BACKGROUND
psychological conditions like attention  <cit>  and hypnotic effects  <cit>  exert powerful influences on human pain sensations.
the anterior cingulate cortex  has crucial roles in conscious perception of pain but not the sensory discriminative aspect of pain  <cit> .
noxious stimulation applied to peripheral tissues evoked nociceptive responses in rabbit and rodent cingulate and prelimbic areas  <cit> , lesions of which impaired place avoidance test responses while pain behavior on the formalin test was normal  <cit> .
the human acc and the rodent prefrontal cortex  receiving inputs from the amygdala are involved in emotional processing  <cit> .
in our previous study  <cit> , nociceptive responses of the pfc were inhibited by inputs from the amygdala.
we also found that a dopamine  d <dig> receptor  blocker impaired the inhibition induced by amygdala stimulation.
direct dopaminergic projections from the ventral tegmental area  to the pfc are responsible for modulatory effects on nociceptive responses  <cit> , suggesting that the mesocortical da system modulates pain responses in the pfc.
recent studies have explored how da modulates pain perception  <cit> .
systemic administration of da antagonists were reported to alter nociceptive responses  <cit> .
symptoms of pain in parkinson disease  imply that da levels in the brain affect pain sensations.
in clinical reports on pd, patients frequently complain of neuropathic or central pain before motor disorders  <cit> .
these clinical reports indicate that the brain da level modifies pain sensations.
in animal studies, microinjections of da into the acc reduced autotomy scores in a sciatic neurotomy model  <cit> .
behavioral in vivo studies of pain have demonstrated that da depletion induces significant changes in thresholds for noxious stimuli  <cit> .
six-hydroxydopamine  lesions in the vta induced hyperalgesia in acute and chronic pain models  <cit> .
we determined whether lower da concentration changes in the vta induced by 6-ohda lesions alter nociceptive responses in the pfc.
we also examined effects of a κ-opiate receptor agonist injected into vta on nociceptive responses.
dopaminergic projections from vta have two main target areas, pfc and the nucleus accumbens.
a κ-opiate receptor agonist injected into vta inhibited only da neurons projecting to pfc and reduced da level in pfc  <cit> .
the rodent pfc receives da projections from the vta.
the present study demonstrated vta-pfc projections to directly affect nociceptive responses recorded in the pfc.
vta projections terminated at synapses within the soma, dendritic shafts, and spines of pyramidal cells in layers ii to v of the pfc  <cit> , while nociceptive information was transmitted through the medial pain pathways from the periphery to the pfc  <cit> .
the crucial roles of da in pfc function were discussed in the review by seamans  <cit> .
the mesocortical da pathways from the vta have modifying effects on cognition  <cit>  in the pfc.
the vta-pfc pathways may affect activities of the medial pain pathways, of which the pfc is the center.
we analyzed the effects of dopaminergic inputs from the vta to the prelimbic and cingulate areas on nociceptive responses in the pfc.
we also clarified which of the da receptor subtypes is related to the modulatory effects on nociceptive responses in the pfc.
RESULTS
mechanical noxious stimulation induced nociceptive responses in the pfc
mechanical noxious stimulation induced excitatory responses in pfc neurons, which persisted during and frequently after stimulation.
two types of nociceptive responses, a wide dynamic range type and a specific high threshold  type, were recorded  <cit> .
in this study, we used sht neurons to monitor the response to noxious stimulation.
the spontaneous background discharges of neurons, which usually showed the spindle bursts characteristic urethane anesthesia, continued for more than two hours, if the anesthetic level was maintained like we had previously reported  <cit> .
we administered additional urethane when typical spindle bursts disappeared.
electrocorticography  also showed typical high amplitude slow waves and was changed to low amplitude fast waves by mechanical stimulation .
anesthetic levels were also detected with ecog patterns.
the nociceptive responses were recorded without adaptation, if mechanical stimulation was applied every  <dig> s.
hfs delivered to the vta decreased nociceptive responses in the pfc
high frequency stimulation  delivered to the vta impaired pfc nociceptive responses evoked by mechanical stimulation applied to the rat tail  .
durations of nociceptive responses were  <dig>  ±  <dig>  s pre-hfs, and  <dig>  ±  <dig>  s  at  <dig> min ,  <dig>  ±  <dig>  s  at  <dig> min  and  <dig>  ±  <dig>  s  at  <dig> min post-hfs .
frequencies of discharge were  <dig>  ±  <dig>  pre-hfs, and  <dig>  ±  <dig>   at  <dig> min ,  <dig>  ±  <dig>   at  <dig> min and  <dig>  ±  <dig>   at  <dig> min post-hfs .
long-lasting suppression  of nociceptive responses appeared within  <dig> min and persisted for  <dig> min after hfs.
