Behavioral action of ethanol in Porsolt's forced swim test: modulation by 3 alpha-hydroxy-5 alpha-pregnan-20-one

Ethanol is known to increase cortical and plasma content of GABAergic neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) which is responsible for some of its behavioral and electrophysiological effects. We have previously demonstrated the antidepressant like effect of 3alpha,5alpha-THP in mice. This study investigated the role of 3alpha,5alpha-THP in acute, chronic and withdrawal effects of ethanol using mouse forced swim test (FST) paradigm. While acute systemic ethanol (2 or 2.5 g/kg) administration exhibited an antidepressant like effect, its prolonged consumption produced tolerance to this effect and its withdrawal, on the other hand, elicited enhanced behavioral despair (depression). The antidepressant like effect of ethanol was potentiated by GABA(A) receptor agonist, muscimol (0.5 mg/kg, i.p.), 3alpha,5alpha-THP (0.5, 1 or 2 microg/mouse, i.c.v.) and by neurosteroidogenic drugs viz. selective serotonin reuptake inhibitor (SSRI), fluoxetine (5 or 20 mg/kg, i.p.), agonist at mitochondrial diazepam binding inhibitor receptor, FGIN 1-27 (0.5 or 1 microg/mouse, i.c.v.), or 11beta-hydroxylase inhibitor, metyrapone (0.5 or 1 microg/mouse, i.c.v.) which are known to increase endogenous 3alpha,5alpha-THP content. Furthermore, inhibition of the endogenous neurosteroid biosynthesis by drugs like 5alpha-reductase inhibitor, finasteride (50 mg/kg, s.c.), 3beta-hydroxysteroid dehydrogenase inhibitor, trilostane (30 mg/kg i.p.) or 3alpha-hydroxysteroid dehydrogenase inhibitor, indomethacin (5 mg/kg, i.p.) and GABA(A) receptor antagonist, bicuculline (1 mg/kg, i.p.) blocked the antidepressant like effect of ethanol. Withdrawal of ethanol from mice consuming it chronically displayed enhanced behavioral despair and elicited tolerance to antidepressant like action of acute ethanol (2.5, 3 or 3.5 g/kg). Moreover, sub-antidepressant doses (0.25 or 0.5 microg/mouse, i.c.v.) of 3alpha,5alpha-THP and fluoxetine (5 mg/kg, i.p.) but not imipramine (1 mg/kg, i.p.) reversed the depression associated with ethanol withdrawal indicating sensitization to their antidepressant action. Thus, 3alpha,5alpha-THP plays a pivotal role in the actions of ethanol and in the depression associated with ethanol withdrawal. These findings may be of potential ramification to contribute to the depression associated with alcoholism and its treatment using neurosteroids.     

Subunit-dependent interaction of the general anaesthetic etomidate with the γ-aminobutyric acid type A receptor

1. The GABA modulating and GABA-mimetic actions of the general anaesthetic etomidate were examined in voltage-clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant γ-aminobutyric acid_A (GABA_A) receptor subunits.
2. Currents mediated by recombinant receptors with the ternary subunit composition α_xβ_yγ_2L (where x=1,2,3 or 6 and y=1 or 2), in response to GABA applied at the appropriate EC₁₀, were enhanced by etomidate in a manner that was dependent upon the identity of both the α and β subunit isoforms.
3. For the β₂-subunit containing receptors tested, the EC₅₀ for the potentiation of GABA-evoked currents by etomidate (range 0.6 to 1.2 μM) was little affected by the nature of the α subunit present within the hetero-oligomeric complex. However, replacement of the β₂ by the β₁ subunit produced a 9–12 fold increase in the etomidate EC₅₀ (6 to 11 μM) for all α-isoforms tested.
4. For α₁, α₂ and α₆, but not α₃-subunit containing receptors, the maximal potentiation of GABA-evoked currents by etomidate was greater for β₂- than for β₁-subunit containing receptors. This was most clearly exemplified by receptors composed of α₆β₁γ_2L compared to α₆β₂γ_2L subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC₁₀ to 28±2% and 169±4% of the maximal GABA response, respectively.
5. For α₁ subunit-containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the β subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5α-pregnan-3α-ol-20-one was marginally higher for β₁ rather than the β₂ subunit-containing receptor, although its maximal effect was similar at the two receptor isoforms.
6. The GABA-mimetic action of etomidate was supported by β₂- but not β₁-subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either β isoform. For β₂-subunit containing receptors, both the agonist EC₅₀ and the maximal current produced by etomidate were additionally influenced by the α isoform.
7. It is concluded that the subtype of β-subunit influences the potency with which etomidate potentiates GABA-evoked currents and that the β isoform is a crucial determinant of the GABA-mimetic activity of this compound. The nature of the α-subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced influences may aid the identification of the site that recognises etomidate. More generally, these results provide a clear example of structural specificity in anaesthetic action.

