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1.
bioRxiv ; 2023 Dec 07.
Article in English | MEDLINE | ID: mdl-38106086

ABSTRACT

Numerous studies of hippocampal synaptic function in learning and memory have established the functional significance of the scaffolding A-kinase anchoring protein 150 (AKAP150) in kinase and phosphatase regulation of synaptic receptor and ion channel trafficking/function and hence synaptic transmission/plasticity, and neuronal excitability. Emerging evidence also suggests that AKAP150 signaling may play a critical role in brain's processing of rewarding/aversive experiences. Here we focused on an unexplored role of AKAP150 in the lateral habenula (LHb), a diencephalic brain region that integrates and relays negative reward signals from forebrain striatal and limbic structures to midbrain monoaminergic centers. LHb aberrant activity (specifically hyperactivity) is also linked to depression. Using whole cell patch clamp recordings in LHb of male wildtype (WT) and ΔPKA knockin mice (with deficiency in AKAP-anchoring of PKA), we found that the genetic disruption of PKA anchoring to AKAP150 significantly reduced AMPA receptor (AMPAR)-mediated glutamatergic transmission and prevented the induction of presynaptic endocannabinoid (eCB)-mediated long-term depression (LTD) in LHb neurons. Moreover, ΔPKA mutation potentiated GABAA receptor (GABAAR)-mediated inhibitory transmission postsynaptically while increasing LHb intrinsic neuronal excitability through suppression of medium afterhyperpolarizations (mAHPs). Given that LHb is a highly stress-responsive brain region, we further tested the effects of corticotropin releasing factor (CRF) stress neuromodulator on synaptic transmission and intrinsic excitability of LHb neurons in WT and ΔPKA mice. As in our earlier study in rat LHb, CRF significantly suppressed GABAergic transmission onto LHb neurons and increased intrinsic excitability by diminishing small-conductance potassium (SK) channel-mediated mAHPs. ΔPKA mutation-induced suppression of mAHPs also blunted the synaptic and neuroexcitatory actions of CRF in mouse LHb. Altogether, our data suggest that AKAP150 complex signaling plays a critical role in regulation of AMPAR and GABAAR synaptic strength, glutamatergic plasticity and CRF neuromodulation possibly through AMPAR and potassium channel trafficking and eCB signaling within the LHb.

2.
Mar Pollut Bull ; 131(Pt A): 130-141, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29886929

ABSTRACT

Research into sediment-seagrass tissue metal relationships has been undertaken in Sydney estuary due to the recognized role contamination plays in threats to seagrass health. Seagrass (Halophila ovalis) leaf tissue concentrations are elevated in Cu, Pb and Zn and contain the highest reported root Cr concentrations. Seagrass metal concentrations were significantly different between species H. ovalis and Zostera capricorni; between root and leaf tissue; and between sampling locations. Greatest tissue enrichment was for Pb, however metals were not enriched in seagrass relative to surficial sediment. Fine and total sediment metal concentrations were temporally consistent between collection years 2013/15, whereas root tissue metals changed between years and sites and leaf metal contents were temporally inconsistent. Extractable metal concentrations in fine sediment (<62.5 µm) showed moderate significant correlation with root tissue and a weak significant relationship with leaf tissue, whereas total sediment metal showed no such relationships. Management implications are provided.


Subject(s)
Geologic Sediments/analysis , Hydrocharitaceae/chemistry , Metals/analysis , Water Pollutants, Chemical/analysis , Australia , Ecosystem , Ecotoxicology , Environmental Monitoring , Estuaries , Hydrocharitaceae/drug effects , Hydrocharitaceae/metabolism , Metals/pharmacokinetics , Metals, Heavy/analysis , Plant Leaves/chemistry , Plant Leaves/drug effects , Plant Roots/chemistry , Plant Roots/drug effects , Species Specificity , Water Pollutants, Chemical/pharmacokinetics , Zosteraceae/chemistry , Zosteraceae/drug effects , Zosteraceae/metabolism
3.
Mar Pollut Bull ; 128: 72-81, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29571414

ABSTRACT

Seagrass (Zostera capricorni) tissue metal concentrations in Sydney estuary are some of the highest reported for urban environments. A strong declining metal (As, Co, Cr, Cu, Mn, Pb and Zn) concentration gradient in fine sediment from highly modified to the near-pristine estuarine environments was matched by a concurrent and statistically significant, moderately strong, relationship with declining metal concentrations for leaf, rhizomes and root tissue types. Leaf tissue more consistently expressed metal concentration uptake from sediment. Rhizome tissue contained the highest concentrations for all metals, except Mn, while leaf concentrations were higher for Cr and Zn and roots were more elevated in Cr and Pb. Tissue Cr, Pb and Zn were close to background for reference sites for leaves. Maximum tissue enrichment over background was 25, 19, 47 and 8 times for Cr, Cu, Pb and Zn. Bio-sediment accumulation factors were generally <1 for all metals.


