Tonic pain time-dependently affects beta-endorphin-like immunoreactivity in the ventral periaqueductal gray matter of the rat brain

beta-Endorphin-like immunoreactivity (B-EP-LI) levels have been investigated in the ventral periaqueductal gray matter (vPAG) of rats killed 30, 60 or 120 min after the subcutaneous injection of dilute formalin (0.08 ml, 5%) in one fore- or hindpaw, or comparable handling. B-EP-LI was estimated by radioimmunoassay, using an anti-camel B-EP serum directed against the C-terminal portion of B-EP molecule. In both fore- and hindlimb groups vPAG B-EP-LI values were significantly increased 60 and 120 min after the injection relative to controls. Values from animals killed 120 min after formalin injection were higher than the ones at 30 and 60 min, forelimb effects being quantitatively more pronounced. The increase in B-EP-LI appeared distributed along the whole rostrocaudal extent of the region.     

Repeated electroconvulsive shock promotes the sprouting of serotonergic axons in the lesioned rat hippocampus

This study reports the effect of repeated electroconvulsive shock on the sprouting of 5-hydroxytryptamine neurons in the partly lesioned rat dorsal hippocampus. We have adopted a 5-hydroxytryptamine homotypic collateral sprouting model to examine whether electroconvulsive shock administration altered the rate of 5-hydroxytryptamine axonal reinnervation of the dorsal hippocampus. The 5-hydroxytryptamine innervation of hippocampus originates from the median raphe via the cingulum bundle and the fimbria-fornix. Lesioning of the cingulum bundle has previously been shown to cause sprouting of intact 5-hydroxytryptamine afferents originating from the unharmed fimbria-fornix. Rats were unilaterally injected with the 5-hydroxytryptamine neurotoxin, 5,7-dihydroxytryptamine, into the right cingulum bundle and 5-hydroxytryptamine immunoreactivity in the dorsal hippocampus was investigated 1, 3, 6 and 12weeks after the injection. The lowest level of 5-hydroxytryptamine-immunoreactivity in the hippocampus was detected at three weeks after the lesion. At six weeks, 5-hydroxytryptamine immunoreactive fibres started to reappear, and at 12weeks the level of 5-hydroxytryptamine immunoreactivity was similar to that observed on the unlesioned side. Based on this time-course, six weeks was chosen as the time-point to investigate the action of a course of repeated electroconvulsive shock administrations. Repeated electroconvulsive shock (five shocks over 10days) doubled the number of sprouting 5-hydroxytryptamine-immunoreactive fibres and significantly increased levels of the 5-hydroxytryptamine metabolite, 5-hydroxyindoleacetic acid.The present data provide the first direct evidence that electroconvulsive shock enhances 5-hydroxytryptamine axon sprouting in the partly lesioned hippocampus. This is an effect which may contribute to the therapeutic effect of electroconvulsive therapy in major depression.     

Efferent projections of the periaqueductal gray in the rabbit.

The efferent projections of the periaqueductal gray in the rabbit have been described by anterograde tract-tracing techniques following deposits of tritiated leucine, or horseradish peroxidase, into circumscribed sites within dorsal, lateral or ventral periaqueductal gray. No attempts were made to place labels in the fourth, extremely narrow (medial), region immediately surrounding the aqueduct whose size and disposition did not lend itself to confined placements of label within it. These anatomically distinct regions, defined in Nissl-stained sections, corresponded to the same regions into which deposits of horseradish peroxidase were made in order for us to describe afferent projections to the periaqueductal gray. In this present study distinct ascending and descending fibre projections were found throughout the brain. Terminal labelling was detected in more than 80 sites, depending somewhat upon which of the three regions of the periaqueductal gray received the deposit. Therefore, differential projections with respect to both afferent and efferent connections of these three regions of the periaqueductal gray have now been established. Ventral deposits disclosed a more impressive system of ramifying, efferent fibres than did dorsal or lateral placements of labels. With ventral deposits, ascending fibres were found to follow two major pathways from periaqueductal gray. The periventricular bundle bifurcates at the level of the posterior commissure to form hypothalamic and thalamic components which distribute to the anterior pretectal region, lateral habenulae, and nuclei of the posterior commissure, the majority of the intralaminar and midline thalamic nuclei, and to almost all of the hypothalamus. The other major ascending pathway from the periaqueductal gray takes a ventrolateral course from the deposit site through the reticular formation or, alternatively, through the deep and middle layers of the superior colliculus, to accumulate just medial to the medial geniculate body. This contingent of fibres travels more rostrally above the cerebral peduncle, distributing terminals to the substantia nigra, ventral tegmental area and parabigeminal nucleus before fanning out and turning rostrally to contribute terminals to ventral thalamus, subthalamus and zona incerta, then continuing on to supply amygdala, substantia innominata, lateral preoptic nucleus, the diagonal band of Broca and the lateral septal nucleus. Caudally directed fibres were also observed to follow two major routes. They either leave the periaqueductal gray dorsally and pass through the gray matter in the floor of the fourth ventricle towards the abducens nucleus and ventral medulla, or are directed ventrally after passing through either the inferior colliculus or parabrachial nucleus. These ventrally directed fibres merge just dorsal to the pons on the ventral surface of the brain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tolerance to the antinociceptive effect of morphine microinjections into the ventral but not lateral-dorsal periaqueductal gray of the rat.

