The combination of NMDA antagonism and morphine produces profound antinociception in the rat dorsal horn.

Dorsal horn nociceptive neurones exhibit wind up, a frequency dependent potentiation of their responses to repeated C-fibre stimulation. Intrathecal morphine (5 micrograms) significantly reduces the initial responses of the neurones but not wind up whereas the reverse is true for N-methyl-D-aspartate (NMDA) antagonists. The combination of intrathecal morphine (5 micrograms) and 7-chlorokynurenate (2.5 micrograms), an antagonist at the glycine site of the NMDA receptor, abolishes both the input and wind up of these neurones.     

Measurement of cholecystokinin release in vivo in the rat spinal dorsal horn.

The microdialysis technique, used to monitor extracellular levels of transmitter substances in the central nervous system of laboratory animals as a reflection of transmitter release, is based on the ability of neurotransmitters to diffuse in the extracellular fluid from the site of release and to cross a semipermeable dialysis membrane. Even though the surgical procedure is not very complicated, the detection of released substances in the recovered dialysate may be difficult. Especially, the measurement of neuropeptide release is limited by the low extracellular concentration and of low recovery as compared to, for example, monoamines. Thus, for example, cholecystokinin (CCK), which is the most abundant neuropeptide in the central nervous system, is found at concentrations that are several orders of magnitude lower than those of classical transmitters. Therefore a highly sensitive detection method is of utmost importance. In the dorsal horn of the spinal cord CCK is found mainly in interneurons and in terminals of descending fibers. CCK seems to be involved in nociceptive transmission and CCK attenuates morphine-induced antinociception. We here describe in vivo microdialysis in the lumbar dorsal horn of the rat with subsequent quantification of the level of CCK-like immunoreactivity (-LI) by a highly sensitive radioimmunoassay.     

Axon terminals possessing the alpha 2c-adrenergic receptor in the rat dorsal horn are predominantly excitatory

In this study we used confocal microscopy to show that most (83.67%) alpha(2C)-adrenergic receptor (alpha(2C)-AR)-immunoreactive terminals in the superficial dorsal horn contain the vesicular glutamate transporter 2 and hence are glutamatergic. Few (11.33%) terminals contain glutamic acid decarboxylase (a marker for GABAergic axons) and none were associated with the B subunit of cholera toxin (a marker for myelinated primary afferents) or the vesicular glutamate transporter 1. These data indicate that most dorsal horn axons possessing the alpha(2C)-AR are excitatory and add further support to the suggestion that they originate principally from spinal interneurons.     

Recording of long-term potentiation in single dorsal horn neurons in vivo in the rat.

We have published several reports on long-term potentiation (LTP) in single spinal wide dynamic range (WDR) neurons (responding to both innocuous and noxious stimuli) in urethane-anaesthetised rats. The protocol presented here, with single unit recordings of dorsal horn neurons before and after a nociceptive conditioning stimulation, may be useful in many electrophysiological studies of plastic changes in the spinal cord, such as LTP. We invite others to use this protocol for the study of spinal plasticity. Findings using this technique may be relevant for the understanding of changes in nociceptive transmission, induction of central sensitisation and maybe even in mechanisms of pathological pain and chronic pain states. We describe modified and alternative protocols for the study of LTP mechanisms under different conditions in intact and in spinalised animals, and after natural noxious stimuli. We present a novel method minimising peripheral influence of afferent input induced by antidromic neurogenic inflammation or inflammatory changes following a natural noxious stimulation. This is made possible by dissection of the sciatic nerve at two separate locations and local anaesthetic block distal to the stimulation site.     

Distribution of alpha 2-adrenergic receptor mRNAs in the rat CNS.

alpha 2-Adrenergic receptors (ARs) are involved in central nervous system (CNS) control of blood pressure. It is now known that there are three human genes that encode subtypes of alpha 2-ARs, but little is known regarding the distribution of these subtypes throughout the CNS. The availability of receptor clones allows the mapping of mRNAs encoding the individual alpha 2-AR subtypes in the CNS. In this communication, we report that there are three, closely related rat alpha 2-AR genes. We have developed subtype-specific hybridization probes from each of these genes and have used these reagents to measure alpha 2-AR subtype mRNA accumulation in extracts of discrete regions of the rat CNS. We found that mRNAs encoding the alpha 2A-AR and alpha 2C-AR subtypes are distributed widely, but unevenly, throughout the rat CNS. The A subtype is prominent in the midbrain, brainstem, spinal cord, pituitary and diencephalon while the C subtype predominates in basal ganglia and cerebellum. The cortex, olfactory bulb and hippocampus contain roughly equal amounts of the alpha 2A- and alpha 2C-AR mRNAs. A third subtype's (alpha 2B-AR) mRNA is far less abundant in brain tissues, and is only found in the diencephalon.     

