Co-localization of substance P and Met-enkephalin-Arg-Gly-Leu in the intraspinal neurons of the rat, with special reference to the neurons in the substantia gelatinosa

A double-labeling immunofluorescence technique was employed to investigate the co-localization of the functionally antagonistic neuropeptides, substance P and enkephalins, within intraspinal neurons of the rat. Anti-Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Enk-8) antiserum was used as a marker of the preproenkephalin A neuron system. The observations were focused on the lumbar spinal cord. Co-localization was most prominent within neurons in the substantia gelatinosa, in which more than 95% of substance P-like immunoreactivity neurons showed Enk-8-like immunoreactivity. These double-labeled cells corresponded to 45% of Enk-8-like immunoreactive neurons in the same area. This suggests that SP/Enk-8 interaction occurs at the axon terminals of the substantia gelatinosa neurons. In deeper layers of the dorsal horn (laminae III, IV), only 14% and 6% of SP-like immunoreactive and Enk-8-like immunoreactive neurons were double labeled, respectively. Co-localization was also observed in neurons located in the laminae I, V, VII and X, suggesting concomittant involvement of these peptides in a variety of spinal cord functions.     

Localization of Met-enkephalin-Arg6-Gly7-Leu8 immunoreactivity in rat duodenum

The distribution of Met-enkephalin-Arg6-Gly7-Leu8 (Met-ENK-Arg6-Gly7-Leu8) in the rat duodenum was determined using specific antibodies against Met-ENK-Arg6-Gly7-Leu8 and the immunofluorescence microscope technique. Met-ENK-Arg6-Gly7-Leu8 immunoreactive perikarya have been detected in the myenteric plexus. These neuronal cell bodies were large in diameter and round in shape. Met-ENK-Arg6-Gly7-Leu8 immunostained nerve fibres were noted in both the circular muscle layer and, more abundantly, in interconnecting myenteric plexus nerve fibre bundles. These findings might indicate that Met-ENK-Arg6-Gly7-Leu8 has important physiological roles as neurotransmitter and/or neuromodulator in the human and mammalian gastrointestinal tract.     

Distribution of immunoreactive metorphamide (adrenorphin) in discrete regions of the rat brain: comparison with Met-enkephalin-Arg6-Gly7-Leu8

The distribution of immunoreactive (ir)-metorphamide (adrenorphin) in 101 microdissected rat brain and spinal cord regions was determined using a highly specific radioimmunoassay. The highest concentration of metorphamide in brain was found in globus pallidus (280.1 fmol/mg protein). High concentrations of ir-metorphamide (greater than 120 fmol/mg protein) were found in 9 nuclei, including central amygdaloid nucleus, lateral preoptic area, anterior hypothalamic nucleus, hypothalamic paraventricular nucleus, interpeduncular nucleus, periaqueductal grey matter and nucleus of the solitary tract. Moderate concentrations of the peptide (between 60 and 120 fmol/mg protein) were found in 47 brain nuclei such as nucleus accumbens, bed nucleus of stria terminalis, several septal and amygdaloid nuclei, most of the hypothalamic nuclei, ventral tegmental area, red nucleus, raphe nuclei, lateral reticular nucleus, area postrema and others. Low concentrations or ir-metorphamide (less than 60 fmol/mg protein) were measured in 41 nuclei, e.g., cortical structures, hippocampus, caudate nucleus, thalamic nuclei, supraoptic nucleus, substantia nigra, vestibular nuclei, cerebellum (nuclei and cortex). The olfactory bulb has the lowest metorphamide concentration (5.8 fmol/mg protein). Spinal cord segments exhibit very low peptide concentrations.     