nociceptive responses recovered to pre-hfs levels in  <dig> min.
hfs delivered to the vta clearly inhibited nociceptive responses in the rat pfc.
d2r antagonist blocked the depression of nociceptive responses
a d2r antagonist, sulpiride, which had no effect on control nociceptive responses, significantly blocked the inhibitory effects of hfs .
durations of pain responses were  <dig>  ±  <dig>  s pre-microinjection and  <dig>  ±  <dig>  s post-microinjection.
there was no significant difference in nociceptive responses or background ecog between the two phases  .
after hfs had been delivered to the vta, the durations of nociceptive responses were  <dig>  ±  <dig>  s  at  <dig> min,  <dig>  ±  <dig>  s  at  <dig> min and  <dig>  ±  <dig>  s  at  <dig> min post-hfs .
frequencies of discharge were  <dig>  ±  <dig>  s pre-hfs, post-microinjection, and  <dig>  ±  <dig>   at  <dig> min,  <dig>  ±  <dig>   at  <dig> min and  <dig>  ±  <dig>   at  <dig> min post-hfs .
in the sulpiride-treated group, the durations and frequencies of pain responses showed no statistically significant differences.
d1r antagonist had no effect on nociceptive responses in the pfc
a d <dig> receptor  antagonist, sch <dig>  had no effect on the inhibitory effects of hfs .
durations of pain responses were  <dig>  ±  <dig>  s pre-microinjection and  <dig>  ±  <dig>  s post-microinjection.
there was no significant difference in nociceptive responses or background ecog between the two phases  .
after hfs had been delivered to the vta, the durations of nociceptive responses were  <dig>  ±  <dig>  s  at  <dig> min ,  <dig>  ±  <dig>  s  at  <dig> min and  <dig>  ±  <dig>  s  at  <dig> min post-hfs .
frequencies of discharge were  <dig>  ±  <dig>  pre-hfs, post-microinjection, and  <dig>  ±  <dig>   at  <dig> min ,  <dig>  ±  <dig>   at  <dig> min and  <dig>  ±  <dig>   at  <dig> min post-hfs .
d2r agonist decreased nociceptive responses in pfc
a d2r agonist, quinpirole, decreased nociceptive responses in the pfc  .
durations of pain responses were  <dig>  ±  <dig>  s pre-microinjection, and  <dig>  ±  <dig>  s  at  <dig> min ,  <dig>  ±  <dig>  s  at  <dig> min and  <dig>  ±  <dig>  s  at  <dig> min post-microinjection .
frequencies of discharge were  <dig>  ±  <dig>  pre-microinjection, and  <dig>  ±  <dig>   at  <dig> min ,  <dig>  ±  <dig>   at  <dig> min and  <dig>  ±  <dig>   at  <dig> min post-microinjection .
lls of nociceptive responses appeared within  <dig> min after microinjection.
nociceptive responses recovered to pre-microinjection levels in  <dig> min.
microinjection of a d2r agonist clearly inhibited nociceptive responses in the pfc.
κ-opiate receptor agonist microinjection into the vta
a κ-opiate receptor agonist, u <dig>  microinjected into the vta had no effect on nociceptive responses or background ecog in the pfc  ).
durations of pain responses were  <dig>  ±  <dig>  s pre-microinjection, and  <dig>  ±  <dig>  s  at  <dig> min,  <dig>  ±  <dig>  s  at  <dig> min and  <dig>  ±  <dig>  s  at  <dig> min post-microinjection ).
frequencies of discharge were  <dig>  ±  <dig>  pre-microinjection, and  <dig>  ±  <dig>   at  <dig> min,  <dig>  ±  <dig>   at  <dig> min and  <dig>  ±  <dig>   at  <dig> min post-microinjection ).
da depletion had no effect on nociceptive responses in the pfc
da depletion was established by apomorphine tests three weeks after 6-ohda injection into the medial forebrain bundle .
in apomorphine test-positive animals, background ecog showed low voltage fast waves, as described in a previous report  <cit> .
however, nociceptive responses were normally evoked by mechanical stimulation delivered to the tail.
there was no difference in mean nociceptive responses between the control  and 6-ohda groups   .
discussion
our study examined whether the mesocortical da system directly affects nociceptive responses in the pfc.
hfs delivered to the vta inhibited nociceptive responses recorded in the pfc.
basically, the da concentration in the pfc, which receives direct inputs from the vta  <cit> , is tonically maintained by activities of dopaminergic neurons in the vta.
hfs delivered to the vta  <cit>  and activation of vta neurons by n-methyl-d-aspartate   <cit>  increase the extracellular da concentration, thereby producing inhibitory effects on pfc neurons  <cit> .
higher da concentrations induced by hfs may produce inhibition of nociceptive responses in the pfc.