Tolerance to the antinociceptive effect of morphine is mediated at least in part by morphine's action within the periaqueductal gray (PAG). The objective of the present study was to determine whether both ventral and lateral-dorsal PAG regions contribute to the development of tolerance. It was found that the antinociceptive efficacy of microinjecting morphine (5 microg/0.4 microl) into the ventral but not the lateral-dorsal PAG diminished with successive injections. Control experiments indicated that this decrease was caused by tolerance to morphine and was not a result of cell death caused by repeated microinjections or habituation from repeated behavioral testing. The finding of greater susceptibility of the ventral compared with the lateral-dorsal PAG to the development of tolerance adds to a growing literature distinguishing antinociception from these two regions.     

Midbrain periaqueductal gray and vocal patterning in a teleost fish

Midbrain structures, including the periaqueductal gray (PAG), are essential nodes in vertebrate motor circuits controlling a broad range of behaviors, from locomotion to complex social behaviors such as vocalization. Few single-unit recording studies, so far all in mammals, have investigated the PAG's role in the temporal patterning of these behaviors. Midshipman fish use vocalization to signal social intent in territorial and courtship interactions. Evidence has implicated a region of their midbrain, located in a similar position as the mammalian PAG, in call production. Here, extracellular single-unit recordings of PAG neuronal activity were made during forebrain-evoked fictive vocalizations that mimic natural call types and reflect the rhythmic output of a known hindbrain-spinal pattern generator. The activity patterns of vocally active PAG neurons were mostly correlated with features related to fictive call initiation. However, spike trains in a subset of neurons predicted the duration of vocal output. Duration is the primary feature distinguishing call types used in different social contexts and these cells may play a role in directly establishing this temporal dimension of vocalization. Reversible, lidocaine inactivation experiments demonstrated the necessity of the midshipman PAG for fictive vocalization, whereas tract-tracing studies revealed the PAG's connectivity to vocal motor centers in the fore- and hindbrain comparable to that in mammals. Together, these data support the hypotheses that the midbrain PAG of teleosts plays an essential role in vocalization and is convergent in both its functional and structural organization to the PAG of mammals.     

Lidocaine blockade of amygdala output in fear-conditioned rats reduces Fos expression in the ventrolateral periaqueductal gray

We showed recently that conditioned fear to context induces Fos expression in the ventrolateral periaqueductal gray [Neuroscience (1997) 78, 165-177]. Neurons in this region are thought to play an important role in the expression of freezing during conditioned fear. To test the possibility that this activation comes directly from the amygdala, we looked at changes in Fos expression after a unilateral blockade of the ventral amygdalofugal pathway with lidocaine. The pathway contains fibres originating from the central nucleus of the amygdala that project directly and mainly ipsilaterally to the ventrolateral periaqueductal gray. Conditioned fear was evoked by re-exposing rats to the same box in which they had previously received electric footshocks. The test re-exposure was preceded by a unilateral microinjection of lidocaine (2%, 0.5-1 microl; n = 20) or saline (n = 14). Lidocaine was also tested in non-conditioned animals (n = 13). The results show that, when lidocaine was microinjected in the medial part of the central nucleus of the amygdala or along the ventral amygdalofugal pathway of conditioned rats, fear-induced Fos expression in the ventrolateral periaqueductal gray was reduced on the side ipsilateral to the injection (up to 37% reduction in comparison to the contralateral side). Ipsilateral reductions were also observed with saline, but they were weaker (maximum of 27% reduction). Fos expression remained low on both sides in the non-fear-conditioned animals injected with lidocaine. Finally, although freezing was only partly reduced in the conditioned animals unilaterally injected with lidocaine, it was significantly correlated to the ipsilateral reduction in Fos expression. This study provides direct evidence that the projection from the central nucleus of the amygdala to the ventrolateral periaqueductal gray is activated during fear and that it contributes to the Fos response of the ventrolateral periaqueductal gray.     