Electrophysiological characterization of adrenoceptors in the rat dorsal hippocampus. III. Evidence for the physiological role of terminal alpha 2-adrenergic autoreceptors

The present electrophysiological studies were undertaken to assess the role of terminal alpha 2-adrenergic autoreceptors in regulating noradrenergic synaptic transmission in the rat CNS. The effectiveness of the electrical stimulation of the locus coeruleus (LC) in suppressing the firing activity of pyramidal neurons was determined in the dorsal hippocampus. Intravenous clonidine, an alpha 2-adrenergic agonist, decreased the effectiveness of the LC stimulation, without altering the effect of microiontophoretically applied norepinephrine. The subsequent i.v. administration of low doses of idazoxan, an alpha 2-adrenergic antagonist, reversed this effect of clonidine on the LC stimulation. To ascertain that the effect of clonidine administered i.v. was indeed attributable to its action on noradrenergic terminals, it was applied locally by microiontophoresis; it decreased the effectiveness of the LC stimulation. Another paradigm used to assess the function of terminal alpha 2-adrenoceptors was to increase the frequency of the LC stimulation from 1 to 5 Hz. This resulted in a 5-fold decrease of the effectiveness of the stimulation. That this was attributable to an enhanced activation of terminal alpha 2-adrenoceptors was suggested by the reversal of this effect of increasing the frequency of the LC stimulation by intravenous idazoxan. Furthermore, the degree of enhancement of the effectiveness of the LC stimulation by idazoxan was much greater at 5 than at 1 Hz. These results provide novel electrophysiological evidence for the potent regulatory role of terminal alpha 2-adrenoceptors on noradrenergic neurotransmission.     

Developmentally different onset of alpha 1- and alpha 2-adrenergic responses in the neonatal rat dorsal motor nucleus of the vagus in vitro

Development of noradrenergic responses in the rat dorsal motor nucleus of the vagus (DMV) from postnatal days 0 to 22 was studied by a conventional microelectrode technique using brain slice preparations. Between postnatal days 0 and 6, noradrenaline (NA) caused only alpha 1-adrenoceptor-mediated depolarizations. By postnatal day 14, alpha 2-adrenoceptor-mediated hyperpolarization appeared. During the next postnatal week, the response pattern to NA became similar to that in adults, including both alpha 1- and alpha 2-adrenergic responses. These results suggest that alpha 2-adrenergic responses in DMV neurons are absent at birth but develop rapidly in the 3 weeks after birth. Furthermore, alpha 1-adrenoceptors have already been functioning in the fetal period.     

Long-range oscillatory Ca2+ waves in rat spinal dorsal horn

Synchronous activity of large populations of neurons shapes neuronal networks during development. However, re-emergence of such activity at later stages of development could severely disrupt the orderly processing of sensory information, e.g. in the spinal dorsal horn. We used Ca2+ imaging in spinal cord slices of neonatal and young rats to assess under which conditions synchronous activity occurs in dorsal horn. No spontaneous synchronous Ca2+ transients were detected. However, increasing neuronal excitability by application of 4-aminopyridine after pretreatment of the slice with blockers of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, gamma-aminobutyric acid (GABA)(A) and glycine receptors evoked repetitive Ca2+ waves in dorsal horn. These waves spread mediolaterally with a speed of 1.0 +/- 0.1 mm/s and affected virtually every dorsal horn neuron. The Ca2+ waves were associated with large depolarizing shifts of the membrane potential of participating neurons and were most likely synaptically mediated because they were abolished by blockade of action potentials or N-methyl-D-aspartate (NMDA) receptors. They were most pronounced in the superficial dorsal horn and absent from the ventral horn. A significant proportion of the Ca2+ waves spread to the contralateral dorsal horn. This seemed to be enabled by disinhibition as primary afferent-induced dorsal horn excitation crossed the midline only when GABA(A) and glycine receptors were blocked. Interestingly, the Ca2+ waves occurred under conditions where AMPA/kainate receptors were blocked. Thus, superficial dorsal horn NMDA receptors are able to sustain synchronous neuronal excitation in the absence of functional AMPA/kainate receptors.     