Subject(s)
Environmental Monitoring/methods , Estuaries , Geologic Sediments/chemistry , Metals, Heavy/analysis , Water Pollutants, Chemical/analysis , Zosteraceae/chemistry , Australia , Plant Leaves/chemistry , Plant Roots/chemistry
4.
Mol Psychiatry ; 23(8): 1798-1806, 2018 08.
Article in English | MEDLINE | ID: mdl-29133950

ABSTRACT

Episodic memory, a fundamental component of human cognition, is significantly impaired in autism. We believe we report the first evidence for this problem in the Fmr1-knockout (KO) mouse model of Fragile X syndrome and describe potentially treatable underlying causes. The hippocampus is critical for the formation and use of episodes, with semantic (cue identity) information relayed to the structure via the lateral perforant path (LPP). The unusual form of synaptic plasticity expressed by the LPP (lppLTP) was profoundly impaired in Fmr1-KOs relative to wild-type mice. Two factors contributed to this defect: (i) reduced GluN1 subunit levels in synaptic NMDA receptors and related currents, and (ii) impaired retrograde synaptic signaling by the endocannabinoid 2-arachidonoylglycerol (2-AG). Studies using a novel serial cue paradigm showed that episodic encoding is dependent on both the LPP and the endocannabinoid receptor CB1, and is strikingly impaired in Fmr1-KOs. Enhancing 2-AG signaling rescued both lppLTP and learning in the mutants. Thus, two consequences of the Fragile-X mutation converge on plasticity at one site in hippocampus to prevent encoding of a basic element of cognitive memory. Collectively, the results suggest a clinically plausible approach to treatment.


Subject(s)
Fragile X Syndrome/metabolism , Hippocampus/metabolism , Long-Term Potentiation/physiology , Memory, Episodic , Animals , Arachidonic Acids/metabolism , Discrimination, Psychological/drug effects , Discrimination, Psychological/physiology , Disease Models, Animal , Endocannabinoids/metabolism , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Fragile X Syndrome/drug therapy , Fragile X Syndrome/pathology , Glycerides/metabolism , Hippocampus/drug effects , Hippocampus/pathology , Long-Term Potentiation/drug effects , Male , Mice, Knockout , Nerve Tissue Proteins/metabolism , Neurotransmitter Agents/pharmacology , Olfactory Perception/drug effects , Olfactory Perception/physiology , Patch-Clamp Techniques , Receptor, Cannabinoid, CB1/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Tissue Culture Techniques
5.
Neuroscience ; 169(1): 269-78, 2010 Aug 11.
Article in English | MEDLINE | ID: mdl-20417255

ABSTRACT

Antagonists selectively inhibiting activation of the nociceptin/orphanin FQ (N/OFQ) receptor reduce motor symptoms in experimental models of Parkinson's disease, and genetic deletion of the ppN/OFQ gene offers partial protection of mid-brain dopamine neurons against the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP increased ppN/OFQ mRNA expression in the substantia nigra (SN). We have evaluated the temporal relationship of dopamine cell loss to increased ppN/OFQ mRNA expression in the substantia nigra after MPTP treatment, and characterized the cellular locations in which increased ppN/OFQ mRNA expression was observed after MPTP treatment. MPTP increased by about 5-fold the number of neurons expressing ppN/OFQ mRNA in the pars reticulata of SN (SNr) by 24 h after treatment and the elevation remained significant for at least 7 days. This period coincided with the timing of the loss of dopamine neurons from the pars compacta of substantia nigra (SNc) after MPTP. The increased expression of ppN/OFQ mRNA co-localized with a neuronal marker in the SNr. MPTP treatment resulted in a small increase in the numbers of neurons expressing ppN/OFQ in the SNc in mice from one mouse colony but the increase did not reach statistical significance in mice from another colony. No changes in ppN/OFQ-mRNA expression were observed in the ventral tegmental area (VTA), the caudate-putamen, the subthalamic nucleus, or in two other brains areas. These results demonstrate that increased N/OFQ expression in the SNr is closely associated with the MPTP-induced loss of dopamine neurons in the SNc in a widely used animal model of Parkinson's disease.