Lidocaine blockade of amygdala output in fear-conditioned rats reduces Fos expression in the ventrolateral periaqueductal gray

We showed recently that conditioned fear to context induces Fos expression in the ventrolateral periaqueductal gray [Neuroscience (1997) 78, 165–177]. Neurons in this region are thought to play an important role in the expression of freezing during conditioned fear. To test the possibility that this activation comes directly from the amygdala, we looked at changes in Fos expression after a unilateral blockade of the ventral amygdalofugal pathway with lidocaine. The pathway contains fibres originating from the central nucleus of the amygdala that project directly and mainly ipsilaterally to the ventrolateral periaqueductal gray. Conditioned fear was evoked by re-exposing rats to the same box in which they had previously received electric footshocks. The test re-exposure was preceded by a unilateral microinjection of lidocaine (2%, 0.5–1 μl; n=20) or saline (n=14). Lidocaine was also tested in non-conditioned animals (n=13). The results show that, when lidocaine was microinjected in the medial part of the central nucleus of the amygdala or along the ventral amygdalofugal pathway of conditioned rats, fear-induced Fos expression in the ventrolateral periaqueductal gray was reduced on the side ipsilateral to the injection (up to 37% reduction in comparison to the contralateral side). Ipsilateral reductions were also observed with saline, but they were weaker (maximum of 27% reduction). Fos expression remained low on both sides in the non-fear-conditioned animals injected with lidocaine. Finally, although freezing was only partly reduced in the conditioned animals unilaterally injected with lidocaine, it was significantly correlated to the ipsilateral reduction in Fos expression.This study provides direct evidence that the projection from the central nucleus of the amygdala to the ventrolateral periaqueductal gray is activated during fear and that it contributes to the Fos response of the ventrolateral periaqueductal gray.     

Connections of midbrain periaqueductal gray in the monkey. I. Ascending efferent projections

1. To obtain a comprehensive map of the ascending projection of the midbrain periaqueductal gray, small amounts of [3H]leucine were injected into discrete regions of the periaqueductal gray (PAG) of the monkey. 2. Despite the fact that different regions of the PAG were injected in separate animals, the majority of the PAG's efferent projections to the forebrain remained constant. 3. The diencephalic projection areas include the nucleus reticularis thalami, the nucleus medialis dorsalis, the midline thalamic nuclei, the intralaminar thalamic nuclei, the preoptic area, and the anterior, dorsal, periventricular, ventromedial, periarcurate, lateral, and posterior hypothalamic nuclei. 4. More caudal areas receiving a projection from the PAG include the zona incerta and the mesencephalic reticular formation. 5. The main route for PAG-diencephalic projections is through the periventricular bundle. This implies that if a stimulating electrode was placed in this region, both the diencephalic leads to PAG and PAG leads to diencephalic pathways would be stimulated. This observation may explain the wide variety of side effects often encountered in stimulation-produced analgesia in humans. 6. These results support our hypothesis that the PAG functions as a visceral, nociceptive, and cognitive integrator.     

Repeated electroconvulsive shocks cause transient changes in rat hippocampal somatostatin and neuropeptide Y immunoreactivity and mRNA in situ hybridization signals

