Opioidergic Neurons Research Articles

NNOS immunoreactivity co-localizes with GABAergic and cholinergic neurons, and associates with β-endorphinergic and met-enkephalinergic opioidergic fibers in rostral ventromedial medulla and A5 of the mouse

The present study examined the co-expression of neuronal nitric oxide synthase (nNOS) in the rostral ventromedial medulla (RVM) and A5 regions of the mouse brainstem within several neurochemical populations involved in nociceptive modulation. Double immunohistochemical methods showed that nNOS+ neurons do not co-localize with serotonergic neurons within any of these regions. Within the RVM, the nuclei raphe magnus and gigantocellularis contain a population of nNOS+/GAD67+ neurons, and within the paragigantocellularis lateralis, there is a smaller population of nNOS+/CHAT+ neurons. Further, nNOS+ neurons overlap the region of expression of β-endorphinergic and met-enkephalinergic fibers within the RVM. No co-labeling was found within the A5 for any of these populations. These findings suggest that pain-modulatory serotonergic neurons within the brainstem do not directly produce nitric oxide (NO). Rather, NO-producing neurons within the RVM belong to GABAergic and cholinergic cell populations, and are in a position to modulate or be modulated by local opioidergic neurons.

Interaction between the dopaminergic and opioidergic systems in dorsal hippocampus in modulation of formalin-induced orofacial pain in rats

The hippocampus is a region of the brain that serves several functions. The dopaminergic system acts through D1- and D2-like receptors to interfere in pain modulation and the opioid receptors play major roles in analgesic processes and there are obvious overlaps between these two systems. The present study investigated the interaction between the opioidergic and dopaminergic systems in the dorsal hippocampus (CA1) region for formalin-induced orofacial pain. Two guide cannulae were stereotaxically implanted in the CA1 region and morphine (0.5, 1, 2 and 4μg/0.5μl saline) and naloxone (0.3, 1 and 3μg/0.5μl saline) were used as the opioid receptor agonist and antagonist, respectively. SKF-38393 (1μg/0.5μl saline) was used as a D1-like receptor agonist, quinpirole (2μg/0.5μl saline) as a D2-like receptor agonist, SCH-23390 (0.5μg/0.5μl saline) as a D1-like receptor antagonist and sulpiride (3μg/0.5μl DMSO) as a D2-like receptor antagonist. To induce orofacial pain, 50μl of 1% formalin was subcutaneously injected into the left side of the upper lip. Our results showed that different doses of morphine significantly reduced orofacial pain in both phases induced by formalin. Naloxone (1 and 3μg) reversed morphine induced analgesia in CA1. SKF-38393 and quinpirole with naloxone (1μg) significantly decreased formalin-induced orofacial pain in both phases. SCH-23390 had no effect on the antinociceptive response of morphine in both phases of orofacial pain. Sulpiride reversed the antinociceptive effects of morphine only in the first phase, but this result was not significant. Our findings suggest that there is cross-talk between the opioidergic and dopaminergic systems. Opioidergic neurons also exerted antinociceptive effects by modulation of the dopaminergic system in the CA1 region of the brain.

Enkephalin- and dynorphin-release produced by electrical stimulation of different frequencies in rat brain slices

Endogenous opioid peptides contribute to the process of pain control and. Previous studies, in vivo, have shown that 2 and 100Hz peripheral electrical stimulation evokes the release of endogenous met-enkephalin (Mek) and dynorphin (Dyn) in cerebrospinal fluid, respectively, in a frequency-dependent manner. However, it is unknown whether opioidergic neurons in the brain areas related to analgesia could respond directly to electrical stimulation of differing frequencies. In the present study, the release of Mek and DynA from slices of rat spinal dorsal horn (DH) and arcuate nucleus (Arc), elicited by different frequencies of electrical stimulation, were investigated using radioimmunoassays and in vitro perfusion techniques. Slices were stimulated with electrical field stimulation (600 pulses, 0.2ms duration, and 10mA), in a frequency range of 0.5–200Hz. Frequencies at 2, 15, and 100Hz could increase the releases over basal level of Mek and DynA, but 2Hz was most potently to evoke Mek release in ARC slice, 100Hz was optimal to DynA release in DH slice. This frequency-dependent and region-specific release was unrelated to the ratio of the two peptides content. An intracellular calcium chelator inhibited Mek release induced by 2Hz, but did not affect the release of DynA at 100Hz from DH slices, implying that intracellular calcium ions in part mediate the release of Mek evoked by low frequency stimulation in DH slices. These data indicate that Mek and DynA peptidergic neurons can be stimulated by specific electrical frequencies in a frequency-dependent and region-specific manner, in which intracellular calcium ions may partially play some role. Thus, our findings may provide a new point of understanding about how opioidergic neurons code information.

