Search

Local ENKergic circuits involved in nociceptive modulation within the SDH: ENKergic neurons receive excitatory inputs from primary afferents and send projections toward PBN-projecting neurons. In the condition of pain stimuli, primary afferents may directly activate PBN-projecting neurons within lamina I to transmit pain signals toward upper brain structures, as well as ENKergic interneurons within lamina II to modulate spinal pain processing. ENK might inhibit presynaptic glutamate release via presynaptic DORs through non-synaptic mechanisms and suppress the excitability of projection neurons via postsynaptic DORs. • Non-peptidergic afferents were in close apposition to PBN-projecting neurons within lamina I. • Non-peptidergic afferents were in close apposition to ENKergic neurons within lamina II. • ENKergic terminals were in close apposition to PBN-projecting neurons within lamina I. • ENK might inhibit the glutamatergic transmission towards PBN-projecting neurons via presynaptic and postsynaptic DORs. Enkephalin (ENK) has been implicated in pain modulation within the spinal dorsal horn (SDH). Revealing the mechanisms underlying ENK analgesia entails the anatomical and functional knowledge of spinal ENK-ergic circuits. Herein, we combined morphological and electrophysiological studies to unravel local ENK-ergic circuitry within the SDH. First, the distribution pattern of spinal ENK-ergic neurons was observed in adult preproenkephalin (PPE)-GFP knock-in mice. Next, the retrograde tracer tetramethylrhodamine (TMR) or horseradish peroxidase (HRP) was injected into the parabrachial nucleus (PBN) in PPE-GFP mice. Immunofluorescent staining showed I-isolectin B4 (IB4) labeled non-peptidergic afferents were in close apposition to TMR-labeled PBN-projecting neurons within lamina I as well as PPE-immunoreactivity (-ir) neurons within lamina II. Some TMR-labeled neurons were simultaneously in close association with both IB4 and PPE-ir terminals. Synaptic connections of these components were further confirmed by electron microscopy. Finally, TMR was injected into the PBN in adult C57BL/6 mice. Whole-cell patch recordings showed that δ-opioid receptor (DOR) agonist, [D-Pen2,5]-enkephalin (DPDPE, 1 µM), significantly reduced the frequency of miniature excitatory postsynaptic current (mEPSC) and decreased the activity of TMR-labeled neurons. In conclusion, spinal ENKergic neurons receive direct excitatory inputs from primary afferents, which might be directly recruited to release ENK under the condition of noxious stimuli; ENK could inhibit the glutamatergic transmission towards projecting neurons via presynaptic and postsynaptic DORs. These morphological and functional evidence may explain the mechanisms underlying the analgesic effects exerted by ENK within the SDH. [ABSTRACT FROM AUTHOR]

The intergeniculate leaflet (IGL) is a flat thalamic nucleus implicated in the modulation of circadian rhythmicity. In rat, two main GABAergic subpopulations can be distinguished in the IGL: neurons synthesizing neuropeptide Y (NPY), which directly innervates the suprachiasmatic nuclei, and enkephalinergic cells, which connect contralaterally located leaflets. The aim of this study was to evaluate possible effects of inner IGL neurotransmitters on the spontaneous and synaptic activity of IGL neurons. The data presented in this article provide evidence that enkephalin, and not NPY, could act upon the majority of IGL neurons. Moreover, we investigated the type of opioid receptor activated by enkephalin and showed that the μ-receptor is functionally predominant in the IGL. The application of met-enkephalin not only robustly hyperpolarized IGL neurons (both putatively NPY-synthesizing and putatively enkephalinergic neurons), but it also was able to inhibit GABAergic and glutamatergic synaptic transmission. Based on this and previous studies, we hypothesize that IGL enkephalinergic neurons may act as powerful interneurons that inhibit themselves and NPY-synthesizing neurons, also in the contralaterally located IGL. [ABSTRACT FROM AUTHOR]

Using an immunohistochemical technique, we mapped the immunoreactive structures containing methionine-enkephalin-Arg 6 -Gly 7 -Leu 8 (Met-8) (a marker for the pro-enkephalin system) in the human diencephalon. Compared with previous studies, we observed a more widespread distribution of Met-8 in the human diencephalon. Met-8-immunoreactive cell bodies and fibers exhibited a more widespread distribution in the hypothalamus than in the thalamus. We observed six populations of Met-8-immunoreactive cell bodies. These perikarya were observed in the paratenial thalamic nucleus, ventromedial and dorsomedial hypothalamic nuclei, lateral hypothalamic area, pallidohypothalamic nucleus and in the paraventricular hypothalamic nucleus (posterior part). In the thalamus, Met-8-immunoreactive fibers were primarily observed in the midline region, whereas in the hypothalamus, these fibers were widely distributed. In general, a moderate/low density of Met-8-immunoreactive fibers was observed in the diencephalic nuclei. A moderate density was observed in the paraventricular thalamic nucleus, reuniens thalamic nucleus, lateral and medial geniculate nuclei, dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus (posterior part) and ventromedial hypothalamic nucleus. The present study is the first to demonstrate the presence of clusters of Met-8-immunoreactive cell bodies in the human thalamus and hypothalamus, the distribution of fibers containing neuropeptides in the hypothalamus and the presence of these fibers in several thalamic nuclei. This neuroanatomical study will serve to elucidate the physiological roles of Met-8 in future studies of the human diencephalon. [ABSTRACT FROM AUTHOR]