Increased levels of somatostatin (SS) and neuropeptide Y (NPY) have been demonstrated in the hippocampal formation after kindling. The increase might be specifically associated with kindling, or be an effect of repeated seizures per se. In order to separate these two components we studied the effects of repeated electroconvulsive shocks (ECS) on hippocampal SS-like and NPY-like immunoreactivity and SS mRNA and NPY mRNA in situ hybridization. ECS elicit seizures without having a demonstrable kindling effect. Rats were subjected to 10, 20, or 36 ECS (50 mA, 0.5 s), given as one shock per day, 5 days per week. One, 2 and 30 days after the last ECS, the rats were killed, together with sham-treated control rats, and processed for immunocytochemistry and non-radioactive in situ hybridization. There was a bilateral increase in SS-like and NPY-like immunoreactivity 1 and 2 days after the last ECS in the outer part of the dentate molecular layer. This is the terminal field of the hilar SS-containing and NPY-containing neurons, which displayed both increased immunoreactivity and hybridization signal of the cell bodies. There was also a bilateral de novo expression of NPY-like immunoreactivity in the mossy fiber system, but this was not accompanied by the appearance of a detectable NPY hybridization signal over the parent dentate granule cell bodies. The increase in SS-like immunoreactivity and hybridization signal was most pronounced in the rats that had received the largest number of ECS. This was not observed for the NPY-like immunoreactivity and hybridization signal, where the increase appeared similar after 10, 20 and 36 ECS. One month after the last ECS, both the SS-like and NPY-like immunoreactivity and the in situ hybridization signals had decreased towards normal levels. Since increased SS and NPY levels are also induced by repeated ECS, these changes are accordingly not specific to kindling-induced seizures. In a second experiment, the perforant path to the fascia dentata was transected 1 month prior to the ECS treatment. Removal of such major afferent input did not abolish the ECS-induced increase in hippocampal SS-like and NPY-like immunoreactivity, suggesting that the neuropeptide changes were not caused by afferent stimulation via the perfant path fibers, but rather may be an effect of direct electrical activation of the relevant cells.     

Low dose of morphine microinjected in the ventral periaqueductal gray matter of the rat depresses responses of nociceptive ventrobasal thalamic neurons

The effect of low doses of morphine (2 micrograms in 0.2 microliter) microinjected in the periaqueductal gray matter (PAG) was studied on responses of 11 nociceptive (N) and 3 non-nociceptive (Nn) ventrobasal (VB) thalamic neurons in lightly anesthetized rats. Responses of Nn neurons were unmodified but their receptive field was consistently enlarged during 50-60 min. Responses of N neurons to calibrated pinches were strongly depressed when the microinjection site was located in the PAG or in the dorsal raphe (NDR) (7 cases), and not significantly changed when morphine was applied in the dorsal PAG. For the 7 depressed neurons, the mean value of the responses expressed as a percentage of the control was 20.12 +/- 5.17, 15 min after morphine application. This depressive effect was variably reversed by naloxone (0.1-0.5 mg/kg).     

Afferents to the periaqueductal gray in the rat. A horseradish peroxidase study

Afferent projections to the periaqueductal gray matter in the rat have been studied by use of the retrograde axonal transport of horseradish peroxidase. Iontophoretic injections of horseradish peroxidase were made in dorsal, lateral and medial areas of the periaqueductal gray, primarily at intercollicular levels. The pattern of projections was similar in all of the injections restricted to the periaqueductal gray. Within the brainstem, numerous reticular formation nuclei were labeled, including nucleus reticularis lateralis, nucleus raphe magnus, pallidus and obscurus, the nucleus reticularis pontis oralis and caudalis, the paralemniscal nucleus and the dorsal and ventral parabrachial nuclei. At diencephalic levels, dense projections were seen from the parafascicular nucleus, dorsal premamillary nucleus, zona incerta, dorsomedial and ventromedial nuclei of the hypothalamus and the retrochiasmatic area, in the ventral portion of the anterior hypothalamus. At forebrain levels, occasional cells were seen in the medial preoptic area, lateral septum and the anterior cingulate cortex. Control injections of horseradish peroxidase into structures adjacent to the periaqueductal gray matter included three well localized deposits in the dorsal raphe. Retrogradely-labeled cells were found in lateral reticular nucleus of the medulla, nucleus raphe magnus, nucleus reticularis pontis caudalis, locus ceruleus, dorsal and ventral parabrachial nuclei, substantia nigra and the lateral hypothalamus. No labeled cells were found in the habenular nuclei. It is suggested that many of the descending hypothalamic and forebrain afferents may be relay centers for descending hippocampal formation efferents. Many of the periaqueductal gray afferent systems receive a direct projection from the hippocampal formation and could therefore coordinate influences from this limbic center with information on homeostatic mechanisms controlled by the hypothalamus. The numerous brainstem afferents to the periaqueductal gray could be involved in relay of ascending sensory information important for initiating any of several behavioral responses known to be controlled by the periaqueductal gray. In addition, certain raphe afferents might play a part in a feedback loop of the pain suppression circuit of which the periaqueductal gray is an important component.     