Mechanism of Ghrelin-Induced Gastric Contractions in Suncus murinus (House Musk Shrew): Involvement of Intrinsic Primary Afferent Neurons

Here, we have reported that motilin can induce contractions in a dose-dependent manner in isolated Suncus murinus (house musk shrew) stomach. We have also shown that after pretreatment with a low dose of motilin (10−10 M), ghrelin also induces gastric contractions at levels of 10−10 M to 10−7 M. However, the neural mechanism of ghrelin action in the stomach has not been fully revealed. In the present study, we studied the mechanism of ghrelin-induced contraction in vitro using a pharmacological method. The responses to ghrelin in the stomach were almost completely abolished by hexamethonium and were significantly suppressed by the administration of phentolamine, prazosin, ondansetron, and naloxone. Additionally, N-nitro-l-arginine methylester significantly potentiated the contractions. Importantly, the mucosa is essential for ghrelin-induced, but not motilin-induced, gastric contractions. To evaluate the involvement of intrinsic primary afferent neurons (IPANs), which are multiaxonal neurons that pass signals from the mucosa to the myenteric plexus, we examined the effect of the IPAN-related pathway on ghrelin-induced contractions and found that pretreatment with adenosine and tachykinergic receptor 3 antagonists (SR142801) significantly eliminated the contractions and GR113808 (5-hydroxytryptamine receptor 4 antagonist) almost completely eliminated it. The results indicate that ghrelin stimulates and modulates suncus gastric contractions through cholinergic, adrenergic, serotonergic, opioidergic neurons and nitric oxide synthases in the myenteric plexus. The mucosa is also important for ghrelin-induced gastric contractions, and IPANs may be the important interneurons that pass the signal from the mucosa to the myenteric plexus.

Placebo and other psychological interactions in headache treatment

We present a theory according which a headache treatment acts through a specific biological effect (when it exists), a placebo effect linked to both expectancy and repetition of its administration (conditioning), and a non-specific psychological effect. The respective part of these components varies with the treatments and the clinical situations. During antiquity, suggestions and beliefs were the mainstays of headache treatment. The word placebo appeared at the beginning of the eighteenth century. Controversies about its effect came from an excessive interpretation due to methodological bias, inadequate consideration of the variation of the measure (regression to the mean) and of the natural course of the disease. Several powerful studies on placebo effect showed that the nature of the treatment, the associated announce, the patients’ expectancy, and the repetition of the procedures are of paramount importance. The placebo expectancy is associated with an activation of pre-frontal, anterior cingular, accumbens, and periacqueducal grey opioidergic neurons possibly triggered by the dopaminergic meso-limbic system. In randomized control trials, several arms design could theoretically give information concerning the respective part of the different component of the outcome and control the natural course of the disease. However, for migraine and tension type headache attacks treatment, no three arm (verum, placebo, and natural course) trial is available in the literature. Indirect evidence of a placebo effect in migraine attack treatment, comes from the high amplitude of the improvement observed in the placebo arms (28% of the patients). This figure is lower (6%) when using the harder criterium of pain free at 2 h. But these data disregard the effect of the natural course. For prophylactic treatment with oral medication, the trials performed in the last decades report an improvement in 21% of the patients in the placebo arms. However, in these studies the duration of administration was limited, the control of attacks uncertain as well as the evolution of the co-morbid psycho-pathology. Considering the reviews and meta-analysis of complex prophylactic procedures, it must be concluded that their effect is mostly linked to a placebo and non-specific psychological effects. Acupuncture may have a slight specific effect on tension type headache, but not on migraine. Manual therapy studies do not exhibit difference between manipulation, mobilization, and controls; touch has no proven specific effect. A comprehensive efficacy review of biofeedback studies concludes to a small specific effect on tension type headache but not on migraine. A review of behavioral treatment conclude to an interesting mean improvement but did not demonstrated a specific effect with the exception of a four arm study including a pseudo meditation control group. Expectation-linked placebo, conditioning, and non-specific psychological effects vary according clinical situations and psychological context; likely low in RCT, high after anempathic medical contact, and at its maximum with a desired charismatic healer. The announcements of doctors strongly influence the beliefs of patients, and in consequence their pain and anxiety sensibilities; this modulates the amplitude of the placebo and the non-specific psychological effects and is therefore a major determinant of the therapeutic success. Furthermore, any repetitive contact, even through a placebo, may interfere positively with the psychopathological co-morbidity. One has to keep in mind that the non-specific psychological interactions play a major role in the improvement of the majority of the headache sufferers.

