Probing electrophysiological activity of amphiphilic Dynorphin A in planar neutral membranes reveals both ion channel-like activity and neuropeptide translocation.

Dynorphin A (DynA) is an endogenous neuropeptide that besides acting as a ligand of the κ-opioid receptor, presents some non-opioid pathophysiological properties associated to its ability to induce cell permeability similarly to cell-penetrating peptides (CPPs). Here, we use electrophysiology experiments to show that amphiphilic DynA generates aqueous pores in neutral membranes similar to those reported previously in charged membranes, but we also find other events thermodynamically incompatible with voltage-driven ion channel activity (i.e. non-zero currents with no applied voltage in symmetric salt conditions, reversal potentials that exceed the theoretical limit for a given salt concentration gradient). By comparison with current traces generated by other amphiphilic molecule known to spontaneously cross membranes, we hypothesize that DynA could directly translocate across neutral bilayers, a feature never observed in charged membranes following the same electrophysiological protocol. Our findings suggest that DynA interaction with the cellular membrane is modulated by the lipid charge distribution, enabling either passive ionic transport via membrane remodeling and pore formation or by peptide direct internalization independent of cellular transduction pathways.

Cellular Profiles of Prodynorphin and Preproenkephalin mRNA-Expressing Neurons in the Anterior Olfactory Tubercle of Mice.

The olfactory tubercle (OT) is a striatal region that receives olfactory inputs. mRNAs of prodynorphin (Pdyn) and preproenkephalin (Penk), precursors of dynorphins and enkephalins, respectively, are strongly expressed in the striatum. Both produce opioid peptides with various physiological effects such as pain relief and euphoria. Recent studies have revealed that OT has anatomical and cytoarchitectonic domains that play different roles in odor-induced motivated behavior. Neuronal subtypes of the OT can be distinguished by their expression of the dopamine receptors D1 (Drd1) and D2 (Drd2). Here, we addressed whether and which type of opioid peptide precursors the D1- and D2-expressing neurons in the OT express. We used multiple fluorescence in situ hybridization for mRNAs of the opioid precursors and dopamine receptors to characterize mouse OT neurons. Pdyn was mainly expressed by Drd1-expressing cells in the dense cell layer (DCL) of the OT, whereas Penk was expressed primarily by Drd2-expressing cells in the DCL. We also confirmed the presence of a larger population of Pdyn-Penk-Drd1 co-expressing cells in the DCL of the anteromedial OT compared with the anterolateral OT. These observations will help understand whether and how dynorphins and enkephalins in the OT are involved in diverse odor-induced motivated behaviors.

Dynorphin and Enkephalin Opioid Peptides and Transcripts in Spinal Cord and Dorsal Root Ganglion During Peripheral Inflammatory Hyperalgesia and Allodynia.

Understanding molecular alterations associated with peripheral inflammation is a critical factor in selectively controlling acute and persistent pain. The present report employs in situ hybridization of the 2 opioid precursor mRNAs coupled with quantitative measurements of 2 peptides derived from the prodynorphin and proenkephalin precursor proteins: dynorphin A 1-8 and [Met5]-enkephalin-Arg6-Gly7-Leu8. In dorsal spinal cord ipsilateral to the inflammation, dynorphin A 1-8 was elevated after inflammation, and persisted as long as the inflammation was sustained. Qualitative identification by high performance liquid chromatography and gel permeation chromatography revealed the major immunoreactive species in control and inflamed extracts to be dynorphin A 1-8. In situ hybridization in spinal cord after administration of the inflammatory agent, carrageenan, showed increased expression of prodynorphin (Pdyn) mRNA somatotopically in medial superficial dorsal horn neurons. The fold increase in preproenkephalin mRNA (Penk) was comparatively lower, although the basal expression is substantially higher than Pdyn. While Pdyn is not expressed in the dorsal root ganglion (DRG) in basal conditions, it can be induced by nerve injury, but not by inflammation alone. A bioinformatic meta-analysis of multiple nerve injury datasets confirmed Pdyn upregulation in DRG across different nerve injury models. These data support the idea that activation of endogenous opioids, notably dynorphin, is a dynamic indicator of persistent pain states in spinal cord and of nerve injury in DRG. PERSPECTIVE: This is a systematic, quantitative assessment of dynorphin and enkephalin peptides and mRNA in dorsal spinal cord and DRG neurons in response to peripheral inflammation and axotomy. These studies form the foundational framework for understanding how endogenous spinal opioid peptides are involved in nociceptive circuit modulation.