Maternal consumption of a high-fat diet (HFD) during pregnancy is found to stimulate the genesis of hypothalamic orexigenic peptide neurons in the offspring, while HFD intake in adult animals produces a systemic low-grade inflammation which increases neuroimmune factors that may affect neurogenesis and neuronal migration. Building on this evidence and our recent study showing that the inflammatory chemokine, CCL2, stimulates the migration of hypothalamic neurons and expression of orexigenic neuropeptides, we tested here the possibility that prenatal exposure to a HFD in rats affects this chemokine system, both CCL2 and its receptors, CCR2 and CCR4, and alters its actions on hypothalamic neurons, specifically those expressing the neuropeptides, enkephalin (ENK) and galanin (GAL). Using primary dissociated hypothalamic neurons extracted from embryos on embryonic day 19, we found that prenatal HFD exposure compared to chow control actually reduces the expression of CCL2 in these hypothalamic neurons, while increasing CCR2 and CCR4 expression, and also reduces the sensitivity of hypothalamic neurons to CCL2. The HFD abolished the dose-dependent, stimulatory effect of CCL2 on the number of migrated neurons and even shifted its normal stimulatory effect on migrational velocity and distance traveled by control neurons to an inhibition of migration. Further, it abolished the dose-dependent, stimulatory effect of CCL2 on neuronal expression of ENK and GAL. These results demonstrate that prenatal HFD exposure greatly disturbs the functioning of the CCL2 chemokine system in embryonic hypothalamic neurons, reducing its endogenous levels and ability to promote the migration of neurons and their expression of orexigenic peptides. [ABSTRACT FROM AUTHOR]

The opioid system is involved in infant–mother bonds and adult–adult bonds in many species. We have previously shown that μ opioid receptors (MORs) and κ opioid receptors (KORs) are involved in regulating the adult attachment of the monogamous titi monkey. The present study sought to determine the distribution of MOR and KOR in the titi monkey brain using receptor autoradiography. We used [ 3 H][D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO) to label MORs and [ 3 H]U69,593 to label KORs. MOR binding was heterogeneous throughout the titi monkey brain. Specifically, MOR binding was observed in the cingulate gyrus (CG), striatum, septal regions, diagonal band, amygdala, hypothalamus, hippocampus, and thalamus. Binding was particularly dense in the septum, medial amygdala, paraventricular nucleus of the hypothalamus, mediodorsal thalamus with moderate binding in the nucleus accumbens. Consistent with other primate species, MOR were also observed in “neurochemically unique domains of the accumbens and putamen” (NUDAPs). In general KOR binding was more homogenous. KORs were primarily found in the CG, striatum, amygdala and hippocampus. Dense KOR binding was observed in the claustrum. Relative MOR and KOR binding in the titi monkey striatum was similar to other humans and primates, but was much lower compared to rodents. Relative MOR binding in the titi monkey hypothalamus was much greater than that found in rodents. This study was the first to examine MOR and KOR binding in a monogamous primate. The location of these receptors gives insight into where ligands may be acting to regulate social behavior and endocrine function. [ABSTRACT FROM AUTHOR]

Fat, ethanol, and nicotine share a number of properties, including their ability to reinforce behavior and produce overconsumption. To test whether these substances act similarly on the same neuronal populations in specific brain areas mediating these behaviors, we administered the substances short-term, using the same methods and within the same experiment, and measured their effects, in areas of the hypothalamus (HYPO), amygdala (AMYG), and nucleus accumbens (NAc), on mRNA levels of the opioid peptide, enkephalin (ENK), using in situ hybridization and on c-Fos immunoreactivity (ir) to indicate neuronal activity, using immunofluorescence histochemistry. In addition, we examined for comparison another reinforcing substance, sucrose, and also took measurements of stress-related behaviors and circulating corticosterone (CORT) and triglycerides (TG), to determine if they contribute to these substances’ behavioral and physiological effects. Adult Sprague–Dawley rats were gavaged three times daily over 5 days with 3.5 mL of water, Intralipid (20% v/v), ethanol (12% v/v), nicotine (0.01% w/v) or sucrose (22% w/v) (approximately 7 kcal/dose), and tail vein blood was collected for measurements of circulating CORT and TG. On day five, animals were sacrificed, brains removed, and the HYPO, AMYG, and NAc processed for single- or double-labeling of ENK mRNA and c-Fos-ir. Fat, ethanol, and nicotine, but not sucrose, increased the single- and double-labeling of ENK and c-Fos-ir in precisely the same brain areas, the middle parvocellular but not lateral area of the paraventricular nucleus, central but not basolateral nucleus of the AMYG, and core but not shell of the NAc. While having little effect on stress-related behaviors or CORT levels, fat, ethanol, and nicotine all increased circulating levels of TG. These findings suggest that the overconsumption of these three substances and their potential for abuse are mediated by the same populations of ENK-expressing neurons in specific nuclei of the hypothalamus and limbic system. [ABSTRACT FROM AUTHOR]

Highlights: [•] Intra-CeA losartan suppressed sodium intake induced by intra-CeA DAMGO in WD–PR rats. [•] Intra-CeA losartan reduced sodium intake induced by intra-CeA DAMGO in FURO/CAP rats. [•] Natriorexigenic effect of DAMGO is reduced by AT1 receptor blockade in the CeA. [ABSTRACT FROM AUTHOR]