Metabotropic glutamate receptors in superficial laminae of the rat dorsal horn.

Light and electron microscopic immunocytochemistry were employed here to show the distribution of metabotropic glutamate receptors (mGluRs) mGluR2/3 and mGluR5 in laminae I and II of the dorsal horn, to identify their pre- and postsynaptic location, and to test colocalization with gamma-aminobutyric acid (GABA). mGluR2/3 was mainly in the inner part of lamina II; mGluR5 was mainly in laminae I and II. Electron microscopy showed that both mGluR2/3 and mGluR5 were in perikarya, dendrites, and vesicle-containing profiles. Two main morphological types of primary afferent terminals can be distinguished in the superficial laminae: C1, likely to be endings of unmyelinated fibers, and C2, of small myelinated fibers. Quantitative data show that only a small fraction of C2s stained for either receptor; more common were immunopositive dendrites postsynaptic to these terminals, and most common were appositions between C2s and mGluR5 immunopositive dendrites. Vesicle-containing profiles were characteristically apposed to primary afferent terminals, mainly C2s. Immunopositivity for mGluRs, especially mGluR2/3, was present in vesicle-containing profiles apposed to C2, none to C1, and about half of the profiles immunostained for either receptor were also stained for GABA. The presence of presynaptic and postsynaptic mGluRs in both inhibitory and excitatory interneurons may contribute to complex processing of fast and slow responses to peripheral input in superficial laminae. As selective agonists of mGluRs may modulate GABA release, the present demonstration of mGluRs in GABAergic terminals of presumed interneurons suggests that facilitatory effects may involve a mechanism of disinhibition.     

Effects of tramadol on alpha2-adrenergic receptors in the rat brain

In recent years, it has been postulated that tramadol, used mainly for the treatment of moderate to severe pain, might display a potential as an antidepressant drug. The present study investigated the effects of acute and repeated tramadol administration on the binding of [³H]RX 821002, a selective α₂-adrenergic receptor ligand, in the rat brain. Male Wistar rats were used. Tramadol (20 mg/kg, i.p.) administered acutely (single dose), at 24 h after dosing, induced a significant decrease in the α₂-adrenergic receptors in all brain regions studied. The most pronounced effects were observed in all subregions of the olfactory system, nucleus accumbens and septum, thalamus, hypothalamus, amygdala, and cerebral cortex. Repeated treatment with tramadol (20 mg/kg, i.p., once daily for 21 days) also induced statistically significant downregulation of [³H]RX 821002 binding sites in the rat brain. However, the effect—although statistically significant—was less pronounced than in the group treated acutely with the drug. Since drugs such as mianserin and mirtazapine are potent antagonists of central α₂-adrenergic receptors and are effective antidepressants, it is tempting to suggest that, in addition to other alterations induced by tramadol, downregulation of these receptors may represent a potential antidepressant efficacy. On the other hand, one should be careful to avoid the treatment of chronic pain with tramadol in patients already receiving antidepressant drugs. Tramadol-induced downregulation of α₂-adrenergic receptors—when combined with ongoing antidepressant therapy with drugs, which themselves inhibit serotonin reuptake or are antagonists of α₂-adrenergic receptors—might cause threatening complications.     

Reappearance of calcitonin gene-related peptide-like immunoreactivity in the dorsal horn in the long-term dorsal root transected rat.

Calcitonin gene-related peptide (CGRP)-immunoreactive (IR) fibers in the rat dorsal horn superficial laminae vanish almost completely 3 weeks following unilateral dorsal rhizotomy. After a prolonged survival (20 weeks) of dorsal rhizotomy there is, however, a reappearance of CGRP-IR fibers in the corresponding laminae of the dorsal horn. The density of such IR fibres showed a clear gradient with the lowest number found in the midlesion region and an increase in density towards the neighboring, intact segments. In normal as well as lesioned rats, no neurons intrinsic to the dorsal horn contained detectable levels of CGRP-like immunoreactivity (LI). Furthermore, no cells could, by use of in situ hybridization, be demonstrated to contain detectable levels of mRNA encoding for CGRP in the dorsal horn. Based on these findings, we suggest that the CGRP-IR fibers observed following long-term survival of dorsal rhizotomy derive from proliferating collateral branches of primary afferents of neighboring intact segments.     