the inhibition, which was induced by a hfs of a moderate intensity, persisted for  <dig> min.
in pfc slice studies, bath application of da strongly modulated long-term depression of glutamatergic synapses  <cit> .
our previous in vivo study  <cit>  indicated that da modulated lls induced by glutamate receptor activation.
burst stimulation of vta increased da release in pfc, which induced plasticity of pfc neurons  <cit> .
da release by hfs delivered to vta may induce plastic changes in glutamatergic synapses to receive nociceptive inputs from peripheral tissue.
in contrast, da depletion induced by 6-ohda  <cit>  or a decrease in the da concentration induced by κ-opiate application  <cit>  had no effect on nociceptive responses in the pfc.
da depletion by 6-ohda increased the low amplitude and high frequency components in background ecog, as reported previously  <cit> .
decreased tonic da release suppressed d2-mediated activities followed by augmentation of pfc neuron activities  <cit> .
these reports indicate that a low level of da affects spontaneous activities of pfc neurons, but effects on evoked responses remain unclear.
our results and those of a previous report  <cit>  demonstrated a low da concentration in the pfc to impair plastic changes but not normal nociceptive responses.
in the rodent pfc, local application of da reportedly induced spike activities, while a d2r antagonist impaired suppression of spontaneous discharges  <cit> .
our results from hfs to the vta and local injection of a d2r agonist indicate d2r activation to be responsible for the lls of nociceptive responses induced by hfs of the vta.
a d1r antagonist, however, had minimal effects on nociceptive responses, as reported by godbout  <cit> .
da d1r and d2r are found on both pre- and post-synaptic pyramidal neurons in the pfc  <cit> .
d1r activating adenylate cyclase through interactions with g-proteins   <cit>  enhanced the nmda current  <cit> , which induced long-term potentiation  in the pfc  <cit> .
a question is why hfs did not evoke excitatory effects via d1rs activities.
more d1rs are expressed on gamma-aminobutyric acid  neurons than on pyramidal cells in the pfc  <cit> , suggesting that d1r activities evoked by hfs did not consistently induce excitatory responses.
another possibility is that da d1r produces an inverted-u shaped response, indicating a higher dose to be associated with somewhat lower performance  <cit> .
the da concentrations induced by hfs of the vta may evoke da d2- but not da d1-mediated activities in pfc neurons.
the pfc receives numerous projections from several areas involved in complex higher brain functions.
the medial pain pathways extend from the periphery to the pfc  <cit>  and direct projections from the vta terminate in the pfc.
both inputs converged in the same areas  where unit discharges were recorded in this study.
projections from the amygdala and hippocampus also terminate in the pfc and thereby change pain responses  <cit> .
these areas are related to emotion and memory, suggesting that the pfc unifies affectional information and pain.
according to human brain imaging analyses, the strength of conscious pain, which is related to psychological condition, reflects activities of the pfc  <cit> .
da modified synaptic plasticity  <cit>  and prolonged da-mediated modulation biases the long-term processing dynamics of pfc networks  <cit> .
CONCLUSIONS
mechanical noxious stimulation applied peripherally evoked nociceptive discharges in the pfc.
hfs delivered to the vta inhibited persistently nociceptive responses recorded in the pfc.
this long lasting inhibition was decreased by a da d2r antagonist.
in addition, a d2r agonist alone produced inhibition of nociceptive responses.
the da system  exerts modulatory effects on pain responses recorded in the pfc.
the mesocortical da system may affect complex pain cognition.
materials and methods
animal preparation
adult male wistar rats  were used in all experiments.
the rats were housed under controlled temperature  and humidity  conditions with a 12-h light/dark cycle, and had free access to food and water.
experiments conformed to guidelines issued by the national institutes of health for laboratory animals and all procedures were approved by the animal experimental committee of tokyo women’s medical university.
efforts were made to minimize the number of animals used and their suffering.
all rats were anesthetized with a single injection of urethane  and mounted in a stereotaxic instrument  for the acute experiments.
mechanical stimulation
mechanical pressure was applied to the tail  employing a mechanical stimulator , using a probe with a circular contact area with a  <dig> mm-in-diameter tip.
mechanical stimuli were delivered every  <dig> s at constant force with a feedback system.
stimulus intensity in this experiment was  <dig> gf with a  <dig>  s rising  time to maximum force and a  <dig> s hold time.
the nociceptive stimulus intensity of  <dig> gf has been shown to be adequate  <cit>  to induce c-fiber mediated activity in peripheral nerves  <cit> .
these mechanical stimulus conditions induced stabile nociceptive responses lasting more than two hours under the same anesthetic levels.