Immunocytochemical distribution of met-enkephalin-Arg6-Gly7-Leu8 in the rat lower brainstem

The distribution of methionine-enkephalin-Arg6-Gly7-Leu8, a unique peptide derived from proenkephalin A in the rat brainstem, was studied immunocytochemically by using a highly specific antiserum to this octapeptide sequence. Immunoreactive perikarya with various shapes and sizes were detected in many regions of the rat brainstem. Dense accumulation of immunoreactive perikarya and fibers was seen in the nuclei associated with special sensory and visceral functions, such as the interpeduncular nucleus, the parabrachial nucleus, the nucleus of the solitary tract, and the nucleus of the spinal tract of the trigeminal nerve. Clusters of methionine-enkephalin-Arg6-Gly7-Leu8-like immunoreactive perikarya and fibers were observed in certain areas considered to play a role in nociception and analgesia, such as the central gray of the midbrain central gray and the raphe magnus nucleus. Some methionine-enkephalin-Arg6-Gly7-Leu8-like immunoreactive perikarya were distributed in the lateral reticular nucleus, the nucleus of the solitary tract, and the raphe magnus nucleus, where monoaminergic neurons were also detected. In addition to the previously reported enkephalinergic cells, we found many methionine-enkephalin-Arg6-Gly7-Leu8 containing neurons; the rostral and caudal linear nucleus of raphe, the median raphe nucleus, entire length of the raphe magnus nucleus, the medial longitudinal fasciculus, the cuneate nucleus, the external cuneate nucleus, the gracile nucleus, and the area postrema. The wide distribution of this octapeptide-like immunoreactivity reflected neurons expressing the preproenkephalin A gene distributed more widely than previously reported and that innervated many regions.     

Differential colocalization of neuropeptide Y- and methionine-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in catecholaminergic neurons in the rat brain stem

The present study, using a combination of catecholamine (CA) histofluorescence and peptide immunocytochemistry in the same tissue sections, investigated the coexistence of neuropeptide Y (NPY) and methionine-enkephalin-Arg6-Gly7-Leu8 (MEAGL)-like immunoreactivity (LI) in catecholaminergic neurons of colchicine-treated rat brain stems. Of the total number of catecholaminergic neurons in the A1/C1, A2/C2, A3, A4, and A6 regions approximately 83, 28, 98, 76, and 36%, respectively, contained both NPY-LI and CA. Of the total number of catecholaminergic neurons in A1/C1, A2/C2, A3, and A5 regions, approximately 47, 4, 8, and 17%, respectively, contained both MEAGL-LI and CA. Moreover, about 24% of the catecholaminergic neurons in the A1/C1 region contained both NPY- and MEAGL-LI. Neither the noradrenergic neurons (A7) in the pons nor any of the dopaminergic neurons in the midbrain (A8, A9, A10) contained NPY- or MEAGL-LI. Neurons containing both NPY- and MEAGL-like immunoreactive peptides without CA were not found in the rat brain stem. These findings indicate that catecholaminergic neurons in the brain stem of the rat can be subdivided into distinct subgroups on the basis of the coexistence of specific peptides.     

An immunohistochemical study of methionine-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity-containing neurons in the parasympathetic preganglionic regions of the rat spinal cord

The localization of the methionine-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-Arg-Gly-Leu)-like immunoreactivity-containing neurons in the rat lumbosacral spinal cord was immunohistochemically examined by an antiserum very specific to Met-Enk-Arg-Gly-Leu. The immunoreactive neurons occupied the positions corresponding to the parasympathetic preganglionic nuclei determined by the previous horseradish peroxidase (HRP)-tracing experiments. The present study suggests that the parasympathetic preganglionic neurons in the rat lumbosacral spinal cord produce preproenkephalin A and its related peptides.     

The opioid octapeptide Met5-enkephalin-Arg6-Gly7-Leu8: characterization and distribution in rat spinal cord

The regional quantitation, immunohistochemical localization and molecular heterogeneity of Met5-enkephalin-Arg6-Gly7-Leu8 were examined in rat spinal cord with a specific radioimmunoassay. A rostrocaudal gradient in Met5-enkephalin-Arg6-Gly7-Leu8 content was observed; the highest levels occurred in sacral cord. Dorsal cord content was higher than that of ventral cord at all spinal segments. Immunohistochemical staining supported and refined the latter observation: a dense network of perikarya and fibers was found in Laminae I and II of the dorsal horn. Cell bodies were frequently observed in lamina IV. Additional terminals were seen around the central canal and in the ventral gray matter, often outlining perikarya of motor neurons. Total Met5-enkephalin-Arg6-Gly7-Leu8 immunoreactivity could be fractionated into two main components using gel filtration chromatography. Nearly half of the total immuno-reactivity eluted as a high molecular weight peptide; the other half which co-eluted with Met5-enkephalin-Arg6-Gly7-Leu8 was further identified to be authentic Met5-enkephalin-Arg6-Gly7-Leu8 on reverse phase high pressure liquid chromatography. The present data, in conjunction with our previous study of Met5-enkephalin and Met5-enkephalin-Arg6-Phe7 indicates that all opioid peptides derived from preproenkephalin A are present in spinal cord and most likely are stored in the same neurons. Immunohistochemical localization of Met5-enkephalin-Arg6-Gly7-Leu8 in dorsal and ventral cord suggest a role for this peptide in both sensory and motor integration.     

