Pharmacological targeting of the GABAB receptor alters Drosophila's behavioural responses to alcohol.

When exposed to ethanol, Drosophila melanogaster display a variety of addiction-like behaviours similar to those observed in mammals. Sensitivity to ethanol can be quantified by measuring the time at which 50% of the flies are sedated by ethanol exposure (ST50); an increase of ST50 following multiple ethanol exposures is widely interpreted as development of tolerance to ethanol. Sensitivity and tolerance to ethanol were measured after administration of the gamma-aminobutyric acid receptor B (GABAB ) agonist (SKF 97541) and antagonist (CGP 54626), when compared with flies treated with ethanol alone. Dose-dependent increases and decreases in sensitivity to ethanol were observed for both the agonist and antagonist respectively. Tolerance was recorded in the presence of GABAB drugs, but the rate of tolerance development was increased by SKF 97451 and unaltered in presence of CGP 54626. This indicates that the GABAB receptor contributes to both the sensitivity to ethanol and mechanisms by which tolerance develops. The data also reinforce the usefulness of Drosophila as a model for identifying the molecular components of addictive behaviours and for testing drugs that could potentially be used for the treatment of alcohol use disorder (AUD).

G-protein αq gene expression plays a role in alcohol tolerance in Drosophila melanogaster.

Ethanol is a psychoactive substance causing both short- and long-term behavioural changes in humans and animal models. We have used the fruit fly Drosophila melanogaster to investigate the effect of ethanol exposure on the expression of the Gαq protein subunit. Repetitive exposure to ethanol causes a reduction in sensitivity (tolerance) to ethanol, which we have measured as the time for 50% of a set of flies to become sedated after exposure to ethanol (ST50). We demonstrate that the same treatment that induces an increase in ST50 over consecutive days (tolerance) also causes a decrease in Gαq protein subunit expression at both the messenger RNA and protein level. To identify whether there may be a causal relationship between these two outcomes, we have developed strains of flies in which Gαq messenger RNA expression is suppressed in a time- and tissue-specific manner. In these flies, the sensitivity to ethanol and the development of tolerance are altered. This work further supports the value of Drosophila as a model to dissect the molecular mechanisms of the behavioural response to alcohol and identifies G proteins as potentially important regulatory targets for alcohol use disorders.

Naltrexone Reverses Ethanol Preference and Protein Kinase C Activation in Drosophila melanogaster.

Alcohol use disorder (AUD) is a major health, social and economic problem for which there are few effective treatments. The opiate antagonist naltrexone is currently prescribed clinically with mixed success. We have used naltrexone in an established behavioral assay (CAFE) in Drosophila melanogaster that measures the flies' preference for ethanol-containing food. We have confirmed that Drosophila exposed to ethanol develop a preference toward this drug and we demonstrate that naltrexone, in a dose dependant manner, reverses the ethanol-induced ethanol preference. This effect is not permanent, as preference for alcohol returns after discontinuing naltrexone. Additionally, naltrexone reduced the alcohol-induced increase in protein kinase C activity. These findings are of interest because they confirm that Drosophila is a useful model for studying human responses to addictive drugs. Additionally because of the lack of a closely conserved opiate system in insects, our results could either indicate that a functionally related system does exist in insects or that in insects, and potentially also in mammals, naltrexone binds to alternative sites. Identifying such sites could lead to improved treatment strategies for AUD.

ß3-integrin is required for differentiation in OC-2 cells derived from mammalian embryonic inner ear.

BACKGROUND: The mammalian inner ear contains the organ of Corti which is responsible for the conversion of sound into neuronal signals. This specialised epithelial tissue is the product of a complex developmental process where a common precursor cell type differentiates into the sound transducing hair cells and the non-innervated supporting cells. We hypothesised that integrin proteins, which are involved in cell attachment to extracellular matrix proteins and cellular signalling, play a role in the differentiation process of the precursor inner ear epithelial cells. To test our hypothesis we have utilised a cell line (OC-2) derived from E13 embryonic immortomouse inner ears. In vitro, by switching the incubation temperature from 33°C to 39°C, the OC-2 cells can be induced to differentiate and express hair cells markers, such as Myosin VIIa. The OC-2 cells are thus a useful model system for testing mechanism of hair cells differentiation. RESULTS: We have identified 4 integrin subunits which are expressed in OC-2 cells: α6, αv, ß1 and ß3. Among these, the relative level of expression of the αv, ß1 and ß3 subunits increased in a time dependent manner when the cells were exposed to the differentiating temperature of 39°C, most notably so for ß3 which was not detectable at 33°C. Treatment of fully differentiated OC-2 cells with siRNA against the four integrin subunits reduced the expression of not only the respective integrin proteins but also of the hair cell marker Myosin VIIa. Conversely over-expression of ß3 was sufficient to induce the expression of Myosin VIIa at 33°C. CONCLUSIONS: Our data demonstrate that modulation of integrin expression is associated with the differentiation process of the OC-2 cells. This suggests that the maturation of the organ of Corti, from where OC-2 cells are derived, may also depend on changes of gene expression associated with integrin expression.