Repeated immobilization stress increases uncoupling protein 1 expression and activity in Wistar rats

Repeat immobilization-stressed rats are leaner and have improved cold tolerance due to enhancement of brown adipose tissue (BAT) thermogenesis. This process likely involves stress-induced sympathetic nervous system activation and adrenocortical hormone release, which dynamically enhances and suppresses uncoupling protein 1 (UCP1) function, respectively. To investigate whether repeated immobilization influences UCP1 thermogenic properties, we assessed UCP1 mRNA, protein expression, and activity (GDP binding) in BAT from immobilization-naive or repeatedly immobilized rats (3 h daily for 4 weeks) and sham operated or adrenalectomized (ADX) rats. UCP1 properties were assessed before (basal) and after exposure to 3 h of acute immobilization. Basal levels of GDP binding and UCP1 expression was significantly increased (140 and 140%) in the repeated immobilized group. Acute immobilization increased GDP binding in both naive (180%) and repeated immobilized groups (220%) without changing UCP1 expression. In ADX rats, basal GDP binding and UCP1 gene expression significantly increased (140 and 110%), and acute immobilization induced further increase. These data demonstrate that repeated immobilization resulted in enhanced UCP1 function, suggesting that enhanced BAT thermogenesis contributes to lower body weight gain through excess energy loss and an improved ability to maintain body temperature during cold exposure.     

Repeated methamphetamine treatment increases expression of TRPV1 mRNA in the frontal cortex but not in the striatum or hippocampus of mice

The transient receptor potential vanilloid type 1 (TRPV1) is a non-selective ligand-gated cationic channel. The distribution of TRPV1 mRNA in various regions of the brain has been successfully established. Methamphetamine (MAP) is a psychostimulant and a major drug of abuse in many parts of the world. The powerful rewarding properties of MAP are attributed to multiple pharmacological actions, but the mechanistic association between TRPV1 expression and MAP-induced drug addiction has not established. In the present study, we conducted a time-course analysis of TRPV1 mRNA levels in the frontal cortex, striatum, and hippocampus of mouse brain following repeated MAP (2 mg/kg, i.p.) treatment. Our results demonstrate that expression of TRPV1 mRNA is significantly increased 1, 2, 6, 24, 48 h, and 1 week after the last MAP treatment in the frontal cortex but not in the striatum or hippocampus. These data support a potential role for TRPV1 in the treatment of MAP-induced drug addiction.     

Fluoxetine attenuates the effects of pentylenetetrazol on rat freezing behavior and c-Fos expression in the dorsomedial periaqueductal gray

The aim of the study was to investigate the role of the periaqueductal gray (PAG) in anxiolytic-like actions of fluoxetine in animals treated with an anxiogenic drug, pentylenetetrazol (PTZ), and subjected to fear conditioning procedure. The data showed that PTZ given at the dose of 30 mg/kg 15 min before a retention trial significantly decreased freezing reaction (p<0.01), and potently enhanced rat locomotor activity (p<0.01), in comparison to the control group. These effects were reversed by prior (60 min) administration of fluoxetine (20 mg/kg). Simultaneously, PTZ significantly increased c-Fos expression in the dorsomedial periaqueductal gray (DMPAG), examined 2h after the retention trial, in comparison to the control group (p<0.01). Fluoxetine (20 mg/kg) administered 60 min before PTZ reversed this effect. PTZ given at the same dose and time interval in the open field test did not affect rat locomotor behavior. Importantly, fluoxetine pretreatment did not change PTZ concentration in brain tissue. Our experiment based on PTZ-enhanced aversive conditioning revealed that acutely administered fluoxetine antagonized PTZ-induced panic-like behavior, and this phenomenon was accompanied by inhibition of activity of DMPAG.     

Repeated treatment with amitriptyline or electroconvulsive shock does not affect thyrotropin releasing hormone receptors in discrete rat brain structures

We studied the effect of repeated treatment with amitriptyline (10 mg/kg, p.o., twice daily for 14 days) or electroconvulsive shock (ECS) (once daily for 10 days) on the thyrotropin-releasing hormone (TRH) content and TRH receptors in the cerebral cortex, nucleus accumbens, striatum and septum of the rat. Repeated amitriptyline did not significantly affect the density or affinity of TRH receptors in the examined structures, but caused a marked increase in the TRH content in the striatum and nucleus accumbens. Long-term treatment with ECS reduced the density and affinity of TRH receptors in the septum only, but it increased the TRH concentration in the cerebral cortex and striatum. These results, together with the literature data, indicate that there is no simple relationship between the brain content (and release) of TRH and the functional sensitivity of TRH receptors on one hand, and the density of these receptors on the other.     