Low-picomolar analysis of peptides by on-line coupling of fritless solid-phase extraction to sheathless capillary electrophoresis-mass spectrometry.

A novel fritless solid-phase extraction (SPE) microcartridge was designed for combination with sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) employing a prototype porous-tip capillary for nanoelectrospray ionization (nanoESI). The inlet of the separation capillary (30µm inner diameter (id), 150µm outer diameter (od)) was inserted in a 4mm long SPE microcartridge (150µm id, 365µm od) packed with a C18 sorbent of 55-105µm particle size. Performance of the SPE-CE-MS system was evaluated using diluted solutions of the three opioid peptides dynorphin A (1-7) (DynA), endomorphin 1 (End1) and met-enkephalin (Met). Sample volumes of 1.5µL were loaded on the SPE microcartridge and the retained peptides were eluted with 22nL of an acidic methanol/water (60:40, v/v) solution. Using a pressure of 50mbar during separation to speed up the analysis, good peptide resolution was obtained with acceptable plate numbers (between 53,000 and 92,000). Intraday relative standard deviations (% RSD) for peptide migration times and peak areas were below 4% and 9%, respectively. The SPE-CE-MS method showed good linearity in the 0.05-5ngmL(-1) range and limits of detection (LODs) were 10pgmL(-1). However, loading a larger volume of sample (8µL), LODs could be decreased down to 2pgmL(-1) (2.2-3.5pM). This represents an improvement of up to 5000-fold with respect to the LODs achieved by sheathless CE-MS without on-line preconcentration demonstrating the potential of on-line SPE for further enhancing sensitivity.

Antinociceptive effects of eugenol evaluated in a monoiodoacetate-induced osteoarthritis rat model.

The aim of the present study was to evaluate whether eugenol, the main constituent of clove oil, has the capacity to provide analgesia in the monoiodoacetate-induced rat model of osteoarthritis. Animals (n = 6/group) received either eugenol (20 or 40 mg/kg) or a vehicle by gavage. Daily administrations were initiated 2 days post osteoarthritis induction and continued for the duration of the study (4 weeks). Gait analysis was performed using the CatWalk method and secondary mechanical allodynia was assessed with von Frey filaments. Selected spinal cord peptides (substance P, calcitonin gene-related peptide and dynorphin) were quantified by mass spectrometry. Significant changes were identified in dynamic gait parameters (swing speed, swing phase duration and duty cycle) of the affected limb following 40 mg/kg eugenol treatment compared with the vehicle (p < 0.05). Von Frey results revealed significant differences between the 40 mg/kg treatment and the vehicle group during the first and the third week of the study (p < 0.02). Spinal pain-related peptide analysis revealed a decreased content of substance P and CGRP accompanied by an increase of dynorphin in animals treated with 40 mg/kg eugenol. These results suggest a therapeutic potential of eugenol to alleviate osteoarthritis-related pain.

Enkephalin surges in dorsal neostriatum as a signal to eat.

Compulsive overconsumption of reward characterizes disorders ranging from binge eating to drug addiction. Here, we provide evidence that enkephalin surges in an anteromedial quadrant of dorsal neostriatum contribute to generating intense consumption of palatable food. In ventral striatum, mu opioid circuitry contributes an important component of motivation to consume reward. In dorsal neostriatum, mu opioid receptors are concentrated within striosomes that receive inputs from limbic regions of prefrontal cortex. We employed advanced opioid microdialysis techniques that allow detection of extracellular enkephalin levels. Endogenous >150% enkephalin surges in anterior dorsomedial neostriatum were triggered as rats began to consume palatable chocolates. In contrast, dynorphin levels remained unchanged. Furthermore, a causal role for mu opioid stimulation in overconsumption was demonstrated by observations that microinjection in the same anterior dorsomedial quadrant of a mu receptor agonist ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin; DAMGO) generated intense >250% increases in intake of palatable sweet food (without altering hedonic impact of sweet tastes). Mapping by "Fos plume" methods confirmed the hyperphagic effect to be anatomically localized to the anteromedial quadrant of the dorsal neostriatum, whereas other quadrants were relatively ineffective. These findings reveal that opioid signals in anteromedial dorsal neostriatum are able to code and cause motivation to consume sensory reward.