Peripheral inflammation increases phosphoinositide activity in the rat dorsal horn

Persistent pain leads to changes in the spinal cord that contribute to hyperalgesia and allodynia. The effort to characterize these changes has focused on neurotransmitters and receptors, while relatively little is known about pain-associated modulation of second-messenger responses. Nearly all neurotransmitters can activate the phosphoinositide (PI) second-messenger system which has been investigated using a method that localizes membrane-bound [(3)H]CDP-diacylglycerol (DAG) produced from the precursor [(3)H]cytidine [Science 249 (1990) 802]. The present study applied this method in spinal cord slices from rats injected with complete Freund's adjuvant in one hindpaw and from uninflamed control rats. Two days after the injection, slices were removed and maintained in vitro for pharmacological testing. Some slices were exposed to the acetylcholine agonist carbachol which is antinociceptive in the spinal cord. Inflammation resulted in increased baseline, unstimulated [(3)H]CDP-DAG accumulation, especially in superficial dorsal horn layers, as well as enhanced carbachol-stimulated labeling. These results suggest that persistent pain leads to neurochemical changes within the spinal cord that could potentially enhance responses to a spectrum of pain-modulating transmitters.     

Evidence that substance P selectively modulates C-fiber-evoked discharges of dorsal horn nociceptive neurons.

Previous studies suggest that the undecapeptide substance P (SP) functions as a primary afferent neurotransmitter or neuromodulator of nociception which may mediate the slow temporal summation ('windup') of discharges of dorsal horn nociceptive neurons elicited by repetitive stimulation of C-afferents. The present study tested this hypothesis by investigating the effects of local spinal application of SP and an SP antagonist. [D-Pro2,D-Trp7,9]-SP (DPDT), on A- and C-fiber-evoked firing of dorsal horn neurons in an intact, urethane-anesthetized rat preparation. Extracellular single unit recordings from both wide dynamic range and nociceptive specific neurons during controlled repetitive electrical stimulation of the ipsilateral hind paw indicated that SP enhanced C-evoked firing in an apparent dose-related manner (100 greater than 20 = 4 nmol), whereas DPDT inhibited C-evoked discharges with an apparent bell-shaped dose-response (20 greater than 100 = 4 nmol). Neither agent significantly altered either A-evoked or spontaneous activity. In agreement with previous investigators, morphine sulfate also selectively inhibited C-fiber-evoked firing without altering A-fiber-mediated activity, validating the selectivity of our system. These findings provide additional evidence that SP functions as a neuromodulator of primary afferent nociception, and further suggest that the effects of SP are selective to nociceptive transmission mediated by C-fibers.     

Platelet membrane alpha 2-adrenergic receptors in depression.

The platelet membrane was used as a model system to examine alpha 2-adrenergic receptors in 30 depressed patients and 30 healthy control subjects. The number of binding sites and their affinity for 3H-UK 14304 (5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline), a potent, highly selective alpha 2-adrenergic receptor agonist, was measured. Plasma magnesium and free 3-methoxy-4-hydroxyphenylglycol (MHPG) concentrations were assayed in the same sample. A decreased agonist-receptor affinity was found in depressed patients, whereas receptor density was not significantly altered compared with that in control subjects. In bipolar depressed and dysthymic patients, there was a tendency toward a higher density of alpha 2-adrenergic receptors. This trend was not apparent in unipolar, recurrent depressed subjects. Moreover, a positive correlation between Bmax and Kd values was observed in patients but not in control subjects--a finding that suggests that a compensatory phenomenon occurs in depression. After the patients were treated with antidepressant drugs, an increased affinity (decrease in Kd) was observed, together with a decrease in binding sites. Plasma magnesium concentrations were higher in drug-free depressed patients than in control subjects. In addition, magnesium concentrations were negatively correlated with the density of alpha 2-adrenergic receptor binding sites in depressed patients, both before and during treatment. Lastly, a trend toward a negative correlation between plasma MHPG concentration and the number of binding sites was also observed. These results suggest a complex multifactorial regulation of alpha 2-adrenergic receptors, which are probably hyposensitive in depressive syndromes.     