Kappa-opioid receptors in the caudal nucleus tractus solitarius mediate 100 hz electroacupuncture-induced sleep activities in rats.

Previous results demonstrated that 10 Hz electroacupuncture (EA) of Anmian acupoints in rats during the dark period enhances slow wave sleep (SWS), which involves the induction of cholinergic activity in the caudal nucleus tractus solitarius (NTS) and subsequent activation of opioidergic neurons and μ-receptors. Studies have shown that different kinds of endogenous opiate peptides and receptors may mediate the consequences of EA with different frequencies. Herein, we further elucidated that high-frequency (100 Hz)-EA of Anmian enhanced SWS during the dark period but exhibited no direct effect on rapid eye movement (REM) sleep. High-frequency EA-induced SWS enhancement was dose-dependently blocked by microinjection of naloxone or κ-receptor antagonist (nor-binaltorphimine) into the caudal NTS, but was affected neither by μ- (naloxonazine) nor δ-receptor antagonists (natatrindole), suggesting the role of NTS κ-receptors in the high-frequency EA-induced SWS enhancement. Current and previous results depict the opioid mechanisms of EA-induced sleep.

Impaired Thyroid Function Provoked by Neonatal Treatment with Drugs Affecting the Maturation of Monoaminergic and Opioidergic Neurons*)

The aim of the present work was to study the basal secretion rate and the reactivity of the TSH-thyroid axis in adult rats neonatally exposed to drugs influencing monoaminergic and opioidergic neurons. The early postnatal administration of drugs antagonistic with the dopaminergic or serotoninergic neurons resulted in a persistent higher rate of basal secretion of TSH, while the administration of drugs synergistic with the monoaminergic neuron systems was weakly influential in this respect. The exposure to opioids in the perinatal period resulted in a permanent reduction of serum TSH levels which was even more pronounced when the exposure to morphine was advanced to the fetal period of life. These data raise the possibility that the permanent TSH depressing effect of perinatal administration of opioids is due to their effect exerted on the maturation of the monoaminergic neurons. From the other hand, our results lead to assume that there is a perinatal critical period in the maturation of monoaminergic neurons regulating TSH secretion in the adult age. In accordance with this assumption, the data obtained in rats bearing perinatal neurotoxic destruction of catecholaminergic neurons contribute to the concept that the disturbed maturation of monoaminergic neurons in the supposed critical period of development might lead to permanent deficiency also in the reactivity of the TSH-thyroid axis.

Opioids modulate pain facilitation from the dorsal reticular nucleus

During chronic pain, the supraspinal pain modulatory system undergoes plastic changes with enhancement of facilitation transmission at the spinal cord. The changes induced by chronic pain at descending modulation often affect opioidergic modulation, and were never described for a key facilitatory component of the system, the dorsal reticular nucleus (DRt). Neurochemical characterization of the DRt-spinal pathway showed that δ-opioid receptors are positioned as to indirectly modulate the activity of non-projecting DRt neurons, whereas neurons expressing μ-opioid receptors project to the spinal dorsal horn or act as interneurons, the latter of which co-expressing GABA B receptors. In monoarthritic rats, the expression of μ-opioid receptors decreased in the DRt whereas the levels of endogenous enkephalin remained unaltered. To increase the opioidergic inhibition of the DRt, we locally injected selective agonists of δ- and μ-opioid receptors or a viral vector containing the human preproenkephalin transgene. Injection of the Herpes Simplex viral vector encoding preproenkephalin induced thermal hypoalgesia in non-inflamed animals and hyperalgesia in monoarthritic rats. The opioid agonists [D-Ala 2, Glu 4]-deltorphin (DELT) and [D-Ala 2, NMePhe 4Gly-ol 5]-enkephalin (DAMGO) induced thermal hyperalgesia in both non-inflamed and monoarthritic rats, but with lower doses in the latter group. The present study shows that opioidergic neurons at the DRt are modulated by GABAergic cells herein controlling the descending facilitation of pain transmission. The DRt exhibits plastic changes during chronic inflammatory pain, with decrease opioid receptor expression which may account for increased descending facilitation during chronic pain.