Dynorphin A 1-17 biotransformation in inflamed tissue, serum and trypsin solution analysed by liquid chromatography-tandem mass spectrometry.

Dynorphin A 1-17 (DYN A) is an endogenous neuropeptide that is of interest due to its diverse roles in analgesia, inflammation and addiction. Upon release, DYN A is subject to metabolism by a range of enzymes and its biotransformation is dependent on the site and environment into which it is released. In this study, we investigated the biotransformation of DYN A in rat inflamed tissue at pH 7.4 and 5.5, in rat serum and in trypsin solution. DYN A-porcine was incubated at 37 °C in each matrix over a range of incubation periods. The resultant fragments were separated using a C4 column and detected by mass spectrometry using total ion current mode. Incubation of DYN A in trypsin solution and in rat serum resulted in 6 and 14 fragments, respectively. Incubation in inflamed rat paw tissue occasioned 21 fragments at pH 7.4 and 31 fragments at pH 5.5. Secondary breakdown of some larger primary fragments was also observed in this study.

Determination of specific neuropeptides modulation time course in a rat model of osteoarthritis pain by liquid chromatography ion trap mass spectrometry.

Animal models are useful to evaluate pharmacological therapies to alleviate joint pain. The present study characterized central neuropeptides modulation in the monoiodoacetate (MIA) rat model. Animals receiving a single 3mg MIA injection were euthanized at 3, 7, 14, 21 and 28 days post injection. Spinal cords were analyzed by liquid chromatography ion trap mass spectrometry. Up-regulations of the calcitonin gene-related peptide and substance P were observed starting on days 7 and 28 respectively, whereas big dynorphin(1₋32) content decreased significantly on day 14 in comparison to control animals (P<0.05). Preclinical drug evaluations using this model should be conducted between 7 and 21 days post injection when the lesions resemble most to human osteoarthritis.

L-DOPA-induced dyskinesia is associated with regional increase of striatal dynorphin peptides as elucidated by imaging mass spectrometry.

Opioid peptides are involved in various pathophysiological processes, including algesia, epilepsy, and drug dependence. A strong association between L-DOPA-induced dyskinesia (LID) and elevated prodynorphin mRNA levels has been established in both patients and in animal models of Parkinson's disease, but to date the endogenous prodynorphin peptide products have not been determined. Here, matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) was used for characterization, localization, and relative quantification of striatal neuropeptides in a rat model of LID in Parkinson's disease. MALDI IMS has the unique advantage of high sensitivity and high molecular specificity, allowing comprehensive detection of multiple molecular species in a single tissue section. Indeed, several dynorphins and enkephalins could be detected in the present study, including dynorphin A(1-8), dynorphin B, α-neoendorphin, MetEnkRF, MetEnkRGL, PEnk (198-209, 219-229). IMS analysis revealed elevated levels of dynorphin B, α-neoendorphin, substance P, and PEnk (220-229) in the dorsolateral striatum of high-dyskinetic animals compared with low-dyskinetic and lesion-only control rats. Furthermore, the peak-intensities of the prodynorphin derived peptides, dynorphin B and α-neoendorphin, were strongly and positively correlated with LID severity. Interestingly, these LID associated dynorphin peptides are not those with high affinity to κ opioid receptors, but are known to bind and activate also µ- and Δ-opioid receptors. In addition, the peak intensities of a novel endogenous metabolite of α-neoendorphin lacking the N-terminal tyrosine correlated positively with dyskinesia severity. MALDI IMS of striatal sections from Pdyn knockout mice verified the identity of fully processed dynorphin peptides and the presence of endogenous des-tyrosine α-neoendorphin. Des-tyrosine dynorphins display reduced opioid receptor binding and this points to possible novel nonopioid receptor mediated changes in the striatum of dyskinetic rats. Because des-tyrosine dynorphins can only be detected by mass spectrometry, as no antibodies are available, these findings highlight the importance of MALDI IMS analysis for the study of molecular dynamics in neurological diseases.

Oxytocin in the periaqueductal gray participates in pain modulation in the rat by influencing endogenous opiate peptides.

