system , the control corresponds to the ifn-induced nmda currents recorded immediately after patch clamping .
( b ) bar graphs show the average rate of nmda current enhancement by ifn at the start of patch - clamp recording and 30  min later .
the peak amplitude of the ifn-induced nmda currents did not change compared with the control recording .
this confirmed that ifn enhances neuronal nmda - induced inward current via the microglial ifn receptor .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate ; n.s . : not significant .
as jak - stat signaling was not the intraneuronal signal transducer underlying the effect of ifn on nmda currents , we examined another possible signaling pathway .
first , we added 1  mm gdp--s , a non - hydrolysable analog of gdp that competitively inhibits g proteins , to the pipette solution .
nmda - induced inward currents were significantly suppressed when ifn and nmda were applied 30  min after patch clamping with pipettes containing gdp--s ( 78.7  5.1% , n  =  6 , p  =  0.023 ; figure 5 ) .
these findings suggested that the ifn-induced increase of nmda currents involves the activation of g protein - coupled receptors such as chemokine receptors .
specifically , the chemokine mcp-1 , also known as chemokine ligand 2 , ccl2 and its receptor c - c chemokine receptor type 2 ( ccr2 ) are reportedly involved in the generation of neuropathic pain and are related to ifn. we then investigated the participation of ccl2/ccr2 in this effect of ifn on nmda currents .
ifn-induced enhancement of nmda currents was blocked by a selective antagonist of ccr2 , teijin compound 1 hydrochloride ( 10  m , 5.5  min ) ( 95.0    5.3% , n  =  6 , p  =  0.43 ; figure 6 ) .
in addition , bath - applied ccl2 ( 100  ng / ml ) for 2.5  min enhanced nmda - induced inward current ( 128.3    6.4% , n 
, it is possible that microglia activated by ifn release ccl2 , which binds to ccr2 and enhances neuronal nmda - induced current . 

figure 5.the g protein inhibitor gdp--s significantly inhibited ifn-induced nmda currents . ( a ) in a potassium gluconate pipette solution containing gdp--s ( 1  mm ) , the ifn-induced nmda current after 30  min of recording was significantly lower than the control recording of ifn-induced nmda currents recorded at the start of patch clamping .
( b ) bar graphs show the average rate of ifn-induced enhancement of nmda current in the same neuron at the beginning of the recording and after 30  min .
the value of the right bar is 78.7    5.1% of the left bar .
this finding suggested that the ifn-induced enhancement of nmda currents involves the activation of g protein - coupled receptors . * p  <  0.05 .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate . 

figure 6.the enhancement of nmda - induced inward current by ifn was mediated by ccr2 .
( a ) bath application of the ccr2 antagonist teijin compound 1 hydrochloride ( 10  m , 5.5  min ) blocked the ifn-induced enhancement of nmda currents .
( b ) the peak amplitude of the nmda - induced currents after simultaneous application of teijin compound 1 hydrochloride and ifn was 95.0    5.3% that of baseline .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate ; n.s . : not significant . 

( a ) perfusion of spinal cord slices with ccl2 ( 100  ng / ml , 2.5  min ) increased nmda - induced inward currents .
( b ) the peak amplitude of the nmda - induced currents after simultaneous ccl2 application is 128.3    6.4% compared with baseline .
this suggests that ifn increases the nmda - induced inward current in sg neurons via ccl2/ccr2 signaling .
mcp-1 : monocyte chemoattractant protein 1 ; nmda : n - methyl - d - aspartate .
( a ) in a potassium gluconate pipette solution containing gdp--s ( 1  mm ) , the ifn-induced nmda current after 30  min of recording was significantly lower than the control recording of ifn-induced nmda currents recorded at the start of patch clamping .
( b ) bar graphs show the average rate of ifn-induced enhancement of nmda current in the same neuron at the beginning of the recording and after 30  min .
the value of the right bar is 78.7    5.1% of the left bar .
this finding suggested that the ifn-induced enhancement of nmda currents involves the activation of g protein - coupled receptors . *
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate . the enhancement of nmda - induced inward current by ifn was mediated by ccr2 .
( a ) bath application of the ccr2 antagonist teijin compound 1 hydrochloride ( 10  m , 5.5  min ) blocked the ifn-induced enhancement of nmda currents .
( b ) the peak amplitude of the nmda - induced currents after simultaneous application of teijin compound 1 hydrochloride and ifn was 95.0    5.3% that of baseline .
ifn : interferon - gamma ; nmda : n - methyl - d - aspartate ; n.s .
( a ) perfusion of spinal cord slices with ccl2 ( 100  ng / ml , 2.5  min ) increased nmda - induced inward currents .
( b ) the peak amplitude of the nmda - induced currents after simultaneous ccl2 application is 128.3    6.4% compared with baseline .
this suggests that ifn increases the nmda - induced inward current in sg neurons via ccl2/ccr2 signaling . *
mcp-1 : monocyte chemoattractant protein 1 ; nmda : n - methyl - d - aspartate .
in the present study , ifn significantly enhanced nmda - induced inward currents but not ampa - induced inward currents in sg neurons .
this ifn-induced facilitation of nmda current was inhibited by an ifn receptor - selective antagonist .
the results suggest that ifn enhances nmda receptor signaling by activating ifn receptors in the dorsal horn of the spinal cord .
minocycline , an inhibitor of microglial activation , inhibited the ifn-induced facilitation of nmda current , but inhibition of the neuronal jak - stat signaling did not .
therefore , we conclude that ifn binds its own receptors in microglia but not sg neurons , and the subsequent microglial activation leads to nmda receptor activation .
moreover , ifn-induced enhancement of nmda currents was blocked by gdp--s in the pipette solution or bath application of a ccr2 antagonist .
ccr2 signaling is mediated by a g protein and a typical chemokine involved in central sensitization .
these results suggested that activation of ifn receptors in the spinal microglia leads to ccl2 release and subsequently enhances nmda receptors in dorsal horn neurons ( figure 8) . 