In vivo characterization of the effects of human hemokinin-1 and human hemokinin-1(4-11), mammalian tachykinin peptides, on the modulation of pain in mice

Human hemokinin-1 (h HK-1) and its truncated form h HK-1(4-11) are mammalian tachykinin peptides encoded by the recently identified TAC4 gene in human, and the biological functions of these peptides have not been well investigated. In the present study, an attempt has been made to investigate the effects and mechanisms of action of h HK-1 and h HK-1(4-11) in pain modulation at the supraspinal level in mice using the tail immersion test. Intracerebroventricular (i.c.v.) administration of h HK-1 (0.3, 1, 3 and 6 nmol/mouse) produced a dose- and time-related antinociceptive effect. This effect was significantly antagonized by the NK 1 receptor antagonist SR140333, but not by the NK 2 receptor antagonist SR48968, indicating that the analgesic effect induced by i.c.v. h HK-1 is mediated through the activation of NK 1 receptors. Interestingly, naloxone, β-funaltrexamine and naloxonazine, but not naltrindole and nor-binaltorphimine, could also block the analgesic effect markedly, suggesting that this effect is related to descending μ opioidergic neurons (primary μ 1 subtype). Human HK-1(4-11) could also induce a dose- and time-dependent analgesic effect after i.c.v. administration, however, the potency of analgesia was less than h HK-1. Surprisingly, SR140333 could not modify this analgesic effect, suggesting that this effect is not mediated through the NK 1 receptors like h HK-1. SR48968 could modestly enhance the analgesic effect induced by h HK-1(4-11), indicating that a small amount of h HK-1(4-11) may bind to NK 2 receptors. Furthermore, none of the opioid receptor (OR) antagonists could markedly block the analgesia of h HK-1(4-11), suggesting that the analgesic effect is not mediated through the descending opioidergic neurons. Blocking of δ ORs significantly enhanced the analgesia, indicating that δ OR is a negatively modulatory factor in the analgesic effect of h HK-1(4-11). It is striking that bicuculline (a competitive antagonist at GABA A receptors) effectively blocked the analgesia induced by h HK-1(4-11), suggesting that this analgesic effect is mediated through the descending inhibitory GABAergic neurons. The novel mechanism involved in the analgesic effect of h HK-1(4-11), which is different from that of h HK-1, may pave the way for a new strategy for the investigation and control of pain.

Involvement of spinal Met–enkephalin in nicotine-induced antinociception in mice

Nicotine is neuronal stimulating drug in the central nervous system and elicits various effects through nicotinic acetylcholine receptors. As previously reported, nicotine has an antinociceptive effect through activation of endogenous opioid neurons. However, detailed mechanisms of nicotine-induced antinociception are uncertain. In this study, we focused on spinal cord and investigated the involvement of endogenous opioidergic neurons in nicotine-induced antinociception in mice. In the tail-pinch test, subcutaneously administered nicotine (5 mg/kg) produced maximal antinociception 0.5 h after nicotine administration; this was attenuated by mecamylamine (MEC, 3 mg/kg, s.c.) or naloxone (NLX, 1 mg/kg, s.c.) administration. Intrathecal nicotine (10 μg) produced maximal antinociception at 2 min and this was also attenuated by MEC (3 mg/kg, s.c.) or NLX (1 mg/kg, s.c.) administration. The preproenkephalin (ppENK) mRNA level in spinal cord, but not dorsal root ganglion, was significantly increased 2 h following nicotine administration and recovered to control level 4 h after nicotine (5 mg/kg, s.c.) administration. This increase in ppENK mRNA level was inhibited by MEC (3 mg/kg, s.c.). The mRNA levels of preprodynorphin and preproopiomelanocortin were not increased by nicotine (5 mg/kg, s.c.). In the dorsal horn of the lumbar spinal cord, methionine–enkephalin (Met–ENK)-like IR was remarkably reduced at 0.5 h following nicotine administration and recovered to control levels by 2 h after nicotine (3 mg/kg, s.c.) administration. These results suggest that nicotine has an antinociceptive effect by promoting the release of Met–ENK, but not dynorphins and endorphins, from activated opioidergic neurons in spinal cord.