Amphetamine and pseudoephedrine cross-tolerance measured by c-Fos protein expression in brains of chronically treated rats.

BACKGROUND: Pseudoephedrine is a drug commonly prescribed as a nasal decongestant and bronchodilator and is also freely available in cold remedies and medications. The structural and pharmacological similarity of pseudoephedrine to amphetamine has led to evaluation of its psychomotor stimulant properties within the central nervous system. Previous investigations have shown that the acute responses to pseudoephedrine were similar to those of amphetamine and other psychostimulants. RESULTS: This study examined the effect of chronic administration of pseudoephedrine in rat nucleus accumbens and striatum and identified three further similarities to amphetamine. (i) Chronic exposure to pseudoephedrine reduced the c-Fos response to acute pseudoephedrine treatment suggesting that pseudoephedrine induced tolerance in the animals. (ii) In animals chronically treated with amphetamine or pseudoephedrine the acute c-Fos response to pseudoephedrine and amphetamine was reduced respectively as compared to naïve animals indicating cross-tolerance for the two drugs. (iii)The known involvement of the dopamine system in the response to amphetamine and pseudoephedrine was further confirmed in this study by demonstrating that pseudoephedrine similarly to amphetamine, but with lower potency, inhibited [3H]dopamine uptake in synaptosomal preparations. CONCLUSION: This work has demonstrated further similarities of the effect of pseudoephedrine to those of amphetamine in brain areas known to be associated with drug addiction. The most significant result presented here is the cross tolerance effect of amphetamine and pseudoephedrine. This suggests that both drugs induce similar mechanisms of action in the brain. Further studies are required to establish whether despite its considerable lower potency, pseudoephedrine could pose health and addiction risks in humans similar to that of known psychostimulants.

The existence of opioid receptors in the cochlea of guinea pigs.

Several independent investigations have demonstrated the presence of opioid peptides in the inner ear organ of Corti and in particular in the efferent nerve fibers innervating the cochlear hair cells. However, the precise innervation pattern of opioid fibers remains to be investigated. In the present study the expression of opioid receptors and their peptides is demonstrated in young adult guinea pig cochlea. Opioid receptors are mainly expressed in hair cells of the organ of Corti and in inner and outer spiral bundles with different characteristics for each type of receptor. Co-localization studies were employed to compare the distribution of mu-, delta- and kappa-opioid receptors and their respective peptides, beta-endorphin, leu-enkephalin and dynorphin. Additionally, immunostaining of synaptophysin was used in this study to identify the presynaptic site. Immunoreactivity for enkephalin and dynorphin was found in the organ of Corti. Leu-enkephalin was co-localized with synaptophysin prominently in the inner spiral bundle (ISB). Dynorphin was co-localized with synaptophysin in both inner and outer spiral bundles. Delta-opioid receptor was most prominently co-localized with its peptide in the ISB bundle. Kappa-opioid receptor was seemingly present with dynorphin in both inner and outer spiral bundles. The co-staining of both peptides and receptors with synaptophysin in the same areas suggests that some of the opioid receptors may act as auto-receptors. The results provide further evidence that opioids may function as neurotransmitters or neuromodulators in the cochlea establishing the basis for further electrophysiological and pharmacological investigations to understand better the roles of the opioid system in auditory function.

Opioid modulation of GABA release in the rat inferior colliculus.