Orphanin FQ/nociceptin modulates glutamate- and kainic acid-induced currents in acutely isolated rat spinal dorsal horn neurons

The heptadecapeptide orphanin FQ (OFQ), also known as nociceptin (NOC), is a newly discovered endogenous ligand for the opioid-like G-protein coupled receptor, ORL1. In the present study, the effects of OFQ/NOC on glutamate (Glu), kainic acid (KA) and quisqualic acid (QA) induced currents were examined in isolated rat spinal dorsal horn neurons of young rats using whole-cell patch-clamp techniques. Glu, KA and QA elicited rapid inward currents in 90%, 69%, 83% of tested neurons. OFQ/NOC(0.03300 nM) failed to induce any changes of membrane currents, but modulated Glu-, KA- and QA-elicited currents. OFQ/NOC inhibited and potentiated Glu-induced currents in 40.6% and 27.3% of examined cells (n=106) respectively. In about one third examined neurons, OFQ/NOC had no detectable effects on Glu responses. OFQ/NOC also inhibited and enhanced KA- and QA-induced currents (inhibition: KA, 67.1%, N=76; QA, 50%, N=36. Potentiation: KA, 23.7%, N=76; QA, 16.7%, N=36). In about 10% of tested cells, Gluinduced currents were potentiated after the application of OFQ/NOC, and lasted for 2030 min. The inhibitory effects of OFQ/NOC on KA and QA responses were naloxone-insensitive. The C-terminal fragment OFQ(8–17) presented same effects on EAA-induced responses. Taken together, OFQ/NOC primarily inhibited Glu-, KA- and QA-induced currents in isolated rat spinal dorsal horn neurons via non-opioid mechanism, which might contribute to nociceptive transmission in the spinal level.     

Metabotropic purinoreceptors in rat dorsal horn neurones: predominant dendritic location.

Elevations of cytosolic free Ca2+ concentration ([Ca2+]i) induced by addition of ATP have been compared in rat dorsal horn neurones in slices and after their isolation. ATP application induced in neurones in situ a rise of [Ca2+]i by 201 +/- 12 nM. In Ca2+-free external solution the rise was 156 +/- 14 nM (n = 45 of 76), indicating the presence of active purinergic metabotropic receptors in about 59% of neurones. [Ca2+]i transients induced by 2MeSATP in Ca2+-free external solution were completely abolished by 10 microM PPADS, indicating that some of the corresponding receptors are of the P2Y1 type. In acutely isolated neurones which lost their dendrites, there were no metabotropic response. The results confirm the presence of metabotropic postsynaptic purinoreceptors located in the dendritic tree of dorsal horn neurones.     

Numbers of synapses in laminae I-IV of the rat dorsal horn

The present study determines numerical densities (NVsyn) and total numbers of synaptic discs in laminae I-IV of the rat S2 dorsal horn. Previous methods for NVsyn have the advantage of being relatively simple, but these assume that the discs are round, flat, and of uniform size. In our material, serial reconstructions indicate that these assumptions are not met. Accordingly we use a stereological method that is not as dependent on these assumptions. This method is to divide the surface density of the discs by the mean surface area of a disc (NVsyn = SVsyn/Ssyn). We refer to this as a reconstruction method because synaptic discs are reconstructed from serial sections. We also calculate numerical densities by several previously used standard methods, and the findings are similar but not identical. We find that numerical density and total synaptic numbers are smallest in lamina I, and densities and total numbers are not significantly different when lamina II is compared to laminae III and IV. Thus the intense labeling of terminals with certain compounds that characterize lamina I and II does not imply an increase in total synaptic numbers or in synaptic density. In addition there is a general increase in synaptic densities and numbers as one proceeds from lamina I to lamina IV. Another point is that the numerical density of synapses in the dorsal horn is approximately that of the cerebral cortex. These data will serve as a basis from which to judge the effects of denervations and other manipulations that purportedly change synaptic numbers.     

Connectivity of pacemaker neurons in the neonatal rat superficial dorsal horn

Pacemaker neurons with an intrinsic ability to generate rhythmic burst‐firing have been characterized in lamina I of the neonatal spinal cord, where they are innervated by high‐threshold sensory afferents. However, little is known about the output of these pacemakers, as the neuronal populations that are targeted by pacemaker axons have yet to be identified. The present study combines patch‐clamp recordings in the intact neonatal rat spinal cord with tract‐tracing to demonstrate that lamina I pacemaker neurons contact multiple spinal motor pathways during early life. Retrograde labeling of premotor interneurons with the trans‐synaptic pseudorabies virus PRV‐152 revealed the presence of burst‐firing in PRV‐infected lamina I neurons, thereby confirming that pacemakers are synaptically coupled to motor networks in the spinal ventral horn. Notably, two classes of pacemakers could be distinguished in lamina I based on cell size and the pattern of their axonal projections. Whereas small pacemaker neurons possessed ramified axons that contacted ipsilateral motor circuits, large pacemaker neurons had unbranched axons that crossed the midline and ascended rostrally in the contralateral white matter. Recordings from identified spino‐parabrachial and spino‐periaqueductal gray neurons indicated the presence of pacemaker activity within neonatal lamina I projection neurons. Overall, these results show that lamina I pacemakers are positioned to regulate both the level of activity in developing motor circuits and the ascending flow of nociceptive information to the brain, thus highlighting a potential role for pacemaker activity in the maturation of pain and sensorimotor networks in the central nervous system. J. Comp. Neurol. 523:1038–1053, 2015. © 2015 Wiley Periodicals, Inc.     