Glutamate decarboxylase-immunoreactive neurons and terminals in the periaqueductal gray of the rat

The periaqueductal gray of 5 rats was processed for immunocytochemistry using an antiserum to glutamate decarboxylase. In both colchicine-pretreated (4 rats) and untreated (1 rat) animals, glutamate decarboxylase-positive cell bodies were present in all periaqueductal gray subdivisions, especially in the dorsal and ventrolateral subdivision. The perikaryal cross-sectional area of labelled neurons was smaller than that of periaqueductal gray projecting neurons retrogradely labelled with horseradish peroxidase in separate experiments. The morphology of glutamate decarboxylase-containing neurons resembled that of small polygonal, triangular and fusiform cells described in previous Golgi studies. Glutamate decarboxylase immunoreactivity was also observed in a large number of terminal-like structures, most of which were distributed close to the somata and dendrites of both glutamate decarboxylate-positive and -negative neurons. At all rostrocaudal levels the highest concentration of these elements was observed around the aqueduct. These results suggest that two sub-populations of neurons are present in the periaqueductal gray of rats, one consisting of small-sized glutamate decarboxylase-positive neurons (intrinsic neurons) and the other of large-sized glutamate decarboxylase-negative neurons (projecting neurons). Intrinsic circuits could be present between glutamate decarboxylase-positive and -negative neurons and between glutamate decarboxylase-positive neurons.     

Enkephalinergic involvement in periaqueductal gray control of hypothalamically elicited predatory attack in the cat.

The effects of central infusion of naloxone into the midbrain periaqueductal gray (PAG) upon predatory attack behavior in the cat were studied in 12 cats. Initially, quiet biting attack was elicited by electrical stimulation of sites within the lateral hypothalamus using monopolar electrodes. Then cannula-electrodes were implanted into sites within the PAG from which electrical stimulation facilitated or suppressed the attack response. Following identification of modulatory sites within the PAG, naloxone (1.0 micrograms/0.5 microliter) was microinjected into those sites and the effects upon hypothalamically elicited attack were assessed. At nine of twelve sites in the PAG where suppression was obtained, administration of naloxone served to block those effects. Similarly, at six of eight facilitatory sites within the PAG, naloxone also blocked the modulatory effects of PAG stimulation. However, vehicle (isotonic saline) alone did not alter the modulatory effects of PAG stimulation. Administration of DAME (250 ng/0.3 microliter) into PAG modulatory sites in four cats, two which facilitated and two that suppressed the attack response, reversed the effects of naloxone at these sites. These results demonstrate that opioid peptides within the PAG play a complex role in the expression of predatory attack behavior in the cat.     

Differential expression of NMDA receptors in serotonergic and/or GABAergic neurons in the midbrain periaqueductal gray of the mouse

N-methyl-d-aspartate (NMDA) receptors expressed in the midbrain periaqueductal gray (PAG) exert various physiological functions. The PAG contains various neurotransmitter phenotypes, which include GABAergic neurons and serotonergic neurons. In the present experiments, we made tight-seal whole-cell recordings from GABAergic and/or serotonergic neurons in mouse PAG slices and analyzed NMDA and non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation. The NMDA/non-NMDA ratio of EPSC amplitude was high and the decay time course of NMDA-EPSC was slow in non-serotonergic/GABAergic neurons. In contrast, serotonergic neurons exhibited a low NMDA/non-NMDA ratio and a fast decay time course of NMDA-EPSC. Peripheral nerve ligation-induced chronic pain was associated with an increased NMDA/non-NMDA ratio in serotonergic neurons. Additionally, single-cell real-time RT-PCR analysis showed that peripheral nerve ligation up-regulated NR2B subunit expression in non-serotonergic/non-GABAergic neurons. Such changes in NMDA receptor expression in the PAG result in an alteration of the descending modulation of nociception, which might be an underlying mechanism for peripheral nerve injury-evoked persistent pain. Finally, the expression of NMDA receptors seems differentially regulated among neurons of different neurotransmitter phenotypes in the PAG.