.
electrophysiological recording
recording tungsten needle microelectrodes  were stereotaxically positioned in the cingulate or prelimbic areas of the pfc.
stereotactic coordinates of the pfc were  <dig> - <dig>  mm anterior and  <dig> - <dig>  mm lateral to the bregma  <cit> .
the perpendicular depths of the recording sites were between  <dig>  and  <dig>  mm from the dorsal cortical surface ).
the unit spikes were processed with a multichannel amplifier  and an active filter , respectively.
through a memory oscilloscope , the data were fed into a thermal array recorder  for paper recording and a personal computer  via an integrated system  for recording storage and later off-line analysis.
the spontaneous background discharge patterns and ecog were recorded through the same electrodes.
anesthetic levels and nociceptive responses were determined with ecog.
we administered additional urethane when typical spindle bursts disappeared.
ecog changed from slow waves with spindle bursts to low amplitude fast waves induced by mechanical stimulation .
local application of drugs
a cannula  with a stainless recording electrode  was stereotaxically positioned in the left pfc or vta.
the cannula was connected by polyethylene tubing  to a  <dig> μl syringe .
the drug was microinjected by means of a syringe pump at a rate of  <dig> μl/min .
all drugs were dissolved in  <dig> % saline.
the following barrel concentrations were used:  <dig> μm/ <dig> μl sulpiride ,  <dig> μm/ <dig> μl quinpirole ,  <dig> nm/ <dig> μl sch <dig> ,  <dig> μm/ <dig> μl u- <dig> .
sulpiride, sch <dig> and quinpirole were locally injected into the pfc.
a κ-opiate receptor agonist, u- <dig>  was injected into the vta to decrease da concentrations  <cit> .
hfs applied to the vta
in these experiments, tungsten needle microelectrodes  were positioned in the vta.
stereotactic coordinates of the vta were  <dig> - <dig>  mm posterior and  <dig> - <dig>  mm lateral to the bregma.
the perpendicular depths of the recording sites were between  <dig>  and  <dig>  mm from the dorsal cortical surface ).
electrical hfs  was applied to the vta to increase da concentrations in the pfc  <cit> .
depletion of da
rats were anesthetized with nembutal .
then, five μl of 6-ohda hcl  were injected into the left mfb through a cannula with a microinjection pump at a rate of  <dig> μl/hr, and the cannula was left in place for  <dig> min after the completion of pumping.
stereotactic coordinates of the mfb were  <dig>  mm posterior and  <dig>  mm lateral to the bregma.
the perpendicular depths of the recording sites were  <dig>  mm from the dorsal cortical surface.
at the end of injection, the cannula was removed and the skull skin was sutured.
we assessed motor disturbance  <dig> weeks after 6-ohda injection by circling behavior  with apomorphine  administration.
the same acute experimental procedures as described above  were carried out after da depletion had been established.
nociceptive discharges were recorded in the pfc of the side ipsilateral to 6-ohda injection.
apomorphine induced rotational asymmetry was not observed in the intact group  serving as the control.
before the 6-ohda injections, we performed apomorphine tests  to avoid spontaneous da abnormalities.
unit recording locations
the locations of units were marked with a positive electric current lesion .
at the end of each experiment, the animals were perfused with normal saline and 4% paraformaldehyde.
after overnight post-fixation, the brains were sectioned  and stained with cresyl violet solution to examine the recording sites under light microscopy.
data analysis
a single unit spike was discriminated on the basis of the height and width of each unit from a multi-unit recording obtained  left) and the same single units were determined from clusterized discharges with pattern matching  right).
software labchart  <dig>   was used for unit discharge analysis.
the spike histogram was analyzed using single spikes during a 22s period ).
each bin of histograms consists of spikes during a  <dig> s period.
the durations of responses exceeding double the mean spontaneous discharges on the histogram were assessed as the response durations ).
significant differences in discharges evoked by mechanical stimuli were assessed with the nonparametric paired-test  to compare pre- and post- stimulation values.
data are expressed as means ± standard errors .
a probability level of < <dig>  was considered significant.
abbreviations
acc: anterior cingulate cortex; cg1: cingulate cortex area 1; d1r: d <dig> receptor; d2r: d <dig> receptor; da: dopamine; ecog: electrocorticography; gaba: gamma-aminobutyric acid; hfs: high frequency stimulation; il: infralimbic cortex; lls: long-lasting suppression; ltp: long-term potentiation; mfb: medial forebrain bundle; nmda: n-methyl-d-aspartate; 6-ohda: 6-hydroxydopamine; pd: parkinson disease, pfc, prefrontal cortex; prl: prelimbic cortex; sht: specific high threshold; vta: ventral tegmental area.
competing interests
none of the authors have any conflicts of interest to report.
authors’ contributions
ss, yy and ko carried out the experiments.
ss, yy and ko analyzed the experimental data.
ss wrote the manuscript.
yk conceived the study, designed the experiments and helped to draft the manuscript.
all authors have read and approved the final manuscript.
supplementary material
additional file 1
convergence of vta and medial pain pathways in the superficial layer of the pfc.
click here for file
acknowledgement
this work was supported by a grant-in aid scientific research .