Highlights: [•] Spinal ENKergic neurogenesis was restricted at E9.5 to E14.5. [•] Spinal ENKergic neurons underwent heterogeneous characteristics. [•] The birth date of ENKergic neurons is one determinant for their arrangement. [Copyright &y& Elsevier]

Highlights: [•] The majority of the GHRH perikarya form juxtapositions with enkephalinergic fibers. [•] Endorphinergic-GHRH associations are much less frequent. [•] Dynorphinergic-GHRH juxtapositions are extremely rare in the human hypothalamus. [•] Thus endorphins/dynorphins play a minor role in the regulation of growth via GHRH. [•] Enkephalinergic-GHRH contacts may represent the impact of opiates on growth. [ABSTRACT FROM AUTHOR]

Highlights: [•] CFA injection into hindpaw induced significant conditioned place avoidance CPA). [•] Low doses of systemic morphine inhibited CFA-induced CPA but had no effect on PWL. [•] I.t. morphine did not inhibit the display of CPA but significantly increased the PWL. [•] Intra-CeA DAMGO significantly inhibited the CPA but had no effect on the PWL. [•] The sensory and the affective dimensions are underpinned by different mechanisms. [Copyright &y& Elsevier]

Highlights: [•] Role of the Nacc core in the feed-forward dopaminergic circuitry was studied. [•] Stimulation of μ-opioid receptors in the VTA-induced contraversive circling. [•] This circling is inhibited by the dopamine receptor inhibition in the Nacc shell. [•] Dopamine receptor inhibition in the Nacc core changed this circling to pivoting. [•] The Nacc core is an essential link in the feed-forward circuitry from the Nacc shell. [Copyright &y& Elsevier]

Highlights: [•] Lateral hypothalamic hcrt-1 neurons co-express L-Enk. [•] Hcrt-1/L-Enk neurons innervate caudal NTS. [•] Injections of hcrt-1 and DADL into NTS elicit depressor and bradycardia responses. [•] DADL potentiates hcrt-1 cardiovascular responses. [•] δ- and μ-opioid receptors may be involved in mediating hcrt-1 cardiovascular responses. [ABSTRACT FROM AUTHOR]

Highlights: [•] Diversity of striosomes in mouse striatum was studied by immunohistochemistry. [•] Mu-opioid receptor-rich striosomes were located only in rostral half of the striatum. [•] Substance P-positive striosomes were located in rostral two-thirds of the striatum. [•] Labeling for enkephalin and SP showed dorsoventral differences in rostral striosomes. [•] The results suggest site-dependent regulation of striosomal neurons by opioids and SP. [ABSTRACT FROM AUTHOR]

Abstract: In adult rat striatum the dopamine D1–D2 receptor heteromer is expressed selectively in a subset of medium spiny neurons (MSNs) that coexpress the dopamine D1 and D2 receptors (D1R and D2R) as well as dynorphin (DYN) and enkephalin (ENK), with higher coexpression in nucleus accumbens (NAc) and much lower in the caudate putamen (CP). In the present study we showed that in neonatal striatal cultured neurons >90% exhibited the D1R/D2R–DYN/ENK phenotype. Similarly, in the striatum of juvenile rats (age 26–28days) coexpression of D1R and D2R was also coincident with the expression of both DYN and ENK. Quantification of the number of striatal MSNs exhibiting coexpression of D1R and D2R in juvenile rats revealed significantly lower coexpression in NAc shell, but not core, and CP than in adult rats. However, within MSNs that coexpressed D1R and D2R, the propensity to form the D1–D2 receptor heteromer did not differ between age groups. Consistent with reduced coexpression of the D1R and D2R, juvenile rats exhibited subsensitivity to D1–D2 receptor heteromer-induced grooming following activation by SKF 83959. Given the proposed role of D1R/D2R-coexpressing MSNs in the regulation of thalamic output, and the recent discovery that these MSNs exhibit both inhibitory and excitatory capabilities, these findings suggest that the functional regulation of neurotransmission by the dopamine D1–D2 receptor heteromer within the juvenile striatum may be significantly different than in the adult. [Copyright &y& Elsevier]

Abstract: Exposure to ethanol during the prenatal period contributes to increased alcohol consumption and preference in rodents and increased risk for alcoholism in humans. With studies in adult animals showing the orexigenic peptides, enkephalin (ENK), galanin (GAL) and orexin (OX), to stimulate ethanol consumption, the question addressed here is whether prenatal ethanol alters the development in utero of specific neurons that express these peptides. With reports describing suppressive effects of high doses of ethanol, we examined the offspring of dams gavaged from embryonic day 9 to parturition with a control solution or lower ethanol doses, 1 and 3g/kg/day, known to promote ethanol consumption in the offspring. To understand underlying mechanisms, measurements were taken in postnatal offspring of the expression of ENK in the hypothalamic paraventricular nucleus (PVN) and nucleus accumbens (NAc), GAL in the PVN, and OX in the perifornical lateral hypothalamus (PFLH) using real-time qPCR and in situ hybridization, and also of the cell proliferation marker, 5-bromo-2-deoxyuridine (BrdU), and its double-labeling with either neuronal nuclei (NeuN), a marker of mature neurons, or the peptides. On postnatal day 15 (P15), after two weeks without ethanol, the offspring showed increased expression of ENK in the PVN and NAc core but not shell, GAL in the PVN, and OX in the PFLH. In these same areas, prenatal ethanol compared to control increased the density at birth (P0) of neurons expressing these peptides and at P0 and P15 of neurons double-labeling BrdU and NeuN, indicating increased neurogenesis. These BrdU-positive neurons were found to express ENK, GAL and OX, indicating that prenatal ethanol promotes neurogenesis in these specific peptide systems. There were no changes in gliogenesis or apoptosis. This increase in neurogenesis and density of peptide-expressing neurons suggests the involvement of these hypothalamic and accumbal peptide systems in mediating the increased alcohol consumption observed in prenatal ethanol-exposed offspring. [Copyright &y& Elsevier]