BACKGROUND: The inferior colliculus, which receives almost all ascending and descending auditory signals, plays a crucial role in the processing of auditory information. While the majority of the recorded activities in the inferior colliculus are attributed to GABAergic and glutamatergic signalling, other neurotransmitter systems are expressed in this brain area including opiate peptides and their receptors which may play a modulatory role in neuronal communication. RESULTS: Using a perfusion protocol we demonstrate that morphine can inhibit KCl-induced release of [3H]GABA from rat inferior colliculus slices. DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin) but not DADLE ([D-Ala2, D-Leu5]-enkephalin or U69593 has the same effect as morphine indicating that micro rather than delta or kappa opioid receptors mediate this action. [3H]GABA release was diminished by 16%, and this was not altered by the protein kinase C inhibitor bisindolylmaleimide I. Immunostaining of inferior colliculus cryosections shows extensive staining for glutamic acid decarboxylase, more limited staining for micro opiate receptors and relatively few neurons co-stained for both proteins. CONCLUSION: The results suggest that micro-opioid receptor ligands can modify neurotransmitter release in a sub population of GABAergic neurons of the inferior colliculus. This could have important physiological implications in the processing of hearing information and/or other functions attributed to the inferior colliculus such as audiogenic seizures and aversive behaviour.

The inner ear contains heteromeric channels composed of cx26 and cx30 and deafness-related mutations in cx26 have a dominant negative effect on cx30.

Cx26 and cx30 co-localize in tissues of the mammalian cochlea. Transfected HeLa cells were used to examine interactions between cx26 and cx30 and the effects on cx30 of four point mutations in cx26 that are associated with dominantly inherited hearing loss--W44S, G59A, D66H and R75W. When co-expressed, wtcx26 and wtcx30 trafficked to the same gap junction plaques. Cells transferred neurobiotin but not Lucifer Yellow, which passes freely through cx26 channels, suggesting cx30 affects the properties of cx26. G59A and D66H had a perinuclear localization when expressed alone but trafficked to the membrane when co-expressed with cx30. Co-expression of W44S, G59A or R75W with cx30, significantly reduced neurobiotin transfer in comparison with cells expressing cx30 only. These results indicate that cx26 and cx30 can oligomerize to form heteromeric connexons and demonstrate a dominant negative effect of some cx26 mutants on cx30. Immunogold labeling of thin sections of the cochlea showed both cx26 and cx30 distributed evenly on both sides of individual gap junction profiles. Immunoprecipitation of cochlear membrane proteins, isolated by procedures that preserve connexons, with either cx30 or cx26 antibodies precipitated both cx26 and cx30. Following co-injection of Lucifer Yellow and neurobiotin into individual supporting cells of the organ of Corti in cochlear slices, neurobiotin transferred to many cells, but Lucifer Yellow was retained in the injected cell. These observations are consistent with junctions composed of cx26/cx30 heteromeric connexons in the cochlea. The functional disruption caused by some cx26 mutations upon such heteromeric channels may underlie the non-syndromic nature of their effects on hearing.

The opioid receptors in inner ear of different stages of postnatal rats.

There is increasing evidence that the opioid system has a role in hearing. To provide further evidence for such a role, the expression of opioid receptor mRNAs and proteins in the inner ear of rats was studied during development from birth (P0) to postnatal day 16 (P16). A semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect changes in the expression of delta- (DOR) kappa- (KOR) and mu- (MOR) opioid receptor mRNAs in rat cochleae at P0, P4, P8 and P16. Expression of DOR mRNA levels steadily increased from P0 to P8 with no further increases by P16. KOR mRNA was expressed at a relatively high level at P0 and P4 followed by a decrease while MOR mRNA was expressed at a low level at P0 and P4 followed by an increase by P8 and P16. Immunocytochemical labelling of inner ear sections revealed unique developmental and distribution patterns of opioid receptors. In the organ of Corti DOR immunoreactivity (DOR-IR) was detected in hair cells from P4. In contrast MOR-IR was present only in supporting cells at P0-P16. In the spiral ganglion all three receptor subtypes were expressed from P0 on nerve cell soma and qualitatively appeared to increase with age. Also DOR-IR and MOR-IR were detected at P8 and P16 in nerve fibers within the spiral ganglion. In the limbus DOR-IR was detected at P8 and P16 on cells proximal to the tectorial membrane while MOR-IR was detected more distally. In general these findings demonstrate that within the inner ear each receptor subtype follows specific temporal and spatial developmental patterns, some of which may be associated to the onset of hearing. The data provide further evidence that the opioid system may play a role in the development and functioning of the inner ear.