Periaqueductal gray (PAG) plays a very important role in pain modulation through endogenous opiate peptides including leucine-enkephalin (L-Ek), methionine-enkephalin (M-Ek), ß-endorphin (ß-Ep) and dynorphin A(1-13) (DynA(1-13)). Our pervious study has demonstrated that intra-PAG injection of oxytocin (OXT) increases the pain threshold, and local administration of OXT receptor antagonist decreases the pain threshold, in which the antinociceptive role of OXT can be reversed by pre-PAG administration of OXT receptor antagonist. The experiment was designed to investigate the effect of OXT on endogenous opiate peptides in the rat PAG during the pain process. The results showed that (1) the concentrations of OXT, L-Ek, M-Ek and ß-Ep, not DynA(1-13) in the PAG perfusion liquid were increased after the pain stimulation; (2) the concentrations of L-Ek, M-Ek and ß-Ep, not DynA(1-13) in the PAG perfusion liquid were decreased by the OXT receptor antagonist; (3) the increased pain threshold induced by the OXT was attenuated by naloxone, an opiate receptor antagonist; and (4) the concentrations of L-Ek, M-Ek and ß-Ep, not DynA(1-13) in the PAG perfusion liquid were increased by exogenous OXT administration. The data suggested that OXT in the PAG could influence the L-Ek, M-Ek and ß-Ep rather than DynA(1-13) to participate in pain modulation, i.e. OXT in the PAG participate in pain modulation by influencing the L-Ek, M-Ek and ß-Ep rather than DynA(1-13).

Prodynorphin mutations cause the neurodegenerative disorder spinocerebellar ataxia type 23.

Spinocerebellar ataxias (SCAs) are dominantly inherited neurodegenerative disorders characterized by progressive cerebellar ataxia and dysarthria. We have identified missense mutations in prodynorphin (PDYN) that cause SCA23 in four Dutch families displaying progressive gait and limb ataxia. PDYN is the precursor protein for the opioid neuropeptides, α-neoendorphin, and dynorphins A and B (Dyn A and B). Dynorphins regulate pain processing and modulate the rewarding effects of addictive substances. Three mutations were located in Dyn A, a peptide with both opioid activities and nonopioid neurodegenerative actions. Two of these mutations resulted in excessive generation of Dyn A in a cellular model system. In addition, two of the mutant Dyn A peptides induced toxicity above that of wild-type Dyn A in cultured striatal neurons. The fourth mutation was located in the nonopioid PDYN domain and was associated with altered expression of components of the opioid and glutamate system, as evident from analysis of SCA23 autopsy tissue. Thus, alterations in Dyn A activities and/or impairment of secretory pathways by mutant PDYN may lead to glutamate neurotoxicity, which underlies Purkinje cell degeneration and ataxia. PDYN mutations are identified in a small subset of ataxia families, indicating that SCA23 is an infrequent SCA type (∼0.5%) in the Netherlands and suggesting further genetic SCA heterogeneity.

Effects of early vs. late initiation of levodopa treatment in hemiparkinsonian rats.

We investigated the effect of early vs. late initiation of levodopa treatment on dyskinetic movements, rotational behavior and molecular markers in hemiparkinsonian rats. Male Sprague-Dawley rats received a unilateral 6-hydroxydopamine (6-OHDA) administration in the nigrostriatal pathway. Rats were divided into three groups treated with: (i) levodopa (6 mg/kg) twice daily for 22 days starting at 4 weeks after 6-OHDA (Early group); (ii) levodopa at the same dose, regimen and duration but starting at 12 weeks after 6-OHDA (Late group), and (iii) saline starting at 4 weeks after 6-OHDA and continuing until the Late group finished treatment. Dyskinesias were quantified on days 1 and 22 of levodopa treatment. Striatal expression of preproenkephalin and preprodynorphin mRNAs, subthalamic cytochrome oxidase mRNA, and glutamate decarboxylase 67 mRNA in the pars reticulata of the substantia nigra was measured by in-situ hybridization. After 22 days of levodopa treatment, the percentage of rats showing dyskinesia was lower in the Early group than in the Late group (60% vs. 100%, respectively). No significant differences in total dyskinesia score were observed between both groups with the exception of the orolingual dyskinesias that were significantly less frequent in the Late group (P < 0.01). No significant differences were observed in the molecular markers between the Early and Late groups. Prompt initiation of levodopa treatment might be able to delay some of the basal ganglia molecular and circuitry changes underlying the development of dyskinesia but, once developed, they are behaviorally and molecularly similar to those appearing after late initiation of levodopa.