Abstract: Recent studies show that the non-opioid peptides, galanin (GAL) and orexin (OX), are similar to the opioid enkephalin (ENK) in being stimulated by dietary fat and also in enhancing the consumption of a high-fat diet (HFD). This suggests that, when an HFD is provided, these non-opioids may stimulate the opioid system to promote excess consumption of this diet. Using single- and double-labeling immunohistochemistry, the present study sought to identify possible neuroanatomical substrates for this close relationship. Focusing on the hypothalamic paraventricular nucleus (PVN), and particularly its anterior (aPVN), middle (mPVN) and posterior (pPVN) parts, the experiments examined whether GAL itself or the receptors for GAL and OX are stimulated by an HFD in the same areas and possibly the same neurons as ENK. Compared to animals fed a standard chow diet, rats consuming an HFD exhibited an increased density of medial parvocellular neurons immunoreactive (IR) for GAL in the mPVN and aPVN and for ENK in the mPVN and pPVN, distinguishing the mPVN as an area where both peptides were affected. While showing little evidence for GAL and ENK colocalization with a chow diet, double-labeling studies in HFD-fed rats revealed significant colocalization specifically in medial parvocellular neurons of the mPVN. Immediately posterior to this site, further analyses revealed a similar relationship between the OX 2 receptor (OX 2 R) and ENK in HFD-treated animals. While increasing the density of neurons immunoreactive for OX 2 R as well as for the GAL 1 receptor but not OX 1 receptor, HFD consumption increased the colocalization only of OX 2 R and ENK, specifically in the medial parvocellular neurons of the pPVN. These changes in HFD-fed rats, showing GAL and OX 2 R to colocalize with ENK exclusively in neurons of the medial parvocellular mPVN and pPVN, respectively, suggest possible neural substrates through which the non-opioid peptides may functionally interact with ENK when exposed to an HFD. [Copyright &y& Elsevier]

Abstract: Delta opioid receptor (DOR) is essential for neuronal survival against hypoxic/ischemic damages. However, current understanding on how DOR activation affects astrocytic functions under ischemia remains incomplete. The present study investigated the astroglial responses to [d-Ala2, d-Leu5] enkephalin (DADLE) (a selective DOR agonist)-induced DOR activation after global cerebral ischemia. Adult male rats were preimplanted with intracerebral cannula and subjected to global ischemia for 10 min. The rats were divided into four groups: normal group (without any procedure), sham group (sham procedure with intracerebroventricular injection of ACSF), I/R group (ischemia procedure with intracerebroventricular injection of ACSF) and DAD-treated group (ischemia procedure with intracerebroventricular injection of DADLE). Hippocampal CA1 neuronal survival and activation of astrocytes were measured in the animals at 72 h post-ischemia. The distribution and phenotypes of p-Akt and active caspase-3 were also determined. The ischemic injury resulted in a significant neuronal loss and an increase in the dying astrocytes in the hippocampal CA1 region as compared with those in the sham animals (200.7±22.7/mm2 vs. 6.6±3.1/mm2, P<0.001). Improved neuronal survival in the DAD-treated animals was evident, which was accompanied by less dying astrocytes and enhanced astrocytes reaction with more active astrocytes than that in the I/R group (267.6±13.2/mm2 vs. 157.0±18.1/mm2, P<0.01) and a significantly increased immunoreactivity of p-Akt. However, the active caspase-3 positive cells were also evident in DAD-treated group (313.0±23.1/mm2) and significantly increased as compared with those of the sham group (159.0±15.8/mm2, P<0.001) or I/R group (193.6±26.2/mm2, P<0.01). Most of the active caspase-3-expressing cells were colabeled with glial fibrillary acidic protein (GFAP), an astrocytes marker. We conclude that the post-ischemic treatment with DADLE promotes beneficial astrocytes activation and induces astroglial apoptosis 72 h after reperfusion which may be involved in reducing their harmful effect to neurons survival. [Copyright &y& Elsevier]