Immunocytochemical analysis of [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactive structures in the rat superior cervical ganglion

Indirect immunofluorescence and immunoelectron microscopy were employed to analyze the enkephalinergic systems in the rat superior cervical ganglion (SCG). These systems were identified using specific antiserum against [Met5]Enkephalin-Arg6-Gly7-Leu8 (ENK-8), a peptide which is derived only from proenkephalin A. Abundant ENK-8 like immunoreactive (ENK-8-LI) neurons and fibers were observed in the SCG, but their distribution patterns were heterogenous; ENK-8-LI neurons were localized preferentially in the caudal two-thirds of the SCG, while immunoreactive fibers were found to be distributed more densely in the rostral one-third than in the remaining part of the SCG. Most of the ENK-8-LI neurons were large and had ultrastructural features resembling those of principal cells, some were identified electron microscopically as small intensely fluorescent (SIF) cells. ENK-8-LI fibers were varicose in appearance and surrounded the perikarya of neurons. Since most of these fibers were not detected after experimental decentralization of the SCG and since ENK-8-LI terminals were seen to contain small lucent vesicles, most of the former were thought to be preganglionic fibers. Immunoreactive fibers mainly formed synaptic contacts with the dendrites of non-immunoreactive principal cells, but a small proportion of ENK-8-LI principal cells also received synaptic input from them. Occasionally, immunoreactive fibers formed synapses with the processes or the soma of both ENK-8-LI and non-immunoreactive SIF cells. On the basis of these findings, we conclude that: (1) preganglionic ENK-8-LI fibers terminate mainly on the principal cells, which are devoid of ENK-8-LI structures; (2) the majority of ENK-8-LI neurons are principal cells, while the remainder are SIF cells; (3) inputs to these cells mainly involve structures lacking ENK-8 immunoreactivity; and (4) there are, however, a small number of ENK-8-LI preganglionic fibers which terminate on ENK-8-LI principal cells and SIF cells.     

Immunocytochemical survey of haloperidol-induced immunoreactive changes of [Met]enkephalin-Arg6-Gly7-Leu8 in the rat forebrain.

It has already been demonstrated that chronic treatment with the dopamine receptor blocker, haloperidol, results in an increase of proenkephalin-A-derived peptides in the caudate-putamen (CP). To examine this phenomenon at the cellular level, we used immunocytochemistry to investigate the effects of haloperidol on [Met]enkephalin-Arg6-Gly7-Leu8 (MEAGL) immunoreactivity in the rat forebrain. After daily haloperidol (5 mg/kg, IP, for 6 days) or haloperidol decanoate (70 mg/kg, IM, given once or twice) treatment, immunoreactive neurons appeared diffusely in the whole CP and in the core part of the nucleus accumbens (Acb) and less frequently in the outer shell part of the Acb and the cell-dense layer of the tuberculum olfactorium (TuO). Increase of MEAGL-immunoreactive fibers in the CP, Acb, and TuO was also detected after these treatments, a particularly prominent increase being found in the striopallidal terminals in the globus pallidus and ventral pallidum. Haloperidol or haloperidol decanoate had no effect on MEAGL immunoreactivity in the cerebral cortex, amygdala, or hypothalamus. Reserpine treatment (5 mg/kg, IP, for 6 days) caused similar effects on the dorsal and ventral striopallidal system, and the direct injection of 6-hydroxydopamine (10 micrograms/5 microliters) into the CP led to the appearance of MEAGL-immunoreactive neurons in accordance with the depleted dopaminergic terminal area. These findings suggest that haloperidol influences enkephalinergic neurons region specifically and that in the dorsal and ventral striopallidal enkephalinergic system haloperidol increases MEAGL immunoreactivity in cell bodies, fibers, and terminals by blocking intrastriatal dopaminergic neurotransmission.     