 psychological conditions like attention and hypnotic effects exert powerful influences on human pain sensations. the anterior cingulate cortex has crucial roles in conscious perception of pain but not the sensory discriminative aspect of pain. noxious stimulation applied to peripheral tissues evoked nociceptive responses in rabbit and rodent cingulate and prelimbic areas, lesions of which impaired place avoidance test responses while pain behavior on the formalin test was normal. the human acc and the rodent prefrontal cortex receiving inputs from the amygdala are involved in emotional processing. in our previous study, nociceptive responses of the pfc were inhibited by inputs from the amygdala. we also found that a dopamine d receptor blocker impaired the inhibition induced by amygdala stimulation. direct dopaminergic projections from the ventral tegmental area to the pfc are responsible for modulatory effects on nociceptive responses, suggesting that the mesocortical da system modulates pain responses in the pfc. recent studies have explored how da modulates pain perception. systemic administration of da antagonists were reported to alter nociceptive responses. symptoms of pain in parkinson disease imply that da levels in the brain affect pain sensations. in clinical reports on pd, patients frequently complain of neuropathic or central pain before motor disorders. these clinical reports indicate that the brain da level modifies pain sensations. in animal studies, microinjections of da into the acc reduced autotomy scores in a sciatic neurotomy model. behavioral in vivo studies of pain have demonstrated that da depletion induces significant changes in thresholds for noxious stimuli. six-hydroxydopamine lesions in the vta induced hyperalgesia in acute and chronic pain models. we determined whether lower da concentration changes in the vta induced by 6-ohda lesions alter nociceptive responses in the pfc. we also examined effects of a κ-opiate receptor agonist injected into vta on nociceptive responses. dopaminergic projections from vta have two main target areas, pfc and the nucleus accumbens. a κ-opiate receptor agonist injected into vta inhibited only da neurons projecting to pfc and reduced da level in pfc. the rodent pfc receives da projections from the vta. the present study demonstrated vta-pfc projections to directly affect nociceptive responses recorded in the pfc. vta projections terminated at synapses within the soma, dendritic shafts, and spines of pyramidal cells in layers ii to v of the pfc, while nociceptive information was transmitted through the medial pain pathways from the periphery to the pfc. the crucial roles of da in pfc function were discussed in the review by seamans. the mesocortical da pathways from the vta have modifying effects on cognition in the pfc. the vta-pfc pathways may affect activities of the medial pain pathways, of which the pfc is the center. we analyzed the effects of dopaminergic inputs from the vta to the prelimbic and cingulate areas on nociceptive responses in the pfc. we also clarified which of the da receptor subtypes is related to the modulatory effects on nociceptive responses in the pfc.  mechanical noxious stimulation induced nociceptive responses in the pfc mechanical noxious stimulation induced excitatory responses in pfc neurons, which persisted during and frequently after stimulation. two types of nociceptive responses, a wide dynamic range type and a specific high threshold type, were recorded. in this study, we used sht neurons to monitor the response to noxious stimulation. the spontaneous background discharges of neurons, which usually showed the spindle bursts characteristic urethane anesthesia, continued for more than two hours, if the anesthetic level was maintained like we had previously reported. we administered additional urethane when typical spindle bursts disappeared. electrocorticography also showed typical high amplitude slow waves and was changed to low amplitude fast waves by mechanical stimulation. anesthetic levels were also detected with ecog patterns. the nociceptive responses were recorded without adaptation, if mechanical stimulation was applied every s. hfs delivered to the vta decreased nociceptive responses in the pfc high frequency stimulation delivered to the vta impaired pfc nociceptive responses evoked by mechanical stimulation applied to the rat tail. durations of nociceptive responses were ± s pre-hfs, and ± s at min, ± s at min and ± s at min post-hfs. frequencies of discharge were ± pre-hfs, and ± at min, ± at min and ± at min post-hfs. long-lasting suppression of nociceptive responses appeared within min and persisted for min after hfs. nociceptive responses recovered to pre-hfs levels in min. hfs delivered to the vta clearly inhibited nociceptive responses in the rat pfc. d2r antagonist blocked the depression of nociceptive responses a d2r antagonist, sulpiride, which had no effect on control nociceptive responses, significantly blocked the inhibitory effects of hfs. durations of pain responses were ± s pre-microinjection and ± s post-microinjection. there was no significant difference in nociceptive responses or background ecog between the two phases. after hfs had been delivered to the vta, the durations of nociceptive responses were ± s at min, ± s at min and ± s at min post-hfs. frequencies of discharge were ± s pre-hfs, post-microinjection, and ± at min, ± at min and ± at min post-hfs. in the sulpiride-treated group, the durations and frequencies of pain responses