Subject(s)
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Gene Expression Regulation/drug effects , MPTP Poisoning/genetics , Neurons/drug effects , Parkinsonian Disorders/genetics , Protein Precursors/biosynthesis , RNA, Messenger/biosynthesis , Receptors, Opioid/biosynthesis , Substantia Nigra/drug effects , Animals , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/classification , Neurons/metabolism , Opioid Peptides/biosynthesis , Opioid Peptides/genetics , Protein Precursors/deficiency , Protein Precursors/genetics , RNA, Messenger/genetics , Receptors, Opioid/deficiency , Receptors, Opioid/genetics , Substantia Nigra/metabolism , Ventral Tegmental Area/drug effects , Ventral Tegmental Area/metabolism , Nociceptin
6.
Eur J Pharmacol ; 413(2-3): 207-12, 2001 Feb 16.
Article in English | MEDLINE | ID: mdl-11226394

ABSTRACT

The effect of the selective dopamine uptake inhibitor 1-[2-[bis(4-flourophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine dihydrochloride (GBR 12909) was examined on prodynorphin gene expression. GBR 12909 or vehicle was continuously infused for 7 days via osmotic minipump, or injected daily into male rats. Both continuous infusions and daily injections of GBR 12909 produced significant decreases in prodynorphin expression in the hypothalamus (37% and 31% decreases, respectively). There were no significant changes in the caudate putamen, hippocampus or nucleus accumbens. One injection of GBR 12909 had no effects on prodynorphin expression in any of the brain regions studied, suggesting that the effect in the hypothalamus is not an acute effect. As previously reported for other treatment regimens, continuous infusion of cocaine produced a 35% significant decrease in the hypothalamus, consistent with the effects of GBR 12909. In contrast to GBR 12909, however, cocaine also produced a significant increase in prodynorphin expression in the caudate putamen. Thus, chronic inhibition of dopamine uptake can regulate prodynorphin expression in the hypothalamus. In contrast, the increase in the caudate putamen following cocaine administration may not be related to the inhibition of dopamine uptake, since it was not produced by a selective dopamine uptake inhibitor. These findings suggest that regulation of prodynorphin gene expression by cocaine in the caudate putamen may be mediated by the inhibition of norepinephrine or serotonin uptake, by a combination of effects on two or three monoamine transporters, or by a mechanism unrelated to transporter inhibition.


Subject(s)
Brain/drug effects , Cocaine/pharmacology , Dopamine Uptake Inhibitors/pharmacology , Enkephalins/drug effects , Gene Expression/drug effects , Piperazines/pharmacology , Protein Precursors/drug effects , Animals , Brain/metabolism , Enkephalins/metabolism , Gene Expression/physiology , Male , Protein Precursors/metabolism , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley
7.
J Neurochem ; 74(3): 1068-78, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10693938

ABSTRACT

Antibodies directed against the C-terminal and the N-terminal regions of the mu-opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the mu receptor. Two fusion proteins were constructed: One contained the 50 C-terminal amino acids of the mu receptor, and the other contained 61 amino acids near the N terminus of the receptor. Antisera directed against both fusion proteins were capable of immunoprecipitating approximately 70% of solubilized rat brain mu receptors as determined by [3H][D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin ([3H]DAMGO) saturation binding. The material immunoprecipitated with both of the antisera was recognized as a broad band with a molecular mass between 60 and 75 kDa when screened in a western blot. Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) had an EC50 of 0.4 nM in diminishing [3H]DAMGO binding to the immunoprecipitated pellet. The ratio of G proteins to mu receptors in the immunoprecipitated material was 1:1. When the material immunoprecipitated with affinity-purified antibody was screened for the presence of G protein a subunits, it was determined that G(alpha)o, G(alpha)i1, G(alpha)i3, and to a lesser extent G(alpha)i2, but not G(alpha)s or G(alpha)q11, were coimmunoprecipitated with the mu receptor. Inclusion of GTPgammaS during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins.


Subject(s)
Brain/metabolism , GTP-Binding Proteins/metabolism , Guanine Nucleotides/pharmacology , Receptors, Opioid, mu/metabolism , Animals , Binding, Competitive , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Precipitin Tests , Rats , Solubility
8.
J Neurochem ; 72(5): 1882-9, 1999 May.
Article in English | MEDLINE | ID: mdl-10217264

ABSTRACT

We have identified the gene encoding nociceptin/orphanin FQ (N/OFQ), the novel opioid-like neuropeptide, as responsive to ciliary neurotrophic factor (CNTF). N/OFQ mRNA levels were induced five- and ninefold by CNTF in striatal and cortical neurons. In primary astrocytes CNTF also increased N/OFQ mRNA levels. CNTF is a multifunctional cytokine that mediates the development and differentiation of both neurons and astrocytes and supports the survival of various neurons. CNTF is also an injury-induced factor in the brain playing a crucial role in astrogliosis. The mechanism by which CNTF elicits these effects is not well understood, but it is likely to involve regulation of specific genes. CNTF regulation of N/OFQ expression was sensitive to the kinase inhibitors H-7 and genistein but not to inhibition of protein synthesis. This pharmacological profile is consistent with CNTF activating the Janus protein tyrosine kinase (JAK)/ signal transducers and activators of transcription (STAT) pathway to induce N/OFQ transcription. In nuclear extracts of CNTF-treated striatal neurons DNA binding of STAT proteins was increased. Radioimmunoassays revealed elevated N/OFQ immunoreactivity in striatal neurons after CNTF treatment. Expression of the related proenkephalin gene was not affected by CNTF in either neuronal or glial cultures. Regulation of N/OFQ expression by CNTF might point to a possible function of N/OFQ during development and after neural injury.