Intrahypothalamic neuroendocrine actions of corticotropin-releasing factor.

Most studies of the neuroendocrine effects of corticotropin-releasing factor (CRF) have focused on its role in the regulation of the pituitary-adrenal axis; activation of this axis follows release of the peptide from CRF-containing terminals in the median eminence. However, a sizeable proportion of CRF fibres terminate within the hypothalamus itself, where synaptic contacts with other hypothalamic neuropeptidergic neurons (e.g. gonadotropin-releasing hormone-containing and opioidergic neurons) have been identified. Here, we summarize physiological and pharmacological data which provide insights into the nature and significance of these intrahypothalamic connections. It is now clear that CRF is a potent secretagogue of the three major endogenous opioid peptides (beta-endorphin, Met-enkephalin and dynorphin) and that it stimulates opioidergic neurons tonically. In the case of beta-endorphin, another hypothalamic peptide, arginine vasopressin, appears to be an essential mediator of CRF's effect, suggesting the occurrence of CRF synapses on, or in the vicinity of, vasopressin neurons; morphological support for this assumption is still wanting. Evidence for direct and indirect inhibitory effects of CRF on sexual behaviour and secretion of reproductive hormones is also presented; the indirect pathways include opioidergic neurons. An important conclusion from all these studies is that, in addition to its better known functions in producing adaptive responses during stressful situations, CRF might also contribute to the coordinated functioning of various components of the neuroendocrine system under basal conditions. Although feedback regulation of hypothalamic neuronal activity by peripheral steroids is a well-established tenet of endocrinology, data on modulation of the intrahypothalamic actions of CRF by adrenal and sex steroids are just emerging. Some of these newer findings may be useful in framing questions related to the mechanisms underlying disease states (such as depressive illness) in which CRF has been strongly implicated.

P.3.036 Pregabalin in the treatment of core depressive symptoms in generalized anxiety disorder

Human hemokinin-1 (h HK-1) and its truncated form h HK-1(4-11) are mammalian tachykinin peptides encoded by the recently identified TAC4 gene in human, and the biological functions of these peptides have not been well investigated. In the present study, an attempt has been made to investigate the effects and mechanisms of action of h HK-1 and h HK-1(4-11) in pain modulation at the supraspinal level in mice using the tail immersion test. Intracerebroventricular (i.c.v.) administration of h HK-1 (0.3, 1, 3 and 6 nmol/mouse) produced a dose- and time-related antinociceptive effect. This effect was significantly antagonized by the NK1 receptor antagonist SR140333, but not by the NK2 receptor antagonist SR48968, indicating that the analgesic effect induced by i.c.v. h HK-1 is mediated through the activation of NK1 receptors. Interestingly, naloxone, β-funaltrexamine and naloxonazine, but not naltrindole and nor-binaltorphimine, could also block the analgesic effect markedly, suggesting that this effect is related to descending μ opioidergic neurons (primary μ1 subtype). Human HK-1(4-11) could also induce a dose- and time-dependent analgesic effect after i.c.v. administration, however, the potency of analgesia was less than h HK-1. Surprisingly, SR140333 could not modify this analgesic effect, suggesting that this effect is not mediated through the NK1 receptors like h HK-1. SR48968 could modestly enhance the analgesic effect induced by h HK-1(4-11), indicating that a small amount of h HK-1(4-11) may bind to NK2 receptors. Furthermore, none of the opioid receptor (OR) antagonists could markedly block the analgesia of h HK-1(4-11), suggesting that the analgesic effect is not mediated through the descending opioidergic neurons. Blocking of δ ORs significantly enhanced the analgesia, indicating that δ OR is a negatively modulatory factor in the analgesic effect of h HK-1(4-11). It is striking that bicuculline (a competitive antagonist at GABAA receptors) effectively blocked the analgesia induced by h HK-1(4-11), suggesting that this analgesic effect is mediated through the descending inhibitory GABAergic neurons. The novel mechanism involved in the analgesic effect of h HK-1(4-11), which is different from that of h HK-1, may pave the way for a new strategy for the investigation and control of pain.