Dynorphin immunoreactive fibers contact GnRH neurons in the human hypothalamus.

Dynorphin, an endogenous opioid peptide, mediates progesterone-negative feedback on gonadotropin-releasing hormone (GnRH) neurons in other species. The role of dynorphin in humans is unclear. The objective of this study was to determine if dynorphin fibers have close contacts with GnRH neurons in humans. Dual-label immunocytochemistry was performed on postmortem human hypothalamic tissue. The majority of GnRH neurons, 87.5%, had close contacts with dynorphin fibers and multiple close contacts were common, 62.5%. There were no regional differences between the hypothalamus and preoptic area in the distribution of close contacts. More close contacts were identified on the GnRH dendrites compared to the cell bodies (P < .001), but this difference was not significant when corrected for length. In conclusion, dynorphin fibers form close contacts with GnRH neurons in humans. This neuroanatomical evidence may suggest that dynorphin has effects on GnRH regulation in humans as seen in other species.

Identification, characterization and quantification of specific neuropeptides in rat spinal cord by liquid chromatography electrospray quadrupole ion trap mass spectrometry.

Substance P and CGRP play a central role in neuropathic pain development and maintenance. Additionally, dynorphin A is an endogenous ligand of opioid receptors implicated in the modulation of neurotransmitters including neuropeptides, such as substance P and CGRP. This manuscript proposes a method to characterize, identify and quantify substance P, CGRP and dynorphin A in rat spinal cord by HPLC-ESI/MS/MS. Rat spinal cords were collected and homogenized into a TFA solution. Samples were chromatographed using a microbore C(8) 100 x 1 mm column and a 19 min linear gradient (0:100 --> 40:60; ACN:0.2% formic acid in water) at a flow rate of 75 microL/min for a total run time of 32 min. The peptides were identified in rat spinal cord based on full-scan MS/MS spectra. Substance P, CGRP and dynorphin A were predominantly identified by the presence of specific b CID fragments. Extracted ion chromatogram (XIC) suggested selected mass transitions of 674 --> [600 + 254], 952 --> [1215 + 963] and 717 --> [944 + 630] for substance P, CGRP and dynorphin A can be used for isolation and quantitative analysis. A linear regression (weighted 1/x) was used and coefficients of correlations (r) ranging from 0.990 to 0.999 were observed. The precision (%CV) and accuracy (%NOM) observed were 10.9-14.4% and 8.9-14.2%, 8.8-13.0% and 91.0-110.2% and 97.2-107.3% and 91.8-97.3% for substance P, CGRP and dynorphin A respectively. Following the analysis of rat spinal cords, the mean endogenous concentrations were 110.7, 2541 and 779.4 pmol/g for substance P, CGRP and dynorphin A respectively. The results obtained show that the method provides adequate figures of merit to support targeted peptidomic studies aimed to determine neuropeptide regulation in animal neuropathic and chronic pain models.

Practical aspects of in vivo detection of neuropeptides by microdialysis coupled off-line to capillary LC with multistage MS.

A method using capillary liquid chromatography-triple-stage mass spectrometry (LC-MS(3)) to determine endogenous opioid peptides in microdialysis samples collected in vivo was developed, validated, and applied to measurements in the rat striatum. Peptides in dialysate rapidly degraded when stored at room temperature or -80 degrees C. Adding acetic acid to a final concentration of 5% stabilized the peptides for 5 days allowing storage of fractions and off-line measurements which proved more convenient and reliable than previously used on-line methods. Study of the effect of dialysis flow rate from 0.2 to 2 microL/min and column inner diameter (i.d.) from 25 to 75 microm on the relative signal obtained for peptides revealed that lowest flow rates and smallest column i.d. gave the highest relative signal. The method was tested for 10 different neuropeptides and limits of detection (LODs) were from 0.5 to 60 pM (4 microL samples) for most. beta-Endorphin had an LOD of 5 nM when detected directly, but it could be quantitatively determined by detecting a characteristic peptide produced by tryptic digestion with an LOD of 3 pM. This approach may prove useful for other large neuropeptides as well. The method was used to determine met-enkephalin, leu-enkephalin, dynorphin A(1-8), and beta-endorphin in vivo. Endomorphin 1 and 2 were below the detection limit of the method in vivo. Quantitative determination of leu-enkephalin using external calibration was verified by standard addition experiments. The improvements over previous approaches using capillary LC-MS(n) make in vivo neuropeptide monitoring more practical and feasible for a variety of neuropeptides.