showed no statistically significant differences. d1r antagonist had no effect on nociceptive responses in the pfc a d receptor antagonist, sch had no effect on the inhibitory effects of hfs. durations of pain responses were ± s pre-microinjection and ± s post-microinjection. there was no significant difference in nociceptive responses or background ecog between the two phases. after hfs had been delivered to the vta, the durations of nociceptive responses were ± s at min, ± s at min and ± s at min post-hfs. frequencies of discharge were ± pre-hfs, post-microinjection, and ± at min, ± at min and ± at min post-hfs. d2r agonist decreased nociceptive responses in pfc a d2r agonist, quinpirole, decreased nociceptive responses in the pfc. durations of pain responses were ± s pre-microinjection, and ± s at min, ± s at min and ± s at min post-microinjection. frequencies of discharge were ± pre-microinjection, and ± at min, ± at min and ± at min post-microinjection. lls of nociceptive responses appeared within min after microinjection. nociceptive responses recovered to pre-microinjection levels in min. microinjection of a d2r agonist clearly inhibited nociceptive responses in the pfc. κ-opiate receptor agonist microinjection into the vta a κ-opiate receptor agonist, u microinjected into the vta had no effect on nociceptive responses or background ecog in the pfc ). durations of pain responses were ± s pre-microinjection, and ± s at min, ± s at min and ± s at min post-microinjection ). frequencies of discharge were ± pre-microinjection, and ± at min, ± at min and ± at min post-microinjection ). da depletion had no effect on nociceptive responses in the pfc da depletion was established by apomorphine tests three weeks after 6-ohda injection into the medial forebrain bundle. in apomorphine test-positive animals, background ecog showed low voltage fast waves, as described in a previous report. however, nociceptive responses were normally evoked by mechanical stimulation delivered to the tail. there was no difference in mean nociceptive responses between the control and 6-ohda groups. discussion our study examined whether the mesocortical da system directly affects nociceptive responses in the pfc. hfs delivered to the vta inhibited nociceptive responses recorded in the pfc. basically, the da concentration in the pfc, which receives direct inputs from the vta, is tonically maintained by activities of dopaminergic neurons in the vta. hfs delivered to the vta and activation of vta neurons by n-methyl-d-aspartate increase the extracellular da concentration, thereby producing inhibitory effects on pfc neurons. higher da concentrations induced by hfs may produce inhibition of nociceptive responses in the pfc. the inhibition, which was induced by a hfs of a moderate intensity, persisted for min. in pfc slice studies, bath application of da strongly modulated long-term depression of glutamatergic synapses. our previous in vivo study indicated that da modulated lls induced by glutamate receptor activation. burst stimulation of vta increased da release in pfc, which induced plasticity of pfc neurons. da release by hfs delivered to vta may induce plastic changes in glutamatergic synapses to receive nociceptive inputs from peripheral tissue. in contrast, da depletion induced by 6-ohda or a decrease in the da concentration induced by κ-opiate application had no effect on nociceptive responses in the pfc. da depletion by 6-ohda increased the low amplitude and high frequency components in background ecog, as reported previously. decreased tonic da release suppressed d2-mediated activities followed by augmentation of pfc neuron activities. these reports indicate that a low level of da affects spontaneous activities of pfc neurons, but effects on evoked responses remain unclear. our results and those of a previous report demonstrated a low da concentration in the pfc to impair plastic changes but not normal nociceptive responses. in the rodent pfc, local application of da reportedly induced spike activities, while a d2r antagonist impaired suppression of spontaneous discharges. our results from hfs to the vta and local injection of a d2r agonist indicate d2r activation to be responsible for the lls of nociceptive responses induced by hfs of the vta. a d1r antagonist, however, had minimal effects on nociceptive responses, as reported by godbout. da d1r and d2r are found on both pre- and post-synaptic pyramidal neurons in the pfc. d1r activating adenylate cyclase through interactions with g-proteins enhanced the nmda current, which induced long-term potentiation in the pfc. a question is why hfs did not evoke excitatory effects via d1rs activities. more d1rs are expressed on gamma-aminobutyric acid neurons than on pyramidal cells in the pfc, suggesting that d1r activities evoked by hfs did not consistently induce excitatory responses. another possibility is that da d1r produces an inverted-u shaped response, indicating a higher dose to be associated with somewhat lower performance. the da concentrations induced by hfs of the vta may evoke da d2- but not da d1-mediated activities in pfc neurons. the pfc receives numerous projections from several areas involved in complex higher brain functions. the medial pain pathways extend from the periphery to the pfc and direct projections from the vta terminate in the pfc. both inputs converged in the same areas where unit discharges were recorded in this study. projections from the amygdala and hippocampus also terminate in the pfc and thereby change pain responses. these areas are related to emotion and memory, suggesting that the pfc unifies affectional information and pain. according to human brain imaging analyses, the strength of conscious pain, which is related to psychological condition, reflects activities of the pfc. da modified synaptic plasticity and prolonged da-mediated modulation biases the long-term processing dynamics of pfc networks.  