Brain stem opioidergic and GABAergic neurons mediate the antinociceptive effect of nitrous oxide in Fischer rats.

Recent studies have revealed that N2O exerts its antinociceptive effect by inducing opioid peptide release in the brain stem, thereby activating the descending noradrenergic inhibitory neurons, which modulate pain processing in the spinal cord. However, the precise neuronal pathways that mediate these events remain to be determined. Using immunohistochemical and behavioral techniques in adult male Fischer rats, the authors studied the involvement of brain stem opioidergic and gamma-aminobutyric acid-mediated (GABAergic) neurons in the N2O-induced antinociceptive effect using discrete microinjections of an opioid receptor antagonist or GABAergic activator into the periaqueductal gray area and pontine noradrenergic nuclei. They used c-Fos expression as an immunohistochemical mark of neuronal activation induced by N2O and the plantar test as the behavioral paradigm for nociception. Microinjection of either naloxone (an opioid receptor antagonist) or muscimol (a gamma-aminobutyric acid receptor type A agonist) into the ventrolateral periaqueductal gray area inhibited N2O-induced c-Fos expression in the spinal cord and pontine noradrenergic nuclei, particularly in the A7. Microinjection of either naloxone or muscimol into the A7 nuclei also inhibited N2O-induced c-Fos expression in the spinal cord and the N2O-induced antinociceptive effect by the plantar test. These results support the hypothesis that both opioidergic and GABAergic neurons mediate the antinociceptive effect of N2O at the periaqueductal gray area and A7 in the brain stem. The authors postulate that N2O-induced opioid peptide release leads to inhibition of GABAergic neurons via opioid receptors. The descending noradrenergic inhibitory pathways, which are tonically inhibited by these gamma-aminobutyric acid neurons, are thereby activated (disinhibited) and modulate pain processing in the spinal cord.

Close apposition of dynorphin-positive nerve fibres to lymphocytes in the liver suggests opioidergic neuroimmunomodulation.

The liver is innervated by sympathetic efferent, spinal afferent, vagal afferent and probably also vagal efferent fibres. To assess potential functional roles of the various neuronal subsets, data on transmitter systems are of crucial importance. This study was aimed at elucidating a possible opioidergic system in the mouse and rat liver. In particular relationships of opioidergic neurons to immune cells were emphasised. Material from perfusion-fixed mice ( n=29) of different strains (BALB/c, NMRI, C57Bl6, SV 129 inbred) and Wistar rats ( n=7) was cryosectioned at 12-14 microm and incubated for single or double immunofluorescence. Antibodies directed against dynorphin A, met-enkephalin, endomorphin 1 and 2, mu, kappa- and delta-opioid receptors (MOR, KOR, DOR), tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), CD4, CD8 and macrophages were used. Binding sites were detected using Cy3-, FITC-, DTAF-, Cy2-, Alexa 555- and Texas red-tagged secondary antibodies. Specimens were analysed using confocal laser scanning microscopy (CLSM). Numerous nerve fibres staining for dynorphin were found in periportal areas of both mouse and rat livers. Neither met-enkephalin nor endomorphin could be detected in axons. No immunopositive neuronal cell bodies or other cellular elements were seen. All dynorphin positive fibres costained for TH while not every TH-positive fibre costained for dynorphin. Thus, most if not all dynorphin-positive nerve fibres may be of sympathetic origin. KOR immunostaining could be localised to round mononuclear cells which often costained for CD4, less frequently for CD8 and rarely for the pan-macrophage marker BM8. Altogether, about 45% of KOR-positive cells were identified as T-lymphocytes. In some instances, close appositions of dynorphin-positive axons to KOR-positive cells were revealed by CLSM. No KOR immunoreactivity was detected in nerve fibres. Hence, sympathetic neurons innervating the liver may interfere with inflammatory processes, in addition to their well-established beta(2)-adrenergic effect, via an opioidergic action on immune cells.