Defective neuropeptide processing and ischemic brain injury: a study on proprotein convertase 2 and its substrate neuropeptide in ischemic brains.

Using a focal cerebral ischemia model in rats, brain ischemia-induced changes in expression levels of mRNA and protein, and activities of proprotein convertase 2 (PC2) in the cortex were examined. In situ hybridization analyses revealed a transient upregulation of the mRNA level for PC2 at an early reperfusion hour, at which the level of PC2 protein was also high as determined by immunocytochemistry and western blotting. When enzymatic activities of PC2 were analyzed using a synthetic substrate, a significant decrease was observed at early reperfusion hours at which levels of PC2 protein were still high. Also decreased at these reperfusion hours were tissue levels of dynorphin-A(1-8) (DYN-A(1-8)), a PC2 substrate, as determined by radioimmunoassay. Further examination of PC2 protein biosynthesis by metabolic labeling in cultured neuronal cells showed that in ischemic cells, the proteolytic processing of PC2 was greatly attenuated. Finally, in mice, an intracerebroventricular administration of synthetic DYN-A(1-8) significantly reduced the extent of ischemic brain injury. In mice those lack an active PC2, exacerbated brain injury was observed after an otherwise non-lethal focal ischemia. We conclude that brain ischemia attenuates PC2 and PC2-mediated neuropeptide processing. This attenuation may play a role in the pathology of ischemic brain injury.

Relationship of spinal dynorphin neurons to delta-opioid receptors and estrogen receptor alpha: anatomical basis for ovarian sex steroid opioid antinociception.

Pharmacological and behavioral studies suggest that spinal delta- and kappa-opioid antinociceptive systems are functionally associated with ovarian sex steroids. These interactions can be demonstrated specifically during pregnancy or hormone-simulated pregnancy (HSP). The analgesia associated with both conditions can be abolished by blockade of either spinal kappa-opioid receptors or delta-opioid receptors (DOR). Furthermore, both dynorphin (DYN) release (J Pharmacol Exp Ther 298:1213-1220, 2001) and the processing of the DYN precursor (J Neurochem 65:1374-1380, 1995) are significantly increased in the spinal cord during HSP. We undertook the current study to determine whether DYN, DOR, and estrogen receptor alpha (ERalpha) share anatomical relationships that permit their direct interaction. Coexpression of DOR or ERalpha by DYN neurons was assessed using fluorescence immunohistochemistry and a synaptosomal release assay. Findings show that ERalpha and DYN are coexpressed. Moreover, in the spinal cord of HSP animals, there were significant increases in the number of DYN-immunoreactive (DYN-ir) cells, ERalpha-ir cells, cells double-labeled for DYN-ir and ERalpha-ir and the proportion of DYN-ir cells coexpressing ERalpha. Some varicose fibers in the spinal cord dorsal horn and intermediate gray matter that expressed DYN-ir also expressed DOR-ir. Activation of DORs located on DYN terminals was sufficient to inhibit K(+)-evoked DYN release. These data define, at least in part, the anatomical substrates that may be relevant to the antinociception of gestation and its hormonal simulation. Furthermore, they provide a framework for understanding sex-based nociception and antinociception and suggest novel strategies for treating pain.

Dynorphin and stress-related peptides in rat locus coeruleus: contribution of amygdalar efferents.