mechanical noxious stimulation applied peripherally evoked nociceptive discharges in the pfc. hfs delivered to the vta inhibited persistently nociceptive responses recorded in the pfc. this long lasting inhibition was decreased by a da d2r antagonist. in addition, a d2r agonist alone produced inhibition of nociceptive responses. the da system exerts modulatory effects on pain responses recorded in the pfc. the mesocortical da system may affect complex pain cognition. materials and methods animal preparation adult male wistar rats were used in all experiments. the rats were housed under controlled temperature and humidity conditions with a 12-h light/dark cycle, and had free access to food and water. experiments conformed to guidelines issued by the national institutes of health for laboratory animals and all procedures were approved by the animal experimental committee of tokyo women’s medical university. efforts were made to minimize the number of animals used and their suffering. all rats were anesthetized with a single injection of urethane and mounted in a stereotaxic instrument for the acute experiments. mechanical stimulation mechanical pressure was applied to the tail employing a mechanical stimulator, using a probe with a circular contact area with a mm-in-diameter tip. mechanical stimuli were delivered every s at constant force with a feedback system. stimulus intensity in this experiment was gf with a s rising time to maximum force and a s hold time. the nociceptive stimulus intensity of gf has been shown to be adequate to induce c-fiber mediated activity in peripheral nerves. these mechanical stimulus conditions induced stabile nociceptive responses lasting more than two hours under the same anesthetic levels.. electrophysiological recording recording tungsten needle microelectrodes were stereotaxically positioned in the cingulate or prelimbic areas of the pfc. stereotactic coordinates of the pfc were - mm anterior and - mm lateral to the bregma. the perpendicular depths of the recording sites were between and mm from the dorsal cortical surface ). the unit spikes were processed with a multichannel amplifier and an active filter, respectively. through a memory oscilloscope, the data were fed into a thermal array recorder for paper recording and a personal computer via an integrated system for recording storage and later off-line analysis. the spontaneous background discharge patterns and ecog were recorded through the same electrodes. anesthetic levels and nociceptive responses were determined with ecog. we administered additional urethane when typical spindle bursts disappeared. ecog changed from slow waves with spindle bursts to low amplitude fast waves induced by mechanical stimulation. local application of drugs a cannula with a stainless recording electrode was stereotaxically positioned in the left pfc or vta. the cannula was connected by polyethylene tubing to a μl syringe. the drug was microinjected by means of a syringe pump at a rate of μl/min. all drugs were dissolved in % saline. the following barrel concentrations were used: μm/ μl sulpiride, μm/ μl quinpirole, nm/ μl sch, μm/ μl u-. sulpiride, sch and quinpirole were locally injected into the pfc. a κ-opiate receptor agonist, u- was injected into the vta to decrease da concentrations. hfs applied to the vta in these experiments, tungsten needle microelectrodes were positioned in the vta. stereotactic coordinates of the vta were - mm posterior and - mm lateral to the bregma. the perpendicular depths of the recording sites were between and mm from the dorsal cortical surface ). electrical hfs was applied to the vta to increase da concentrations in the pfc. depletion of da rats were anesthetized with nembutal. then, five μl of 6-ohda hcl were injected into the left mfb through a cannula with a microinjection pump at a rate of μl/hr, and the cannula was left in place for min after the completion of pumping. stereotactic coordinates of the mfb were mm posterior and mm lateral to the bregma. the perpendicular depths of the recording sites were mm from the dorsal cortical surface. at the end of injection, the cannula was removed and the skull skin was sutured. we assessed motor disturbance weeks after 6-ohda injection by circling behavior with apomorphine administration. the same acute experimental procedures as described above were carried out after da depletion had been established. nociceptive discharges were recorded in the pfc of the side ipsilateral to 6-ohda injection. apomorphine induced rotational asymmetry was not observed in the intact group serving as the control. before the 6-ohda injections, we performed apomorphine tests to avoid spontaneous da abnormalities. unit recording locations the locations of units were marked with a positive electric current lesion. at the end of each experiment, the animals were perfused with normal saline and 4% paraformaldehyde. after overnight post-fixation, the brains were sectioned and stained with cresyl violet solution to examine the recording sites under light microscopy. data analysis a single unit spike was discriminated on the basis of