Prenatal Δ 9-tetrahydrocannabinol exposure modifies proenkephalin gene expression in the fetal rat brain: sex-dependent differences

Perinatal Δ 9-tetrahydrocannabinol (Δ 9-THC) exposure in rats resulted in enhanced morphine self-administration behavior, naloxone-precipitated withdrawal signs or changes in pain sensitivity, which have been related to changes in μ-opioid receptor binding and/or proenkephalin mRNA levels in several brain regions. However, despite exposure of these animals to Δ 9-THC from fetal ages, the effects were studied only when animals matured, whereas there is no study on possible changes caused by this cannabinoid during the prenatal ontogeny of opioidergic neurons. The purpose of the present study was to examine the changes in proenkephalin mRNA levels, measured by using in situ hybridization, in several brain nuclei of rat fetuses that had been daily exposed to Δ 9-THC from the 5th day of gestation. Results were as follows. Prenatal Δ 9-THC exposure altered proenkephalin mRNA levels in most of the brain areas studied at different fetal ages, but the effects were different between sexes. Thus, proenkephalin mRNA levels increased in females, but decreased in males that had been prenatally exposed to Δ 9-THC. This was observed in the caudate–putamen, hypothalamic paraventricular and ventromedial nuclei and cerebral cortex. No changes were observed, however, in the subventricular zones of the caudate–putamen, neocortex and nucleus accumbens. In summary, prenatal Δ 9-THC exposure produced a sex-dependent effect in proenkephalin mRNA levels in several brain structures of rat fetuses.

Ontogeny of the Diurnal Rhythm of Tuberoinfundibular Dopaminergic Neuronal Activity in Peripubertal Female Rats: Possible Involvement of Cholinergic and Opioidergic Systems

The ontogeny of the diurnal change of tuberoinfundibular dopaminergic (TIDA) neuronal activity in female rats and its control mechanism were the foci of this study. Intact pre (28- and 35-day-old)-, peri (39- and 42-day-old)- and post (49- and 56-day-old)-pubertal female, as well as pre (28-day-old)- and post (56-day-old)-pubertal male Sprague-Dawley rats were used. Basal TIDA neuronal activity, determined by measuring 3,4-dihydroxyphenylalanine or 3,4-dihydroxyphenylacetic acid in the median eminence, increased 3- to 4-fold in the female and less than 2-fold in the male rats from day 28 to 49; so did serum prolactin levels. The diurnal rhythm of TIDA neuronal activity was not evident until day 42 in the female, but not male, rats. Neither nigrostriatal, nor mesolimbic DA neurons exhibited any significant change between pre- and postpubertal stages. In postpubertal (56-day-old) female rats, injection of nicotine (10 µg/kg) in the morning, but not in the afternoon, inhibited TIDA neuronal activity while similar injections of mecamylamine (1 mg/kg) or naloxone (2.5 mg/kg) stimulated these neurons in the afternoon, but not in the morning. Serum prolactin levels changed accordingly. In contrast, none of the injections had any effect in prepubertal (28-day-old) female rats. These results indicate that the genesis of the diurnal rhythm of TIDA neuronal activity in female rats occurs during the peripubertal stage, and endogenous cholinergic and opioidergic neurons may play a significant role.

The effects of steroid hormones on electrical activity of excitable cells

Steroid hormones influence the electrical activity of many neurons and effectors by regulating the transcription of their ion channels and neurotransmitter receptors, or by modulating the activity of their channels and receptors through second messenger-coupled membrane receptors, or both. In this article, four cell types with known functions and distinct electrical activities are focused on to illustrate how different steroids act synergistically with, or in opposition to, each other to modulate specific electrical phenomena such as spontaneous regular firing (GH3 cells, a pituitary cell line), action potential duration (electric organ cells), and intrinsic excitability and sensitivity to neurotransmitters (GnRH and opioidergic neurons). These examples illustrate how steroids might influence electrical activity in neurons involved in more complex central circuits.

The endogenous feeding suppressant, 2-buten-4-olide, impairs the pulsatile secretion of luteinizing hormone through endogenous opioid peptides.