Subject(s)
Astrocytes/physiology , Cytokines/physiology , Gene Expression Regulation/physiology , Nerve Growth Factors/physiology , Neurons/physiology , Opioid Peptides/genetics , Animals , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , Ciliary Neurotrophic Factor , Corpus Striatum/cytology , Corpus Striatum/metabolism , Cytokines/pharmacology , DNA/metabolism , DNA-Binding Proteins/genetics , Enkephalins/genetics , Gene Expression Regulation/drug effects , Inflammation Mediators/pharmacology , Nerve Growth Factors/pharmacology , Nerve Tissue Proteins/pharmacology , Opioid Peptides/metabolism , Protein Kinases/physiology , Protein Precursors/genetics , RNA, Messenger/metabolism , Rats/embryology , Receptors, Opioid, mu/genetics , STAT1 Transcription Factor , Trans-Activators/genetics , Nociceptin
9.
Alcohol Clin Exp Res ; 22(6): 1262-71, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9756041

ABSTRACT

There is convincing evidence that genetic factors contribute to the predisposition to alcoholism. In this respect, alcohol-preferring (like C57BL/6 mice) and alcohol-avoiding lines (like DBA/2 mice) of animals served as models in the search for neurobiological substrates of excessive ethanol consumption. One of the systems that is thought to be associated with the incidence of alcoholism is the endogenous opioid system. In the first experiment, basal mRNA levels of mu- and delta-opioid receptors, and of opioid-degrading enzymes enkephalinase (neutral endopeptidase 24.11; NEP) and angiotensin-converting enzyme (ACE) in the brain regions of C57BL/6 and DBA/2 mice did not reveal genetically determined differences in these parameters between the two strains. Furthermore, in the brain regions studied, the corresponding enzyme activities of NEP and ACE did not differ significantly between the lines of mice, except for a higher NEP activity in the striatum and olfactory bulb of DBA/2 mice (p < 0.01). In the second experiment, C57BL/6 and DBA/2 mice were offered a free choice between water and 10% ethanol solution for 4 weeks and were killed thereafter; from another group, ethanol was removed for 3 days and from a third group ethanol was removed for 3 weeks before killing. In the striatum, a highly significant increase in the ACE mRNA amount was detected after 3 weeks of removal of ethanol in C57BL/6 mice, whereas in DBA/2 mice the delta-opioid receptor mRNA level was increased at this time when compared with the corresponding ethanol treatment group. The most striking changes were seen in the hypothalamus, where mu-opioid receptor, ACE, and NEP mRNA amounts markedly decreased after ethanol treatment in both strains. Thus, chronic ethanol intake caused significant changes in the gene expression of distinct components of the endogenous opioid system. These findings further underline an involvement of the opioid system in the effects of ethanol.


Subject(s)
Alcohol Drinking/genetics , Alcoholism/genetics , Genotype , Neprilysin/genetics , Peptidyl-Dipeptidase A/genetics , Receptors, Opioid/genetics , Alcohol Drinking/adverse effects , Animals , Brain/drug effects , Brain/enzymology , Gene Expression/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Neprilysin/drug effects , Peptidyl-Dipeptidase A/drug effects , RNA, Messenger/drug effects , RNA, Messenger/genetics , Receptors, Opioid/drug effects
10.
J Neurochem ; 71(2): 556-63, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9681445

ABSTRACT

The regulation of nociceptin/orphanin FQ (N/OFQ) gene expression by neuronal activity and by activation of the cyclic AMP signaling pathway in primary neuronal and astroglial cultures is described. Neuronal activity mimicked by veratridine-mediated depolarization profoundly increased N/OFQ gene expression in primary striatal neurons. Calcium entry through L-type, but not N-type, voltage-sensitive calcium channels activated by depolarization appears to be involved, because nitrendipine and nifedipine, but not omega-conotoxin, reduced the induction of N/OFQ expression by veratridine. A selective inhibitor of calcium/calmodulin kinases (KN-62) also antagonized the depolarization-induced increase in N/OFQ mRNA levels, suggesting a role for these enzymes in the activity-dependent induction of N/OFQ gene expression. Constitutively expressed transcription factors may mediate N/OFQ gene expression levels, because veratridine induction of N/OFQ transcription was insensitive to the protein synthesis inhibitor cycloheximide. Regulation of N/OFQ gene expression by depolarization and cyclic AMP is not restricted to striatal neurons, because similar regulation was also observed in neuronal cultures derived from the cerebral cortex. Veratridine did not increase N/OFQ mRNA levels in primary astrocyte cultures; however, elevated intracellular cyclic AMP levels lead to a dramatic, 30-fold induction of N/OFQ mRNA levels in these cells.