Abstract: In Na+ appetite neurobiology, it is essential to investigate whether endogenous opioids modulate the output of the neural substrates that are involved in both the detection and integration of Na+ deficiency and the motivational aspect of Na+ intake. Thus, evaluating the recruitment dynamics of enkephalin (ENK)-containing and/or mu-opioid receptor (μ-OR)-expressing neurons in close correlation with the hydromineral state of the rat might provide useful information regarding the role of the opioid system in regulating the central network that controls water and Na+ intake. Furosemide was used to deplete both fluid volume and the Na+ content of the extracellular fluid (ECF) compartment when combined with water repletion and a short-term Na+-free diet. Na+ restoration in the ECF compartment was achieved by providing unrestricted access to both saline (0.3 M NaCl) and water. Combining in situ hybridization (against ENK and μ-OR mRNA) and immunohistochemistry (against Fos) revealed a specific pattern of hypovolemia-induced Fos expression in the enkephalinergic subpopulations of the central amygdala, in the oval nucleus of the bed nucleus of the stria terminalis and in the nucleus accumbens shell. Hypovolemia also induced transient Fos expression in μ-OR-expressing neurons in the same nuclei and in the median preoptic nucleus and subfornical organ. However, this specific hydromineral state did not activate the ENK and/or μ-OR-expressing neurons in the lateral parabrachial nucleus or in the medial nucleus of the solitary tract. These results implicate the ENK/μ-OR system as a putative facilitator of Na+ intake in discrete regions of the forebrain, possibly by modulating the hedonic and reward value of Na+ by increasing ENK release in these regions. [Copyright &y& Elsevier]

Abstract: Chemical coding of sympathetic preganglionic neurons (SPN) suggests that the chemical content of subpopulations of SPN can define their function. Since neuropeptides, once synthesized are transported to the axon terminal, most demonstrated chemical coding has been identified using immunoreactive terminals at the target organ. Here, we use a different approach to identify and quantify the subpopulations of SPN that contain the mRNA for pituitary adenylate cyclase activating polypeptide (PACAP) or enkephalin. Using double-labeled immunohistochemistry combined with in situ hybridization (ISH) we firstly identified the distribution of these mRNAs in the spinal cord and determined quantitatively, in Sprague–Dawley rats, that many SPN at the T4-T10 spinal level contain preproPACAP (PPP+, 80±3%, n=3), whereas a very small percentage contain preproenkephalin (PPE+, 4±2%, n=4). A similar neurochemical distribution was found at C8-T3 spinal level. These data suggest that PACAP potentially regulates a large number of functions dictated by SPN whereas enkephalins are involved in few functions. We extended the study to explore those SPN that control adrenal chromaffin cells. We found 97±5% of adrenally projecting SPN (AP-SPN) to be PPP+ (n=4) with only 47±3% that were PPE+ (n=5). These data indicate that adrenally projecting PACAPergic SPN regulate both adrenal adrenaline (Ad) and noradrenaline (NAd) release whereas the enkephalinergic SPN subpopulation must control a (sub) population of chromaffin cells – most likely those that release Ad. The sensory innervation of the adrenal gland was also determined. Of the few adrenally projecting dorsal root ganglia (AP-DRG) observed, 74±12% were PPP+ (n=3), whereas 1±1% were PPE+ (n=3). Therefore, if sensory neurons release peptides to the adrenal medulla, PACAP is most likely involved. Together, these data provide a neurochemical basis for differential control of sympathetic outflow particularly that to the adrenal medulla. [Copyright &y& Elsevier]

Abstract: G protein-coupled opioid receptors undergo desensitization after prolonged agonist exposure. Recent in vitro studies of μ-opioid receptor (MOR) signaling revealed an involvement of phosphoinositide 3-kinases (PI3K) in agonist-induced MOR desensitization. Here we document a specific role of the G protein-coupled class IB isoform PI3Kγ in MOR desensitization in mice and isolated sensory neurons. The tail-withdrawal nociception assay evidenced a compromised morphine-induced tolerance of PI3Kγ-deficient mice compared to wild-type animals. Consistent with a role of PI3Kγ in MOR signaling, PI3Kγ was expressed in a subgroup of small-diameter dorsal root ganglia (DRG) along with MOR and the transient receptor potential vanilloid type 1 (TRPV1) receptor. In isolated DRG acute stimulation of MOR blocked voltage-gated calcium currents (VGCC) in both wild-type and PI3Kγ-deficient DRG neurons. By contrast, following long-term opioid administration the attenuating effect of MOR was strongly compromised in wild-type DRG but not in PI3Kγ-deficient DRG. Our results uncover PI3Kγ as an essential modulator of long-term MOR desensitization and tolerance development induced by chronic opioid treatment in sensory neurons. [Copyright &y& Elsevier]

Abstract: The delta opioid receptor (DOR) agonist [d-Ala2, d-Leu5] enkephalin (DADLE) has been implicated as a novel neuroprotective agent in the CNS. The current study was designed to evaluate the effects of intracerebroventricular (ICV) application of DADLE on neurological outcomes following asphyxial cardiac arrest (CA) in rats. Male Sprague-Dawley rats were randomly assigned to four groups: Sham group, CA group, DADLE group (DADLE+CA), and Naltrindole group (Naltrindole and DADLE+CA). All drugs were administered into the left cerebroventricle 30 min before CA. CA was induced by 8-min asphyxiation and the animals were resuscitated with a standardized method. DOR protein expression in the hippocampus was significantly increased in the CA group at 1 h after restoration of spontaneous circulation (ROSC) compared with the Sham group. As time progressed, expression of DOR proteins decreased gradually in the CA group. Treatment with DADLE alone or co-administration with Naltrindole reversed the down-regulation of DOR proteins in the hippocampus induced by CA at 24 h after ROSC. Compared with the CA group, the DADLE group had persistently better neurological functional recovery, as assessed by neurological deficit score (NDS) and Morris water maze trials. The number of surviving hippocampal CA1 neurons in the DADLE group was significantly higher than those in the CA group. However, administration of Naltrindole abolished most of the neuroprotective effects of DADLE. We conclude that ICV administration of DADLE 30 min before asphyxial CA has significant protective effects in attenuating hippocampal CA1 neuronal damage and neurological impairments, and that DADLE executes its effects mainly through DOR. [Copyright &y& Elsevier]