Multiple effects of phorbol esters in the rat spinal dorsal horn

Spinal cord slice preparation and intracellular recording techniques were used to examine the effects of phorbol esters on the sodium- and calcium-dependent action potentials, the excitatory synaptic transmission, the basal (resting) and the dorsal root stimulation-evoked release of 9 endogenous amino acids, including glutamate and aspartate, and the responsiveness of the rat dorsal horn neurons to excitatory amino acids (glutamic, kainic, quisqualic, and N-methyl-D-aspartic). 4-beta-Phorbol-12, 13-dibutyrate and 4-beta-phorbol-12, 13-diacetate produced minor alterations in membrane potential and resistance, but they broadened the sodium-dependent action potential and reduced the duration of the calcium-dependent action potential. In addition, phorbol esters caused a marked and long-lasting increase in the amplitude and the duration of excitatory postsynaptic potentials (EPSPs) evoked in dorsal horn neurons by orthodromic stimulation of a lumbar dorsal root. Phorbol esters produced a brief increase in the basal and electrically evoked release of endogenous excitatory (glutamic, aspartic) and inhibitory amino acids (glycine, GABA). In addition, the rates of release of alanine, serine, and threonine were also elevated. In the presence of TTX, phorbol esters selectively enhanced, in a reversible manner, the depolarizing responses of dorsal horn neurons to N-methyl-D-aspartic acid and L-glutamate but not the responses to kainic or quisqualic acids. The potentiation of the NMDA response was blocked by APV, a specific NMDA receptor antagonist. Thus, phorbol esters appear to enhance excitatory synaptic transmission in the rat spinal dorsal horn slice preparation by acting both at pre- and postsynaptic sites. Phorbol esters could potentiate excitatory synaptic transmission by acting predominantly at a postsynaptic site (NMDA receptor), since the duration of the increased responsiveness of dorsal horn neurons to glutamate and NMDA correlates better with the enhancement of EPSPs than with the increased release of the stimulation-evoked glutamate and aspartate. The increased release of endogenous amino acids is consistent with a presynaptic (terminal) site of action, but it could also be explained by enhanced interneuronal activity. Although our results suggest that in the rat spinal dorsal horn protein kinase C may have a role in controlling the release of putative excitatory and inhibitory neurotransmitters and may also be involved in the regulation of postsynaptic NMDA receptors, the identity of endogenous substance(s) participating in these effects is presently unknown.     

Dual localization of neuropeptide FF receptors in the rat dorsal horn

Although neuropeptide FF (NPFF) is generally considered an anti-opioid, its intrathecal administration produces analgesia. In the present study, the stable analog 1DMe ([D.Tyr(1), (NMe)Phe(3)]neuropeptide FF) was used in quantitative autoradiographic experiments in combination with surgical and chemical lesions to precisely localize NPFF receptors in the rat spinal cord. Ligation of lumbar dorsal spinal roots revealed the presence of NPFF receptors in dorsal root fibers and it induced a significant accumulation of [(125)I]1DMe-specific binding on the side peripheral to the ligature, demonstrating that a population of NPFF receptors is synthesized in dorsal root ganglia and migrates anterogradely towards primary afferent nerve endings. Complete mid-thoracic spinal cord transection failed to modify the [(125)I]1DMe labeling density in the dorsal horn, indicating that NPFF receptors are not located on the descending fiber terminals. In contrast, unilateral microinjections of kainic acid into the dorsal horn dramatically reduced [(125)I]1DMe-specific binding in the superficial layers, revealing localization of a population of NPFF receptors on the spinal intrinsic neurons. NPFF receptor binding was not modified during the development of spinal opioid tolerance. The pre- and postsynaptic localization of spinal NPFF receptors provide further support for heterogeneity in the pain modulation by NPFF and related agonists.