Subject(s)
Astrocytes/enzymology , Cyclic AMP/metabolism , Gene Expression Regulation, Enzymologic/physiology , Mitogen-Activated Protein Kinases , Neurons/enzymology , Opioid Peptides/genetics , Animals , Bucladesine/pharmacology , Calcium/metabolism , Calcium/pharmacology , Calcium Channels/physiology , Calcium Channels, L-Type , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Cerebral Cortex/cytology , Corpus Striatum/cytology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Membrane Potentials/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Nerve Tissue Proteins/physiology , RNA, Messenger/analysis , Rats , Receptors, Opioid, mu/genetics , Signal Transduction/drug effects , Signal Transduction/physiology , Transcription, Genetic/physiology , Nociceptin
11.
Can J Physiol Pharmacol ; 76(3): 278-83, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9673791

ABSTRACT

Chronic continuous cocaine administration for 3 days has been shown to upregulate the level of mu opioid receptor (MOR) mRNA in the nucleus accumbens (n. acc.) of rat brain. Dopamine (DA) antagonists, SCH 23390, eticlopride, and nafadotride, blocked, and DA agonists, SKF 38393, R(+)-6-bromo-APB hydrobromide, and bromocriptine, mimicked the cocaine-induced upregulation of MOR mRNA, suggesting involvement of both subfamilies of DA receptors in the effect of cocaine. In the present study the time course of cocaine-induced and DA agonist induced alterations in the level of MOR mRNA in n. acc. has been determined and compared with the changes in the level of MOR binding sites. Male Sprague-Dawley rats were treated with saline, cocaine (50 mg.kg-1.day-1), or DA agonists for periods between 24 and 336 h. Expression of MOR mRNA in n. acc. was estimated using quantitative competitive polymerase chain reaction assays following reverse transcription. The cocaine-induced upregulation of MOR mRNA in n. acc. was transient, developing 2 days after exposure, and peaking at 3 days with return to baseline levels by 4 days of chronic continuous cocaine treatment. The temporal characteristics of DA agonist induced increase in the levels of MOR mRNA in n. acc. were similar to those of cocaine, with maximum effect after 3 days of treatment. The density of [3H]DAMGO binding sites in n. acc. was 30% higher after 3 days of cocaine administration than in saline-treated control animals, but returned toward baseline levels after 4 days of cocaine treatment. No changes in the binding of [3H]DAMGO were detected after 7 or 14 days exposure to cocaine. The affinity of [3H]DAMGO to n. acc. membranes (approximately 2.0 nM) was unchanged during the cocaine treatment.


Subject(s)
Cocaine/pharmacology , Nucleus Accumbens/drug effects , Receptors, Opioid, mu/metabolism , 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine/analogs & derivatives , 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine/pharmacology , Analgesics, Opioid/pharmacology , Animals , Benzopyrans/pharmacology , Bromocriptine/pharmacology , Cocaine/administration & dosage , Dopamine Agonists/pharmacology , Dopamine Uptake Inhibitors/administration & dosage , Dopamine Uptake Inhibitors/pharmacology , Enkephalin, Ala(2)-MePhe(4)-Gly(5)- , Enkephalins/pharmacology , Gene Expression Regulation , Male , Nucleus Accumbens/metabolism , Oxazines/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Time Factors , Up-Regulation/drug effects
12.
J Neurochem ; 70(1): 105-12, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9422352

ABSTRACT

Regulation of gene expression is one of the mechanisms by which neuronal activity elicits long-term changes in neuronal phenotype and function. Although activity-dependent induction of immediate-early genes has been extensively studied, much less is known about the late-response genes. We have investigated the activity-dependent regulation of delta-opioid receptor (DOR) mRNA levels in NG108-15 cells. Transsynaptic activation was mimicked by depolarization with 55 mM KCl or veratridine. Both treatments lead to a time-dependent increase of DOR mRNA levels. Ca2+ entry through L-type voltage-dependent Ca2+ channels activated by depolarization appears to be involved, because L-type channel blockers reduced the induction of DOR expression. Ca2+ binding to calmodulin is the next step in the signal transduction pathway, because a calmodulin antagonist, W7, reduced the effect of veratridine. A selective inhibitor of calmodulin kinases (KN-62) and cyclosporin, an inhibitor of calcineurin, also antagonized the depolarization-induced increase in DOR mRNA levels, which indicates that both calcium/calmodulin-dependent enzymes are involved in the activity-dependent induction of DOR gene expression. Induction of DOR gene expression by an activity-dependent increase in intracellular Ca2+ concentration may serve as a feedback regulatory mechanism because activation of DOR leads to hyperpolarization and lower excitability of neurons.