Abstract: Sympathetic preganglionic neurons (SPN) coexpress the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase and different peptides in their cell bodies, but can express them independently in separate varicosities, indicating that SPN segregate transmitters to different synapses. Consequently, there are populations of preganglionic varicosities (peptidergic and noncholinergic) that store peptides but not ACh. We studied in the cell bodies and axon processes of the rat SPN the expression and the proportional coexpression of the vesicular ACh transporter-like immunoreactivity (VAChT), a specific marker of cholinergic synaptic vesicles or ChAT-like immunoreactivity (ChAT), and the peptide methionine enkephalin-like immunoreactivity (mENK), and confirmed the presence of a population of SPN peptidergic, noncholinergic varicosities. We characterized these varicosities by exploring the occurrence of synaptophysin-like immunoreactivity (Syn), a marker of small clear vesicles, and synaptotagmin-like immunoreactivity (Syt), a preferential marker of large dense core vesicles. We found that (i) VAChT and mENK, like ChAT–mENK, were coexpressed in only 59% of the mENK-containing varicosities, although they colocalized in the SPN cell bodies; and (ii) almost 60% of the population of mENK-containing varicosities did not express Syn or Syt, and over 80% of the mENK-containing varicosities negative for VAChT also lacked Syn. These data prove that SPN segregate mENK from VAChT and ChAT, and show that most of the subset of mENKergic varicosities negative for VAChT also does not express Syn, suggesting the presence of a different vesicular pattern in these sympathetic preganglionic varicosities. [Copyright &y& Elsevier]

Abstract: Leu- and Met-enkephalin were the first endogenous opioid peptides identified in different mammalian species including the human. Comparative biochemical and bioinformatic evidence indicates that enkephalins are not limited to mammals. Various prodynorphin (PDYN) sequences in lower vertebrates revealed the presence of other enkephalin fingerprints in these precursor polypeptides. Among the novel enkephalins Ile-enkephalin (Tyr-Gly-Gly-Phe-Ile) was primarily observed in the African clawed frog (Xenopus laevis) PDYNs, while the structure of Phe-enkephalin (Tyr-Gly-Gly-Phe-Phe) was predicted by analyzing brain cDNA sequences encoding a PDYN of the African lungfish (Protopterus annectens). Ile-enkephalin can also be found in the PDYNs of four other fish species including the eel, bichir, zebrafish and tilapia, but no further occurrence for the Phe-enkephalin motif is available as yet. Based on sequencing data, the biological relevance of Phe- and Ile-enkephalin is suggested, because both of them can arise by regular posttranslational enzymatic processing of the respective neuropeptide precursors. In various receptor binding assays performed on rat brain membrane preparations both of the new peptides turned out to be moderate affinity opioids with a weak preference for the δ-opioid receptor (DOP) sites. Phe-enkephalin of the lungfish displayed rather unexpectedly low affinities toward the μ-opioid receptor (MOP) and DOP, while exhibiting moderate affinity toward the κ-opioid receptor (KOP). In receptor-mediated G-protein activation assays measured by the stimulation of [35S]GTPγS binding, Met-enkephalin produced the highest stimulation followed by Leu-enkephalin, Ile-enkephalin and Phe-enkephalin, whereas the least efficacious among these endogenous peptides was still more effective than the prototype opiate agonist morphine in these functional tests. [Copyright &y& Elsevier]

Abstract: We previously demonstrated that naloxone administration produces a robust conditioned place aversion (CPA) in opiate-naive rodents by blocking the action of enkephalins at μ opioid receptors (MORs). The aversive response to naloxone is potentiated by prior exposure to morphine. Morphine-induced MOR constitutive activity is hypothesized to underlie this enhanced effect of naloxone, an inverse agonist at the MOR. We sought additional evidence for the role of constitutively active MORs in this morphine-induced enhancement using the pro-enkephalin knockout (pENK−/−) mouse, which is devoid of naloxone CPA in the morphine-naive state. Naloxone, but not the neutral antagonist, 6-β-naloxol, produced CPA and physical withdrawal signs in pENK−/− mice when administered 2 h, but not 20 h, after morphine administration. Naloxone-precipitated physical withdrawal signs were attenuated in the pENK−/− mice relative to wild-type (WT) animals. In both WT and pENK−/− mice, naloxone-precipitated withdrawal jumping was greatest when naloxone was administered 2 h after morphine treatment and diminished at 3 h, in agreement with previous estimates of the time course for morphine-induced MOR constitutive activity in vitro. However, naloxone regained an ability to precipitate physical withdrawal in the WT, but not the pENK−/− mice when administered 4.5 h after morphine administration. Taken together, the data suggest that a compensatory increase in enkephalin release during spontaneous morphine withdrawal promotes a second period of MOR constitutive activity in WT mice that is responsible for the enhanced naloxone aversion observed in such animals even when naloxone is administered 20 h after morphine. The endogenous enkephalin system and MOR constitutive activity may therefore play vital roles in hedonic homeostatic dysregulation following chronic opiate administration. [Copyright &y& Elsevier]