when nerve injury occurs , ifn levels are increased in the dorsal horn of the spinal cord and binds to ifn receptors on microglia .
ccl2 subsequently enhances the neuronal nmda - induced inward current in lamina ii neurons via ccr2 .
ccl2 : chemokine ligand 2 ; ccr2 : c - c chemokine receptor type 2 ; ifn : interferon - gamma ; sg : substantia gelatinosa .
when nerve injury occurs , ifn levels are increased in the dorsal horn of the spinal cord and binds to ifn receptors on microglia .
ccl2 subsequently enhances the neuronal nmda - induced inward current in lamina ii neurons via ccr2 .
ccl2 : chemokine ligand 2 ; ccr2 : c - c chemokine receptor type 2 ; ifn : interferon - gamma ; sg : substantia gelatinosa .
importantly , this is the first study to investigate the function of ifn using blind whole - cell patch - clamp methods . interestingly ,
inward currents induced by exogenously applied nmda , but not ampa , were clearly enhanced by the application of ifn in sg neurons of adult rat spinal cord slices .
this postsynaptic effect of ifn on nmda receptor could explain the pain - related behaviors caused by intrathecal ifn. in fact , there is considerable evidence that chronic pain hypersensitivity depends on nmda receptors .
it is commonly known that nmda receptor activation is an essential step in initiating and maintaining activity - dependent central sensitization , and it critically contributes to the development of pain hypersensitivity after peripheral tissue damage or nerve injury . on the other hand , no acute changes in ampa - induced current
clearly demonstrated that the ifn/ifn receptor system is critical in activating resting spinal microglia , thereby linking them to tactile allodynia .
they indicated that spinal microglia are directly stimulated by intrathecal ifn and contribute to ifn receptor - dependent pain hypersensitivity .
our findings also showed that ifn-induced enhancement of nmda currents was microglial ifn receptor - dependent .
notably , ccr2 blockade inhibited the ifn-induced enhancement of nmda - induced inward currents and ccl2 increases nmda current in sg neurons .
demonstrated electrophysiologically that ccl2 can very rapidly ( within 2  min ) increase nmda currents in dorsal horn neurons .
they showed that incubation of the mouse spinal cord slices with 100  ng / ml ccl2 induced substantial activation of extracellular regulated kinase ( erk ) in lamina i  ii , and erk phosphorylation was primarily induced in neun - positive neurons , indicating that ccl2 may directly act on neurons .
for instance , exogenous spinal administration of ccl2 induces mechanical allodynia in wildtype but not ccr2 knockout mice , and in a model of neuropathic pain the development of mechanical allodynia is totally abrogated in ccr2 knockout mice .
these results are consistent with ours , and in addition , we found that ccl2 lies downstream of ifn in terms of nmda - induced currents .
in summary , our results show that ifn enhanced nmda - induced inward currents in sg neurons through the activation of ifn receptors in microglia , and ccl2 was one of the mediators between microglia and neurons .
thus , ifn plays an important role as one of the initiating agents of glia  neuron interactions that lead to neuropathic pain due to central sensitization .
ms performed or contributed to all the experiments , analyzed data , and drafted the paper .
my , tn , and wt contributed to experiment conception and design and edited the manuscript .
the author(s ) declared no potential conflicts of interest with respect to the research , authorship , and/or publication of this article .
the author(s ) disclosed receipt of the following financial support for the research , authorship , and/or publication of this article : this work was supported in part by mext kakenhi 26462253 to wt , mext kakenhi 25460730 to tn , and mext kakenhi 25462303 to st .
 backgroundglia  neuron interactions play an important role in the development of neuropathic pain . 
 expression of the pro - inflammatory cytokne cytokine interferon - gamma ( ifn ) is upregulated in the dorsal horn after peripheral nerve injury , and intrathecal ifn administration induces mechanical allodynia in rats . 
 a growing body of evidence suggests that ifn might be involved in the mechanisms of neuropathic pain , but its effects on the spinal dorsal horn are unclear . 
 we performed blind whole - cell patch - clamp recording to investigate the effect of ifn on postsynaptic glutamate - induced currents in the substantia gelatinosa neurons of spinal cord slices from adult male rats.resultsifn perfusion significantly enhanced the amplitude of nmda - induced inward currents in substantia gelatinosa neurons , but did not affect ampa - induced currents . 
 the facilitation of nmda - induced current by ifn was inhibited by bath application of an ifn receptor - selective antagonist . adding the janus activated kinase inhibitor tofacitinib to the pipette solution did not affect the ifn-induced facilitation of nmda - induced currents . 
 however , the facilitatory effect of ifn on nmda - induced currents was inhibited by perfusion of the microglial inhibitor minocycline . 
 these results suggest that ifn binds the microglial ifn receptor and enhances nmda receptor activity in substantia gelatinosa neurons . 
 next , to identify the effector of signal transmission from microglia to dorsal horn neurons , we added an inhibitor of g proteins , gdp--s , to the pipette solution . in a gdp--s  containing pipette solution , ifn-induced potentiation of the nmda current 
 was significantly suppressed after 30  min . 
 in addition , ifn-induced potentiation of nmda currents was blocked by application of a selective antagonist of ccr2 , and its ligand ccl2 increased nmda - induced currents.conclusionour findings suggest that ifn enhance the amplitude of nmda - induced inward currents in substantia gelatinosa neurons via microglial ifn receptors and ccl2/ccr2 signaling . 
 this mechanism might be partially responsible for the development of persistent neuropathic pain . 