The interaction between the stress axis and endogenous opioid systems has gained substantial attention, because it is increasingly recognized that stress alters individual sensitivity to opiates. One site at which opiates and stress substrates may interact to have global effects on behavior is within the locus coeruleus (LC). We have previously described interactions of several opioid peptides [e.g., proopiomelanocortin, enkephalin (ENK)] with the stress-related peptide corticotropin-releasing factor (CRF) in the LC. To examine further the interactions among dynorphin (DYN), ENK, and CRF in the LC, sections were processed for detection of DYN and CRF or DYN and ENK in rat brain. DYN- and CRF-containing axon terminals overlapped noradrenergic dendrites in this region. Dual immunoelectron microscopy showed coexistence of DYN and CRF; 35% of axon terminals containing DYN were also immunoreactive for CRF. In contrast, few axon terminals contained both DYN and ENK. A potential DYN/CRF afferent is the central nucleus of the amygdala (CeA). Dual in situ hybridization showed that, in CeA neurons, 31% of DYN mRNA-positive cells colocalized with CRF mRNA, whereas 53% of CRF mRNA-containing cells colocalized with DYN mRNA. Finally, to determine whether limbic DYN afferents target the LC, the CeA was electrolytically lesioned. Light-level densitometry of DYN labeling in the LC showed a significant decrease in immunoreactivity on the side of the lesion. Taken together, these data indicate that DYN- and CRF-labeled axon terminals, most likely arising from amygdalar sources, are positioned dually to affect LC function, whereas DYN and ENK function in parallel.

Kisspeptin neurons in the arcuate nucleus of the ewe express both dynorphin A and neurokinin B.

Kisspeptin is a potent stimulator of GnRH secretion that has been implicated in the feedback actions of ovarian steroids. In ewes, the majority of hypothalamic kisspeptin neurons are found in the arcuate nucleus (ARC), with a smaller population located in the preoptic area. Most arcuate kisspeptin neurons express estrogen receptor-alpha, as do a set of arcuate neurons that contain both dynorphin and neurokinin B (NKB), suggesting that all three neuropeptides are colocalized in the same cells. In this study we tested this hypothesis using dual immunocytochemistry and also determined if kisspeptin neurons contain MSH or agouti-related peptide. To assess colocalization of kisspeptin and dynorphin, we used paraformaldehyde-fixed tissue from estrogen-treated ovariectomized ewes in the breeding season (n = 5). Almost all ARC, but no preoptic area, kisspeptin neurons contained dynorphin. Similarly, almost all ARC dynorphin neurons contained kisspeptin. In experiment 2 we examined colocalization of kisspeptin and NKB in picric-acid fixed tissue collected from ovary intact ewes (n = 9). Over three quarters of ARC kisspeptin neurons also expressed NKB, and a similar percentage of NKB neurons contained kisspeptin. In contrast, no kisspeptin neurons stained for MSH or agouti-related peptide. These data demonstrate that, in the ewe, a high percentage of ARC kisspeptin neurons also produce dynorphin and NKB, and we propose that a single subpopulation of ARC neurons contains all three neuropeptides. Because virtually all of these neurons express estrogen and progesterone re-ceptors, they are likely to relay the feedback effects of these steroids to GnRH neurons to regulate reproductive function.

Influence of acute and chronic treadmill exercise on rat plasma lactate and brain NPY, L-ENK, DYN A1-13.

This study was designed to investigate the effect of acute and chronic high-intensity treadmill exercise on changes in plasma lactate and brain neuropeptide (NPY), leucine-enkephalin (L-ENK), and dynorphin A(1-13) (DYN A(1-13)). Avidin-biotin complex (ABC) immunohistochemistry and image pattern analysis were used to observe the effect of chronic (total 7 weeks) and acute treadmill exercise (an initial speed of 15 m min(-1) gradually increased to 35 m min(-1) with 0 degrees, 20-25 min per day duration) on the changes of NPY, L-ENK, and DYN A(1-13) in different areas of rat brain. Plasma lactate was also measured in response to such exercise. Compared with preexercise control (P < 0.01), plasma lactate concentration significantly increased in the immediate postexercise; but it returned to the normal level soon after the 30 min postexercise. The content of NPY in paraventricular (PVN), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei continued to increase in 0, 30, and 180 min postexercise compared with preexercise control (P < 0.01). The content of L-ENK in caudate-putamen (CPu) significantly increased in the immediate postexercise compared with preexercise control (P < 0.01), but it gradually returned to the normal level after the 180 min postexercise. However, the content of DYN A(1-13) in PVN rose substantially only in 30 min postexercise in comparison with the preexercise control (P < 0.01). Thus, different changes of NPY, L-ENK, and DYN A(1-13) in response to such high-intensity exercise depend on the brain region and the time examined, especially, the contents of NPY in different brain regions continuously remain at a high level after such high-intensity exercise. And this high level might reduce energy expenditure and thus contribute to the stimulation of brain NPY neurons.