Abstract: Phosphorylation of specific sites in the second intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo, affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the second intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. Pretreatment with the antagonist naloxone blocked the fentanyl-induced increase. Furthermore, mutant mice lacking MOP-R showed only non-specific nuclear MOR-P2-IR before or after fentanyl treatment, confirming the specificity of the MOR-P2 antibodies. To assess whether MOP-R phosphorylation occurs following endogenous opioid release, we induced chronic neuropathic pain by partial sciatic nerve ligation (pSNL), which caused a significant increase in MOR-P2-IR in the striatum. pSNL also induced signs of mu opioid receptor tolerance demonstrated by a rightward shift in the morphine dose response in the tail withdrawal assay and by a reduction in morphine conditioned place preference (CPP). Mutant mice selectively lacking all forms of the β-endorphin peptides derived from the proopiomelanocortin (Pomc) gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid β-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance. [Copyright &y& Elsevier]

Abstract: Complex behaviors such as those associated with reward to unconditioned positive reinforcers are polygenic processes. In studies using genetically modified mice specific for the endogenous opioid systems an observed phenotype in a complex behavior is likely to be dependent on interacting genes which, in inbred mouse lines, influence that phenotype. To address this issue we examined operant responding for palatable food reinforcers in mice lacking the expression of β-endorphin, enkephalin or both peptides congenic to two different genetic backgrounds; C57BL/6J and DBA/2J. These two inbred strains were chosen because their endogenous opioid states differ and they respond differently to exogenous opioids in many behavioral assays. We found that wildtype and mutant C57BL/6J mice acquired operant responding for food reinforcers faster than DBA/2J mice, regardless of their opioid genotype. Although wildtype DBA/2J mice had a significant deficit in acquisition of bar-pressing behavior to reach a pre-established performance criterion, no subsequent deficit was observed under two different schedules of reinforcement. Additionally, we found that mice lacking enkephalin had decreased motivation to bar press for palatable food reinforcers under a progressive ratio regardless of sex or background strain. In contrast, the only subset of β-endorphin-deficient mice that had decreased motivation to bar press under a progressive ratio was males on the C57BL/6J background. Of the two classical endogenous opioid peptides with preferential activation of the mu opioid receptor, the knockout models would suggest that enkephalins play a more consistent role than β-endorphin in mediating the motivation for food reward when tested under a progressive ratio. [Copyright &y& Elsevier]

Abstract: The endogenous pentapeptide enkephalins bind to the mu and delta opioid receptors, with a slightly higher affinity for the latter. It remains a controversy regarding the respective physiological role of mu and delta opioid receptors in the control of emotion and motivation. One of the difficulties to investigate this problem is the low tonic extracellular release of enkephalins in various brain structures. To overcome this problem the synaptic levels of these pentapeptides were enhanced by inhibition of enzymes involved in their catabolism with the selective inhibitor H3N-CH(CH2-CH2-S-CH3)-CH2-S-S-CH2-CH(CH2ϕ)-CONH-CH(CH2ϕ)-COOCH2ϕ (RB101). This compound was shown to increase the extracellular levels and lifetime of endogenous enkephalins. Similar responses were obtained in wild-type and mu opioid receptor knockout mice following RB 101 administration in behavioral tests measuring locomotor activity, anxiety (elevated O-maze), and motivation (forced swim test and conditioned suppression of motility). In contrast, RB 101 led to antinociceptive responses only in wild-type animals using hot plate and tail immersion tests. These results clearly demonstrate the critical role of delta opioid receptors activated by the endogenous opioid peptides, in the physiological control of emotion- and motivation-related behaviors. In contrast, antinociceptive modulation, at least with respect to thermal nociceptive stimuli, involves enkephalin-activated mu opioid receptors. These findings could open new perspectives in the treatment of mood disorders using either inhibitors of enkephalin catabolism or delta opioid agonists. [Copyright &y& Elsevier]

This study examines the distribution of prostaglandin E2 receptors of subtype EP3 and EP4 among brain stem parabrachial neurons that were characterized with respect to their neuropeptide expression. By using a dual-labeling in situ hybridization method, we show that preprodynorphin mRNA expressing neurons in the dorsal and central lateral subnuclei express EP3 receptor mRNA. Such receptors are also expressed in preproenkephalin, calcitonin gene related peptide and preprotachykinin mRNA positive neurons in the external lateral subnucleus, whereas preprodynorphin mRNA expressing neurons in this subnucleus are EP receptor negative. In addition, EP3 receptor expression is seen among some enkephalinergic neurons in the Kölliker-Fuse nucleus. Neurons in the central part of the cholecystokininergic population in the regions of the superior lateral subnucleus express EP4 receptor mRNA, whereas those located more peripherally express EP3 receptors. Taken together with previous findings showing that discrete peptidergic cell groups mediate nociceptive and/or visceral afferent information to distinct brain stem and forebrain regions, the present results suggest that the processing of this information in the parabrachial nucleus is influenced by prostaglandin E2. Recent work has shown that prostaglandin E2 is released into the brain following peripheral immune challenge; hence, the parabrachial nucleus may be a region where humoral signaling of peripheral inflammatory events may interact with neuronal signaling elicited by the same peripheral processes. [Copyright &y& Elsevier]