Subject(s)
Calcium/physiology , Calmodulin/physiology , Gene Expression Regulation/physiology , Receptors, Opioid, delta/genetics , Transcription, Genetic/physiology , Binding Sites/physiology , Calcineurin/physiology , Calcium Channels/physiology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Electrophysiology , Enzyme Activation/physiology , Membranes/physiology , RNA, Messenger/metabolism , Time Factors , Tumor Cells, Cultured , Up-Regulation/physiology , Veratridine/pharmacology
13.
J Pharmacol Exp Ther ; 283(2): 557-65, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9353370

ABSTRACT

Morphine treatment of rats (60-70 mg/kg/day, 7 days) reduced delta opioid receptor-mediated inhibition of adenylyl cyclase activity in caudate putamen without any change in regulation by mu receptors. Earlier studies suggested that dopamine D1 and mu opioid receptors that regulate adenylyl cyclase are expressed preferentially by striato-nigral neurons, whereas adenosine A2a and delta1 opioid receptors are expressed preferentially by striato-pallidal neurons. Chronic morphine treatment also resulted in a reduction of dopamine D2 receptor-mediated inhibition of A2a receptor-stimulated adenylyl cyclase. Treatment with a D2 receptor antagonist (eticlopride; 1 mg/kg/day) for 7 days reduced D1 receptor stimulation of adenylyl cyclase. In contrast, chronic treatment with a D1 receptor antagonist R(+)-7-chloro-8-dihydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepine HCL (SCH 23390; 2.5 mg/kg/day) resulted in a reduction of delta1 and delta2 opioid inhibition of adenylyl cyclase, with no change in the inhibitory activity of a mu agonist. The inhibitory activity of the D2 agonist quinelorane against adenosine A2a-activated enzyme was also reduced by this treatment. Thus chronic D1 blockade, like chronic morphine treatment, appears to cause a selective impairment of the regulation of adenylyl cyclase in A2a receptor-expressing striato-pallidal neurons. D2 receptor activation appears to play an important role in the desensitization of delta receptors, because concurrent administration of the D2 antagonist eticlopride with morphine prevented the densitization of delta and D2 receptors. Similar results were obtained in nucleus accumbens, which suggests a role for D2 receptor desensitization in the adaptive response of this brain region to chronic morphine.


Subject(s)
Morphine/pharmacology , Nucleus Accumbens/drug effects , Putamen/drug effects , Receptors, Dopamine D2/physiology , Receptors, Opioid, delta/drug effects , 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine/pharmacology , Adenosine/analogs & derivatives , Adenosine/pharmacology , Adenylyl Cyclase Inhibitors , Animals , Benzazepines/pharmacology , Male , Phenethylamines/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Opioid, delta/physiology
14.
J Neurosci Methods ; 75(2): 207-14, 1997 Aug 22.
Article in English | MEDLINE | ID: mdl-9288654

ABSTRACT

The release of endogenous glutamate and gamma-aminobutyric acid (GABA) from rat brain tissue slices was studied using a tissue slice assay in which detectable amounts of the amino acids were released from 1-2 mg of tissue. An improved method of high performance liquid chromatography (HPLC) with electrochemical detection was employed to measure both glutamate and GABA after derivatization with o-phthalaldehyde and sulfide in a single isocratic HPLC analysis. The non-endogenous amino acid, homoglutamine, was used as an internal standard in verifying the consistent derivatization of amino acids and in quantifying amounts of glutamate and GABA released from the caudate-putamen tissue. The derivatized amino acids (1-30 pmol) were detected as chromatographic peaks eluting at baseline level and free of significant interfering co-eluates in a 25-30 min analysis time.


Subject(s)
Glutamic Acid/metabolism , Neostriatum/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Caudate Nucleus/cytology , Caudate Nucleus/metabolism , Chromatography, High Pressure Liquid , Electrochemistry , Hydrogen-Ion Concentration , In Vitro Techniques , Potassium/pharmacology , Putamen/cytology , Putamen/metabolism , Rats , o-Phthalaldehyde
16.
J Neurochem ; 68(2): 610-5, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9003047