the height and width of each unit from a multi-unit recording obtained left) and the same single units were determined from clusterized discharges with pattern matching right). software labchart was used for unit discharge analysis. the spike histogram was analyzed using single spikes during a 22s period ). each bin of histograms consists of spikes during a s period. the durations of responses exceeding double the mean spontaneous discharges on the histogram were assessed as the response durations ). significant differences in discharges evoked by mechanical stimuli were assessed with the nonparametric paired-test to compare pre- and post- stimulation values. data are expressed as means ± standard errors. a probability level of < was considered significant. abbreviations acc: anterior cingulate cortex; cg1: cingulate cortex area 1; d1r: d receptor; d2r: d receptor; da: dopamine; ecog: electrocorticography; gaba: gamma-aminobutyric acid; hfs: high frequency stimulation; il: infralimbic cortex; lls: long-lasting suppression; ltp: long-term potentiation; mfb: medial forebrain bundle; nmda: n-methyl-d-aspartate; 6-ohda: 6-hydroxydopamine; pd: parkinson disease, pfc, prefrontal cortex; prl: prelimbic cortex; sht: specific high threshold; vta: ventral tegmental area. competing interests none of the authors have any conflicts of interest to report. authors’ contributions ss, yy and ko carried out the experiments. ss, yy and ko analyzed the experimental data. ss wrote the manuscript. yk conceived the study, designed the experiments and helped to draft the manuscript. all authors have read and approved the final manuscript. supplementary material additional file 1 convergence of vta and medial pain pathways in the superficial layer of the pfc. click here for file acknowledgement this work was supported by a grant-in aid scientific research.
the mesocortical da system may modify pfc nociceptive responses via d activity. in this study, we examined whether direct projections from the ventral tegmental area to the pfc affect nociceptive responses in the pfc. in contrast, da depletion by a 6-hydroxydopamine injection or a low concentration of da induced by a κ-opiate receptor agonist injected into the vta had minimal effect on nociceptive responses in the pfc. high frequency stimulation delivered to the vta produced long-lasting suppression of nociceptive responses in the rat pfc including cingulate and prelimbic areas. the rodent prefrontal cortex including cingulate areas is also related to the affective dimension of pain. we previously reported pfc nociceptive responses inhibited by inputs from the amygdala, such as with dopamine d receptor blockers, to show decreased effect on amygdala projections. nociceptive responses evoked by mechanical pressure stimulation applied to the tails of urethane-anesthetized rats were recorded using extracellular unit recording methods in the pfc. hfs delivered to the vta, which has been reported to increase da concentrations in the pfc, significantly suppressed nociceptive responses. the lls of nociceptive responses persisted for about minutes and recovered to the control level within min after hfs. we also demonstrated local microinjection of a selective d agonist of da receptors to induce lls of mechanical nociceptive responses, while a d but not a d antagonist impaired the lls evoked by hfs. hfs delivered to vta inhibited nociceptive responses for a long period in pfc. da d2r activation mediated by local d agonist injection also induced lls of mechanical nociceptive responses. psychological conditions affect pain responses in the human anterior cingulate cortex according to brain imaging analysis.
 psychological conditions affect pain responses in the human anterior cingulate cortex according to brain imaging analysis. the rodent prefrontal cortex including cingulate areas is also related to the affective dimension of pain. we previously reported pfc nociceptive responses inhibited by inputs from the amygdala, such as with dopamine d receptor blockers, to show decreased effect on amygdala projections. in this study, we examined whether direct projections from the ventral tegmental area to the pfc affect nociceptive responses in the pfc.  high frequency stimulation delivered to the vta produced long-lasting suppression of nociceptive responses in the rat pfc including cingulate and prelimbic areas. nociceptive responses evoked by mechanical pressure stimulation applied to the tails of urethane-anesthetized rats were recorded using extracellular unit recording methods in the pfc. hfs delivered to the vta, which has been reported to increase da concentrations in the pfc, significantly suppressed nociceptive responses. the lls of nociceptive responses persisted for about minutes and recovered to the control level within min after hfs. we also demonstrated local microinjection of a selective d agonist of da receptors to induce lls of mechanical nociceptive responses, while a d but not a d antagonist impaired the lls evoked by hfs. in contrast, da depletion by a 6-hydroxydopamine injection or a low concentration of da induced by a κ-opiate receptor agonist injected into the vta had minimal effect on nociceptive responses in the pfc.  hfs delivered to vta inhibited nociceptive responses for a long period in pfc. da d2r activation mediated by local d agonist injection also induced lls of mechanical nociceptive responses. the mesocortical da system may modify pfc nociceptive responses via d activity. accpfcdopamined2rpainparkinson diseasevta 