Distribution of Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactive peptides in rat brain: presence of multiple molecular forms

An antiserum to the opioid octapeptide met5-enk-arg6- gly7 -leu8 was used to measure the distribution and molecular weight heterogeneity of met5-enk-arg6- gly7 -leu8-immunoreactive (IR) peptides in rat brain. High concentrations of met5-enk-arg6- gly7 -leu8-IR peptides were found in the striatum and hypothalamus; low concentrations were observed in the cortex and cerebellum. Intermediate levels of immunoreactivity were found in other brain regions. Thus the distribution of met5-enk-arg6- gly7 -leu8-IR peptides closely parallels the distribution of met5-enkephalin. The immunoreactivity present in brain regions was characterized by gel filtration and reverse-phase high pressure liquid chromatography. In the striatum, more than 85% of the met5-enk-arg6- gly7 -leu8-IR peptides eluted from a Biogel P-30 column at the position of the authentic octapeptide; the identity of this material was further confirmed by HPLC. Gel filtration of extracts prepared from the medulla-pons resulted in two peaks of immunoreactive material of equal size; one eluted at approximately 8000 daltons and one eluted in the position of the octapeptide. Enzymatic digestion (trypsin + carboxypeptidase B) of the 8000 dalton-IR peptide resulted in the generation of met5-enkephalin-IR. Extracts prepared from other brain regions contained varying amounts of this high molecular weight form of met5-enk-arg6- gly7 -leu8.     

Met5-enkephalin-Arg6-Gly7-Leu8 immunoreactivity in rat and human cerebrospinal fluid: influence of neuroleptic drugs and electroconvulsive shock

Met5-Enkephalin-Arg6-Gly7-Leu8 immunoreactivity was quantitated in both rat and human cerebrospinal fluid (CSF) by radioimmunoassay with a carboxy-terminal directed antiserum. The immunoreactivity in CSF was chromatographically characterized in both species and was found to consist almost exclusively of high molecular weight forms. In human CSF there was approximately 300 fmol/ml and in the rat 1,500 fmol/ml of immunoreactivity. The possibility of a rostro-caudal gradient was examined in the human by analyzing the first and the twenty-fifth ml of CSF drawn during a lumbar puncture: none was found. The immunoreactivity was fairly stable; no loss of immunoreactivity was observed after 24 h of incubation of rat CSF at 37 degrees C. Electroconvulsive shock (ECS) produced a significant elevation in CSF content but only after a course of chronic administration; a single acute ECS produced no increase. Human subjects with schizophrenia who were being treated with antipsychotic drugs had elevated levels of immunoreactivity in comparison to non-mediated patients and normals. The high levels of this immunoreactivity in CSF, its stability and the evidence that the content can change with physiological and pharmacological manipulation indicate that Met5-Enkephalin-Arg6-Gly7-Leu8 immunoreactivity can serve as a new and useful CSF marker for investigating the CNS enkephalin system in neurological or psychiatric disorders.     

Met5-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in the pelvic ganglion of the male rat: a light and electron microscopic study

By using both light and electron microscopic immunocytochemical methods, Met5-Enkephalin-Arg6-Gly7-Leu8 (MEAGL)-like immunoreactive structures were detected in the pelvic ganglion of male rats. Denervation studies were carried out to determine the origin of these immunoreactive fibers and the projection of immunoreactive neurons within the pelvic ganglion. MEAGL-like immunoreactivity was found in numerous axon boutons, some small, intensely fluorescent (SIF) cells, and a few principal ganglion neurons. Most of the immunoreactive nerve fibers formed pericellular plexuses surrounding the ganglion cells. In addition, there were a few scattered varicose fibers. These fiber plexuses could be classified into two types: type I (approximately 90% of fibers), which consisted of 80-120 small boutons that synapsed on either the dendrites (80% of cases) or somata (20% of cases) of principal neurons; and type II (approximately 10% of fibers), which consisted of 20-40 larger boutons that formed axodendritic synapses exclusively. After transection of the hypogastric and pelvic nerves, virtually all of the pericellular fiber plexuses disappeared, whereas the scattered varicose fibers remained. According to their ultrastructure, these remaining fibers were considered to arise from SIF cells. Following the injection of Fast Blue into the bladder wall, some of the MEAGL-like immunoreactive principal neurons were retrogradely labeled. The results of this study indicate that there are two origins for the MEAGL-like immunoreactive fibers detected in the pelvic ganglion: most arise from preganglionic neurons in the spinal cord, and a small proportion may originate from intraganglionic MEAGL-like immunoreactive SIF cells or principal neurons. Some MEAGL-like immunoreactive principal neurons may project to the urinary bladder.     