Postnatal touch stimulation acutely alters corticosterone levels and glucocorticoid receptor gene expression in the neonatal rat.

Environmental manipulation early in life can alter the development of the hypothalamic-pituitary-adrenal (HPA) axis by mechanisms that are still unclear. The aim of the present work was to study the acute effects of postnatal touch stimulation, in an attempt to understand the mechanism by which touch stimulation early in life alters the HPA response to stress in adult animals. Rat pups were gently brushed for 15 min daily during the 1st postnatal week. Serum corticosterone levels were determined by radioimmunoassays, while glucocorticoid receptor (GR) gene expression was assayed by semiquantitative reverse transcriptase-polymerase chain reaction. Touch stimulation induced a significant decrease (30-36%) in serum corticosteroid secretion during the 1st and 2nd postnatal day as compared to the unstimulated group. In contrast, GR gene expression in the touch stimulation group was significantly increased in several brain areas such as the hippocampus (19-21%), frontal cortex (26-34%) and midbrain (15-24%). The results thus indicate that neonatal touch stimulation causes acute hormone- secretion and gene-expression changes within the period of stimulation. These changes may be the underlying cause for the permanent changes that have been observed in adult animals touch-stimulated as neonates.

The presence of opioid receptors in rat inner ear.

Opioid peptides have been identified in the inner ear but relatively little information is available about the expression and distribution of their receptors. The aim of the present study was therefore to identify and localize the mu (MOR), delta (DOR) and kappa (KOR) opioid receptor subtypes within the rat cochlea. The expression of these opioid receptor subtypes was determined by reverse transcriptase-polymerase chain reaction followed by nested polymerase chain reaction analysis. Amplification of RNAs from rat cerebral cortex (positive control) and rat cochlea with MOR, DOR and KOR primers resulted in products of the predicted lengths, 564, 356 and 276 bp, respectively. Restriction digestion confirmed the identity of these products. All three receptor subtypes were identified in the cochlea and further characterized by immunocytochemistry. DOR and KOR immunoreactivity was found in inner and outer hair cells, bipolar cells of the spiral ganglion and interdental cells of the limbus. In contrast, no MOR immunoreactivity was observed in the inner and outer hair cells, and interdental cells. All three types of receptor fibers were also detected in the bipolar cells and nerve fibers within the spiral ganglion. In addition, MOR- and KOR-containing nerve fibers were observed in the limbus. These findings are the first report of the presence of all three classical opioid receptors in the inner ear and suggest that these receptors may have both presynaptic and postsynaptic roles.

Mutations in the gene for connexin 26 (GJB2) that cause hearing loss have a dominant negative effect on connexin 30.

Mutations in the gene (GJB2) encoding connexin 26 (cx26) have been linked to sensorineural hearing loss either alone or as part of a syndrome. Here we compare the properties of four cx26 mutants derived from point mutations associated with dominantly inherited hearing loss, either non-syndromic (W44S, R75W) or with various skin disorders (G59A, D66H, R75W). Since cx26 and cx30 are co-localized within the inner ear the effect of the dominant cx26 mutations on both of these wild-type proteins was determined. Communication-deficient HeLa cells were transiently transfected with the various cDNA constructs by microinjection. Dye transfer studies using the gap junction permeant tracer Cascade Blue demonstrated a disruption to the intercellular coupling for all four of the mutant proteins. Immunostaining of the transfected cells revealed that for the G59A and D66H mutants this correlated with impaired intracellular trafficking and targeting to the plasma membrane, as both proteins had a perinuclear localization. The impaired trafficking was rescued by oligomerization both with cx26 and with cx30, suggesting that cx26 and cx30 can form heteromeric connexons. Significantly reduced dye transfer rates were observed between cells co-expressing either cx26 or cx30 together with W44S or R75W compared with the wild-type proteins alone. The dominant actions of the G59A and D66H mutants were only on cx30 and cx26, respectively. We suggest that cx26 and cx30 form heteromeric connexons in vivo, within the inner ear, with particular properties essential for hearing. Disruption of these heteromeric channels by certain mutations may underlie the non-syndromic nature of the deafness.