ABSTRACT

The effects of activation of the adenylyl cyclase-protein kinase A pathway on the expression of delta-opioid receptor mRNA in the NG108-15 neuroblastoma x glioma cell line has been investigated. Activation of prostaglandin E1 (PGE1) receptors, which are positively coupled to adenylyl cyclase, resulted in a reduction in delta-receptor messenger RNA levels. Direct stimulation of adenylyl cyclase by forskolin or treatment of cells with the cyclic AMP analogue dibutyryl cyclic AMP (db-cAMP) mimicked the effect of PGE1. Down-regulation in receptor protein levels, as measured by loss of radioligand binding sites, was also observed and its extent correlated well with the decrease in the amount of delta-opioid receptor transcripts. D-Ser2-Leu-enkephalin-Thr6 (DSLET) inhibition of adenylyl cyclase activity was also diminished after db-cAMP treatment. Inhibitors of protein kinase A (PKA) partially reversed the PGE1- and db-cAMP-mediated repression of the delta-opioid receptor mRNA levels. The rate of degradation of delta-opioid receptor mRNA in the presence of actinomycin D was not altered in response to db-cAMP, suggesting that mRNA stability is not reduced by PKA action. The regulation of delta-opioid receptor mRNA levels by db-cAMP was not sensitive to the protein synthesis inhibitor cycloheximide, suggesting that de novo protein synthesis is not required in this process.


Subject(s)
Adenylyl Cyclases/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Receptors, Opioid, delta/genetics , Adenylyl Cyclase Inhibitors , Analgesics/pharmacology , Animals , Binding Sites/physiology , Bucladesine/pharmacology , Calcium Channels/physiology , Cyclic AMP/analogs & derivatives , Cyclic AMP/metabolism , Diprenorphine/metabolism , Diprenorphine/pharmacology , Enkephalin, Leucine/analogs & derivatives , Enkephalin, Leucine/pharmacology , Gene Expression Regulation, Enzymologic/physiology , Mice , Neuroblastoma , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/metabolism , Tritium , Tumor Cells, Cultured/chemistry , Tumor Cells, Cultured/enzymology
17.
Neuroscience ; 76(2): 479-89, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9015332

ABSTRACT

Competitive polymerase chain reaction assays following reverse transcription have been developed for quantitative analysis of delta and mu opioid receptor gene expression. The assay was used to obtain quantitative measurements of mu and delta opioid receptor expression levels in different brain regions and sensory and sympathetic ganglia in the rat. The assays provide accurate estimates of the relative levels of receptor messenger RNAs by the inclusion in the assays of known amounts of internal standards with the same sequence, except for a small deletion, as the target complementary DNA. The amplification products of target and competitor can be distinguished by size, and their amounts measured by densitometry. Expression of mu and delta opioid receptor messenger RNAs in different regions of the rat brain, somatic and visceral sensory and sympathetic ganglia was investigated using this method. In the brain the highest density of delta receptor messenger RNA was detected in the olfactory bulb, followed by the striatum. The mu receptor was expressed at highest levels in the midbrain-hypothalamic region. All the sensory ganglia studied expressed both mu and delta opioid receptor messenger RNAs. In the nodose ganglion we observed the highest level of mu receptor messenger RNA of any structure studied; in the trigeminal ganglion the level was about 10 times lower than that in the nodose ganglion. Among the dorsal root ganglia, mu receptor messenger RNA density was highest in the lumbar region, followed by the thoracic and cervical regions. The sympathetic superior cervical ganglion expressed a very low level of mu message. Delta receptor messenger RNA was detected only in the sensory ganglia, at levels that were considerably lower than in the striatum. The reverse transcription polymerase chain reaction assay is quantitatively reliable for comparison of messenger RNA levels between different RNA extracts, and sensitive enough to permit the detection and assay of mu and delta opioid receptor gene expression in a single pair of sensory or autonomic ganglia from the rat.


Subject(s)
Brain Chemistry/physiology , Ganglia/metabolism , Gene Expression/physiology , Receptors, Opioid, delta/biosynthesis , Receptors, Opioid, delta/genetics , Receptors, Opioid, mu/biosynthesis , Receptors, Opioid, mu/genetics , Animals , DNA/biosynthesis , Ganglia, Spinal/metabolism , Male , Nodose Ganglion/metabolism , Polymerase Chain Reaction , RNA/biosynthesis , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Superior Cervical Ganglion/metabolism , Trigeminal Ganglion/metabolism
18.
J Neurosci Methods ; 77(2): 211-20, 1997 Dec 01.
Article in English | MEDLINE | ID: mdl-9575046

ABSTRACT

The release of endogenous glutamate and gamma-aminobutyric acid (GABA) from rat brain tissue slices was studied using a tissue slice assay in which detectable amounts of the amino acids were released from 1-2 mg of tissue. An improved method of high performance liquid chromatography (HPLC) with electrochemical detection was employed to measure both glutamate and GABA after derivatization with o-phthalaldehyde and sulphite in a single isocratic HPLC analysis. The non-endogenous amino acid, homoglutamine, was used as an internal standard in verifying the consistent derivatization of amino acids and in quantifying amounts of glutamate and GABA released from the caudate-putamen tissue. The derivatized amino acids (1-30 pmol) were detected as chromatographic peaks eluting at baseline level and free of significant interfering co-eluates in a 25-30 min analysis time.