Alterations in the opioid system in the hippocampal formation and some of the possible functional consequences were investigated in adult male rats that were prenatally exposed to either saline or morphine (10 mg/kg twice daily on gestational days 11–18). In situ hybridization and Northern blots were used to measure proenkephalin and prodynorphin mRNA, and radioimmunoassays quantified proenkephalin- and prodynorphin-derived peptide levels in the dentate gyrus, CA3, and CA1 subfields of the hippocampal formation. Prenatal morphine exposure in male rats decreases proenkephalin and increases prodynorphin mRNA selectively in the granule cell layer of the dentate gyrus. Similarly, met-enkephalin peptide levels are decreased and dynorphin B peptide levels are increased in the dentate gyrus but not CA3 or CA1 of prenatally morphine-exposed males. In addition, there are decreases in dynorphin-derived peptides in the CA3 subfield. Receptor autoradiography revealed increases in the density of μ but not δ receptor labeling in discrete strata of specific hippocampal subfields in morphine-exposed males. Because alterations in the hippocampal opioid system suggest possible alterations in the excitability of the hippocampal formation, changes in opioid regulation of seizures were examined. Morphine exposure, however, does not alter the latency to onset or number of episodes of wet dog shakes or clonic seizures induced by infusion of 10 nmol [d-Ala2, MePhe4, Gly-ol5]enkephalin into the ventral hippocampal formation. Interestingly, a naloxone (5 mg/kg) injection 30 min before bicuculline administration reverses the increased latency to onset of clonic and tonic–clonic seizures in morphine-exposed males. Thus, the present study suggests that exposure of rats to morphine during early development alters the hippocampal opioid system, suggesting possible consequences for hippocampal-mediated functions. [Copyright &y& Elsevier]

Overactivity of the striatopallidal pathway, associated with an enhancement of enkephalin expression, has been suggested to contribute to the development of parkinsonian symptoms. The aim of the present study was to examine whether the blockade of group I metabotropic glutamate receptors: subtypes 1 and 5 (mGluR1/5), or stimulation of group II: subtypes 2 and 3 (mGluR2/3) may normalize enkephalin expression in the striatopallidal pathway in an animal model of parkinsonism. The proenkephalin mRNA level measured by in situ hybridization in the striatum was increased by pretreatments with haloperidol (1.5 mg/kg s.c., three times, 3 h apart). Triple (3 h apart), bilateral, intrastriatal administration of selective antagonists of mGluR1: (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (3×5 μg/0.5 μl) or 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate (3×2.5 μg/0.5 μl), reversed the haloperidol-induced increases in proenkephalin mRNA levels in the rostral and central regions of the striatum. Similarly, repeated (6 times, 1.5 h apart), systemic injections of an antagonist of mGluR5, 2-methyl-6-(phenylethynyl)pyridine (6×10 mg/kg i.p.) counteracted an increase in the striatal proenkephalin mRNA expression elicited by haloperidol. None of the abovementioned antagonists of mGluR1 and mGluR5 per se influenced the proenkephalin expression. Differential effects were induced by agonists of the group II mGluRs, viz. (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine administered intraventricularly (3 times at 0.1–0.2 μg/4 μl, 3 h apart) increased both the normal and haloperidol-increased proenkephalin mRNA level, whereas (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate injected intrastriatally (3 times at 15 μg/0.5 μl, 3 h apart) was ineffective. The present study indicates that the blockade of striatal glutamate receptors belonging to the group I (mGluR1 and mGluR5) but not stimulation of the group II mGluRs may normalize the function of the striatopallidal pathway in an animal model of parkinsonism, which may be important for future antiparkinsonian therapy in humans. [Copyright &y& Elsevier]

Changes in preproenkephalin expression in the caudate and putamen have been linked to the development of L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias in primate models of Parkinson’s disease, although not all investigators have been able to confirm this association. Because nigrostriatal damage per se is associated with increases in striatal preproenkephalin mRNA levels, it is difficult to know if changes in transcript levels are a result of lesioning or concurrent L-DOPA treatment and resulting dyskinesias. To circumvent these difficulties, we measured striatal preproenkephalin mRNA levels in monkeys with L-DOPA-induced dyskinesias both with and without lesions of the nigrostriatal system. The latter model is not confounded by morphological and biochemical changes resulting from nigrostriatal damage. Monkeys were gavaged with L-DOPA (15 mg/kg) twice daily for a 2-week period and killed 3 days after treatment. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment alone resulted in an increase in preproenkephalin mRNA levels as previously shown. However, striatal transcript levels were similarly elevated in dyskinetic MPTP-lesioned animals treated with L-DOPA. In unlesioned animals, preproenkephalin mRNA levels were also similar in control and L-DOPA-treated dyskinetic monkeys. Because drug-induced changes in mRNA may not be sustained for a prolonged period after treatment, a second series of experiments were done in which animals were killed 3–4 h after the last dose of L-DOPA, but the results were similar to those obtained after 3 days. These data show that, while elevations in striatal preproenkephalin mRNA levels are associated with nigrostriatal damage, they are not linked to the development of L-DOPA-induced dyskinesias. These results thus question the importance of preproenkephalin mRNA in the pathogenesis of this disabling complication of L-DOPA therapy in Parkinson’s disease. [Copyright &y& Elsevier]