Action of dexmedetomidine on the substantia gelatinosa neurons of the rat spinal cord

Dexmedetomidine is a highly specific, potent and selective alpha(2)-adrenoceptor agonist. Although intrathecal and epidural administration of dexmedetomidine has been found to produce analgesia, whether this analgesia results from an effect on spinal cord substantia gelatinosa (SG) neurons remains unclear. Here, we investigated the effects of dexmedetomidine on postsynaptic transmission in SG neurons of rat spinal cord slices using the whole-cell patch-clamp technique. In 92% of the SG neurons examined (n = 84), bath-applied dexmedetomidine induced outward currents at -70 mV in a concentration-dependent manner, with the value of effective concentration producing a half-maximal response (0.62 microM). The outward currents induced by dexmedetomidine were suppressed by the alpha(2)-adrenoceptor antagonist yohimbine, but not by prazosin, an alpha(1)-, alpha(2B)- and alpha(2C)-adrenoceptor antagonist. Moreover, the dexmedetomidine-induced currents were partially suppressed by the alpha(2C)-adrenoceptor antagonist JP-1302, while simultaneous application of JP-1302 and the alpha(2A)-adrenoceptor antagonist BRL44408 abolished the current completely. The action of dexmedetomidine was mimicked by the alpha(2A)-adrenoceptor agonist oxymetazoline. Plots of the current-voltage relationship revealed a reversal potential at around -86 mV. Dexmedetomidine-induced currents were blocked by the addition of GDP-beta-S [guanosine-5'-O-(2-thiodiphosphate)] or Cs+ to the pipette solution. These findings suggest that dexmedetomidine hyperpolarizes the membrane potentials of SG neurons by G-protein-mediated activation of K+ channels through alpha(2A)- and alpha(2C)-adrenoceptors. This action of dexmedetomidine might contribute, at least in part, to its antinociceptive action in the spinal cord.     

Taurine activates glycine and gamma-aminobutyric acid A receptors in rat substantia gelatinosa neurons

Taurine has been suggested to modulate nociceptive information at the spinal cord level. In this study, the pharmacological properties of taurine were investigated in adult rat substantia gelatinosa (SG) neurons using whole-cell patch-clamp method. We found that taurine seemed to have higher efficacy than glycine on glycine receptors in SG neurons. An increase in chloride conductance was responsible for taurine-induced currents. Taurine at 0.3 mM activated glycine receptors, whereas at 3 mM activated both glycine and gamma-aminobutyric acid A receptors. The currents activated by coapplication of taurine and glycine are cross inhibitive. Altogether these results show that taurine might represent another important neurotransmitter or modulator in SG neurons, which may be involved in antinociception.     

Baclofen对大鼠脊髓薄片胶状质神经元的抑制作用

用脊髓薄片全细胞电压钳法观察激活γ－氨基丁酸Ｂ亚型受体（ＧＡＢＡＢR)对大鼠脊髓背角胶状质（ＳＧ）神经元活动的影响,选择性ＧＡＢＡＢＲ激动剂baclofen减少所有被记录ＳＧ神经元（n＝１５）的微小兴奋性突触后电流（mEPSGs）的发放频率。同时引起一缓慢的抑制性（外向）膜电流并伴有膜电导增加,细胞内电泳Ｇ蛋白偶联受体抑制剂（ＧＤＰ－β－Ｓ）抑制被钳制神经元的Ｇ蛋白偶联受体后,baclofen引起的缓慢外向电流被抑制,但对mEPSC频率的减低作用依然存在。结果提示脊髓内baclofen敏感的ＧＡＢＡＢ受体被激活后直接引起ＳＧ神经元超极化并能减少突触前递质的释放。     

Axonal and dendritic development of substantia gelatinosa neurons in the lumbosacral spinal cord of the rat