Subject(s)
Glutamic Acid/metabolism , Neostriatum/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Caudate Nucleus/cytology , Caudate Nucleus/metabolism , Chromatography, High Pressure Liquid , Electrochemistry , Hydrogen-Ion Concentration , In Vitro Techniques , Potassium/pharmacology , Putamen/cytology , Putamen/metabolism , Rats , o-Phthalaldehyde
19.
Neurochem Res ; 21(11): 1411-5, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8947931

ABSTRACT

We have previously demonstrated that continuous cocaine treatment for three days induces a marked but transient increase in mu opioid receptor (MOR) mRNA in nucleus accumbens (n. acc.); SCH 23390 and eticlopride, selective antagonists of D1- and D2-like dopamine (DA) receptors, respectively, blocked this cocaine-induced upregulation of MOR mRNA in n. acc. suggesting involvement of both subfamilies of DA receptors in the effect of cocaine (1,2). In the present study the ability of the selective DA D3 receptor antagonist, nafadotride (3,4), to prevent the cocaine-induced upregulation of MOR mRNA in n. acc. has been examined. Also, regulation of MOR mRNA following chronic administration of the DA agonists, SKF 38393, R(+)-6-Bromo-APB hydrobromide, or bromocriptine, has been studied. Male Sprague-Dawley rats were treated for 3 days with saline, cocaine, the DA receptor agonists or antagonist delivered by osmotic minipump. Expression of MOR mRNA in n. acc. was estimated by quantitative competitive polymerase chain reaction (PCR) assays following reverse transcription. Nafadotride (1.0 mg/kg/day) prevented the cocaine-induced upregulation of MOR mRNA in n. acc. When administered alone, nafadotride did not change the expression of MOR mRNA. The levels of MOR mRNA were elevated in n. acc. after 3 days treatment with each of the DA agonists, SKF 38393 (4.0 mg/kg/day), R(+)-6-Bromo-APB hydrobromide (4.0 mg/kg/day), or bromocriptine (5.0 mg/kg/day). Thus, DA agonists mimick the effect of cocaine on the expression of MOR mRNA in n. acc. These data confirm the involvement of dopaminergic mechanisms in the mediation of cocaine effects, indicate the comparability of actions of indirect and direct DA agonists, and point to the usefulness of cocaine as a tool to expose interaction between dopaminergic and opioid systems. The results suggest that activation of more than one type of DA receptor is required for the increased expression of MOR mRNA.


Subject(s)
Dopamine Antagonists/pharmacology , Nucleus Accumbens/metabolism , Receptors, Opioid, mu/biosynthesis , Salicylamides/pharmacology , Up-Regulation/drug effects , Animals , Benzazepines/pharmacology , Bromocriptine/pharmacology , Cocaine/pharmacology , Male , Naphthalenes/pharmacology , Nucleus Accumbens/drug effects , Pyrrolidines/pharmacology , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Receptors, Dopamine D1/physiology , Receptors, Dopamine D2/physiology , Receptors, Dopamine D3 , Transcription, Genetic/drug effects
20.
Brain Res Mol Brain Res ; 40(1): 153-6, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8840025

ABSTRACT

We investigated the effects of sustained administration of cocaine on the regulation of prodynorphin gene expression in rat brain. Intracerebroventricular (i.c.v.) infusion of cocaine hydrochloride (30 micrograms/day) for 7 days, by means of osmotic minipumps, elicited a significant 35% decrease of prodynorphin mRNA levels in rat hypothalamus and increase (22%) in caudate-putamen. At the same time and in the same animals, no significant changes were detected in the hippocampus or in the nucleus accumbens. These results indicate that continuously infused cocaine is able to modulate expression of the prodynorphin gene in opposite directions or has no effect on prodynorphin expression, depending on the brain region analysed. Cocaine, as well as opiates, might activate specific neuronal pathways, shared by different classes of drugs of abuse, involving, at least in part, the endogenous opioid system.


Subject(s)
Caudate Nucleus/metabolism , Cerebral Ventricles/physiology , Cocaine/pharmacology , Enkephalins/biosynthesis , Hypothalamus/metabolism , Protein Precursors/biosynthesis , Putamen/metabolism , Transcription, Genetic/drug effects , Animals , Caudate Nucleus/drug effects , Cerebral Ventricles/drug effects , Cocaine/administration & dosage , Hypothalamus/drug effects , Infusions, Parenteral , Male , Putamen/drug effects , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Rats , Rats, Inbred F344
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