To clarify the mechanism of the suppressive effect of 2-buten-4-olide (2-B4O), an endogenous feeding suppressant, on the pulsatile secretion of luteinizing hormone (LH), by studying whether endogenous opioid peptides are involved in this suppressive effect. Using ovariectomized (ovx) rats, blood samples were taken every 6 min for 2 h after administration of 2-B4O or saline into the third cerebroventricle (3V) and sequential i.v. injection of naloxone (0. 5 mg/kg per h) or saline. Rats were divided into three experimental groups: group 1: 3V saline + i.v. saline (control); group 2: 3V 2-B4O + i.v. saline; group 3: 3V 2-B4O + i.v. naloxone. Serum LH concentrations were determined by double-antibody RIA. To determine whether 2-B4O affected the biosynthetic activity of the opioidergic neurons within the ovx rat arcuate nucleus, we measured the concentrations of pro-opiomelanocortin (POMC) mRNA, a precursor of beta-endorphin, in the rostral arcuate nucleus using non-radioactive in situ hybridization and a computerized image-analysis system. 2-B4O significantly suppressed the pulse frequency of LH (group 2: 1.5+/-0.33 pulses/2 h, group 1: 2.43+/-0.2 pulses/2 h; P < 0.05), but naloxone blocked its suppressive effect and restored the pulse frequency (group 3: 3.29+/-0.36 pulses/2 h, group 2: 1.5+/-0.33 pulses/2 h: P < 0.01). There were no significant changes in the mean LH concentrations and amplitude. Furthermore, 2-B4O significantly stimulated the expression of POMC mRNA in the rostral arcuate nucleus. These results suggest that 2-B4O may impair the pulsatile secretion of LH by activating the opioid pathway within the hypothalamus.

Effect of prior nicotine treatment on drug induced changes in serum LH concentrations in rats

The effect of daily injections of nicotine on drug induced changes in LH secretion was investigated in male rats. Daily administration of nicotine for 7 days resulted in decreased basal serum LH concentrations. Nicotine treatment blocked naloxone induced LH release and reduced LHRH induced increases in serum LH. Clonidine induced increases in serum LH were not altered by nicotine treatment and haloperidol treatment did not alter nicotine induced decreases in serum LH. In an acute study nicotine blocked LH secretion induced by the long acting opioid antagonist naltrexone. Collectively these results indicate that opioidergic neurons are involved in the reduction in serum LH that occurs following nicotine. They also indicate that chronic nicotine treatment can reduce the pituitary gland response to LHRH.

Nicotinic control of tuberoinfundibular dopaminergic neuron activity and prolactin secretion: diurnal rhythm and involvement of endogenous opioidergic system

The possible involvement of cholinergic and opioidergic neurons in the control of diurnal changes of tuberoinfundibular dopaminergic (TIDA) neuronal activity was reported. Adult Sprague-Dawley rats ovariectomized and treated with estrogen were used. All drugs were administered centrally through preimplanted intracerebroventricular cannula, and both TIDA neuronal activity and serum prolactin level were determined. Nicotine (10 ng/3 μl/rat) given at 10:00 h significantly inhibited TIDA neuronal activity from 5 to 30 min and stimulated serum PRL levels at 5 and 15 min. Co-administration of either mecamylamine (1 μg) or naloxone (2.5 μg) prevented both nicotine's effects. A dose-related (0.1–100 ng) effect of nicotine on TIDA neuronal activity and serum PRL level was also observed in the morning when TIDA neuronal activity is high and serum PRL level is low, but not in the afternoon when the former activity is low and the latter is high. When atropine (20 μg), naloxone (25 μg) or Nor-BNI (20 μg) was given at 14:00 h all increased the lowered TIDA neuronal activity in the afternoon. When atropine was co-administered with either naloxone or Nor-BNI, however, no additive effect was observed. Submaximal doses of atropine (0.2 μg), mecamylamine (0.1 μg) or naloxone (0.25 μg) was also effective in stimulating the afternoon levels of TIDA neuronal activity and inhibiting serum PRL, and no additive effect was observed either. Moreover, simultaneous injection of morphine (15 μg) prevented atropine's effect in the afternoon. These results indicate that cholinergic neurons may act through activating the endogenous opioidergic neurons to exhibit an inhibitory effect on TIDA neuronal activity and a stimulatory one on prolactin secretion. A diurnal difference in its endogenous activity between morning and afternoon was also implicated.