In the present study, developing neurons of the substantia gelatinosa (SG) are examined at short interval sequential stages from 15 days of gestation through 20 days postpartum. Rapid Golgi preparations are utilized to examine axonal and dendritic development and toluidine blue preparations are employed to study the overall growth pattern of SG cells by measuring changes in mean cell body area. Results show that there are two maturation periods, which involve two separate groups of SG neurons. The sequence and pattern of development for each group is different. The first period occurs prenatally and involves the axonal and dendritic development of presumptive projection and propriospinal neurons. In classical terminology, these cells can be classified as limiting, large find small central, and transverse cells. These neurons have axons that enter the white matter and their dendritic arbors develop through a relatively simple process of elongation and branching. The second maturation period occurs postnatally and involves the development of presumptive nonprojection intrinsic neurons that have axons which remain within the gray matter. These neurons are identified as islet, stalk, inverted stalklike, and vertical cells. Unlike projection or propriopinal neurons, the intrinsic nonprojection neurons sprout numerous short, beaded dendrites that radiate from the cell body in a starlike fashion. Star-shaped cells undergo a metamorphosis involving a rearrangement of dendrites along adult dendritic patterns. Measurements taken from toluidine blue preparations indicate that the nonprojection intrinsic population makes up the greatest percentage of SG neurons, as evidenced by a marked increase in the size of the average SG nerve cell during the second maturation period.     

Release and depletion of substance P by capsaicin in substantia gelatinosa studied with the antibody microprobe technique and immunohistochemistry.

Using an antibody microprobe technique, we have detected substance P release from the region of the substantia gelatinosa of the cat during the first, but not the second, 30 min of topical application of capsaicin (1-3%) to the tibial nerve. Immunohistochemical analysis also showed that substance P-like immunoreactivity was markedly reduced in the superficial layer of the dorsal horn 30 min after application of capsaicin. These results indicate that substance P is released and then depleted from primary afferent central terminals following acute application of capsaicin to the peripheral sensory nerve.     

A biochemically distinct sub-population of neurons in the human substantia gelatinosa. Study with G-6-PD histochemistry

A method for localization of glucose-6-phosphate dehydrogenase (G-6-PD; D-glucose-6-phosphate: NADP+ oxidoreductase; E.C. 1.1.1.49) activity has been applied to human nervous tissue. Intensely staining cells, not definable by conventional histologic techniques, have been identified in the human spinal cord, with highest numbers present in the substantia gelatinosa of the sacral region. The cells have a neuron-like morphology and express neuronal-specific antigen but are heterogeneous in size and shape. They are not detectable in infant spinal cord, but stain heavily in adults. We propose that these cells are homologous to the G-6-PD-active dorsal medullary cells first noted by Sakharova et al. (1979) and together with the latter group, may comprise a hitherto unrecognized system of neurons in the human central nervous system.     

Cell type-specific postsynaptic effects of neuropeptide Y in substantia gelatinosa neurons of the rat spinal cord

Cellular mechanisms of antinociceptive action of neuropeptide Y were investigated in substantia gelatinosa (SG) neurons in rat spinal cord slices. Somatic and synaptic effects of NPY were compared in two subpopulations of cells with different firing patterns, tonic (TFNs), and delayed firing (DFNs) neurons. For the study, TFNs were selected on morphological basis: they had appearance of central and radial but not islet cells, and are likely excitatory interneurons in dorsal horn networks. In their turn, DFNs were classified as radial and vertical cells. 0.3 μM NPY via Y1 receptors activated hyperpolarizing postsynaptic current of GIRK type in majority of TFNs (～77%) but not DFNs (～8%). Miniature synaptic currents in all neurons were seen as a mixture of excitatory (mEPSCs) and inhibitory (mIPSCs), the frequency of the former being ～5 times greater. The mEPSCs were mediated by glutamate receptors of AMPA subtype, while the dominant part of mIPSCs--by glycine receptors. In all cell types, NPY moderately depressed the frequency of both mEPSCs and mIPSCs; the effects occurred via Y2 and Y1 receptors, respectively. The data suggest that behavioral NPY-evoked antinociception is achieved via postsynaptic hyperpolarization of majority of TFNs (assumingly, excitatory interneurons) via Y1 receptors and depression of the mEPSCs via Y2 receptors.