     

Effects of chronic citalopram treatment on 5-HT1A and 5-HT2A receptors in group- and isolation-housed mice

Selective serotonin reuptake inhibitors (SSRI) are characterized by high clinical effectiveness and good tolerability. A 2-3 week delay in the onset of effects is caused by adaptive mechanisms, probably at the serotonergic (5-HT) receptor level. To analyze this in detail, we measured 5-HT(1A) and 5-HT(2A) receptor bindings in vitro after 3 weeks of citalopram treatment (20 mg/kg i.p. daily) in group-housed as well as isolation-housed mice, reflecting neurobiological aspects seen in psychiatric patients. Isolation housing increased somatodendritic (+52%) and postsynaptic (+30-95%) 5-HT(1A) as well as postsynaptic 5-HT(2A) receptor binding (+25-34%), which confirms previous findings. Chronic citalopram treatment did not induce alterations in raphe 5-HT(1A) autoreceptor binding, independent of housing conditions. Housing-dependent citalopram effects on postsynaptic 5-HT(1A) receptor binding were found with increases in group- (+11-42%) but decreases in isolation-housed (-11 to 35%) mice. Forebrain 5-HT(2A) receptor binding decreased between 11 and 38% after chronic citalopram administration, independent of housing conditions. Citalopram's long-term action comprises alterations at the postsynaptic 5-HT(1A) and 5-HT(2A) receptor binding levels. Housing conditions interact with citalopram effects, especially on 5-HT(1A) receptor binding, and should be more strongly considered in pharmacological studies. In general, SSRI-induced alterations were more pronounced and affected more brain regions in isolates, supporting the concept of a higher responsiveness in "stressed" animals. Isolation-induced receptor binding changes were partly normalized by chronic citalopram treatment, suggesting the isolation housing model for further analyses of SSRI effects, especially at the behavioral level.     

Chronic progesterone treatment augments while dehydroepiandrosterone sulphate prevents tolerance to ethanol anxiolysis and withdrawal anxiety in rats

We have recently shown that the neurosteroid allopregnanolone modulates anxiolytic effect of ethanol. In the present report, we attempted to examine whether neurosteroids progesterone and dehydroepiandrosterone sulphate (DHEAS), which modulate gamma-aminobutyric acid (GABA(A)) receptor function, affects development of tolerance to ethanol anxiolysis and withdrawal anxiety. Rats on ethanol (6% v/v in nutritionally balanced liquid diet) for prolong period (10 days) were injected twice daily either with vehicle, progesterone (a precursor of allopregnanolone, positive GABA(A) receptor modulator), finasteride (5alpha-reductase inhibitor) or DHEAS (negative GABA(A) receptor modulator). During this period, rats were acutely challenged periodically with ethanol (2 g/kg, i.p., 8% w/v) and subjected to the elevated plus maze test. For withdrawal studies, similar treatment protocols (except ethanol challenge) were employed and on day 11, rats were subjected to the elevated plus maze test at different time intervals post-ethanol withdrawal. While progesterone significantly advanced the development of tolerance to ethanol anxiolysis and enhanced withdrawal anxiety, DHEAS and finasteride prevented such behavioral alterations. These data highlight the important role played by GABAergic neurosteroids progesterone and DHEAS in the development of tolerance to ethanol anxiolysis and withdrawal anxiety in rats. Moreover, it points to the potential usefulness of specific neurosteroids as targets in the treatment of alcoholism.     

Functional properties of recombinant GABAA receptors composed of single or multiple β subunit subtypes

GABA_A receptor (GABAR) isoforms in the central nervous system are composed of combinations of α(1–6), β(1–4), γ(1–4), δ(1) and (1) subunit subtypes arranged in a pentamer. Many regions of the brain express high levels of mRNA encoding several different subunits and even multiple subunit subtypes. The stoichiometry of GABAR isoforms is unclear, and the number and identity of individual subunit subtypes that are coassembled remain uncertain. To examine the role of β subunit subtypes in the functional properties of GABARS and to determine whether multiple β subtypes can be coassembled in functional GABARs, plasmids containing cDNAs encoding rat β1 and/or β3, α5 and γ2L subtypes were cotransfected into L929 fibroblasts. The properties of the expressed receptor populations were determined using whole-cell and single-channel recording techniques. The α5β1γ2L isoform was less sensitive to GABA than the α5β3γ2L isoform. α5β1γ2L isoform currents were also insensitive to the allosteric modulator loreclezole, while α5β3γ2L isoform currents were strongly potentiated by loreclezole. Fibroblasts transfected with plasmids containing cDNAs for both β1 and β3 subtypes along with α5 and γ2L subtypes produced a receptor population with an intermediate sensitivity to GABA which was insensitive to loreclezole. These results suggest that functional GABARs can be formed that contain two different β subunit subtypes with properties different from receptors that contain only a single β subtype and that the β subunit subtypes influence the response of GABARs to GABA and to the allosteric modulator loreclezole.     

The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor

1. The γ-aminobutyric acid (GABA)-modulatory and GABA-mimetic actions of etomidate at mammalian GABA_A receptors are favoured by β₂- or β₃- versus β₁-subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (β_{2 N290}, β_{3 N289}, β_{1 S290}). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two-point voltage clamp technique to determine the influence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate-insensitive invertebrate GABA receptor (Rdl) of Drosophila melanogaster.
2. Complementary RNA-injected oocytes expressing the wild type Rdl GABA receptor and voltage-clamped at −60 mV responded to bath applied GABA with a concentration-dependent inward current response and a calculated EC₅₀ for GABA of 20±0.4 μM. Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine (Rdl_M314N) or a serine (Rdl_M314S) also exhibited a concentration-dependent inward current response to GABA, but in both cases with a reduced EC₅₀ of 4.8±0.2 μM.
3. Utilizing the appropriate GABA EC₁₀, etomidate (300 μM) had little effect on the agonist-evoked current of the wild type Rdl receptor. By contrast, at Rdl_M314N receptors, etomidate produced a clear concentration-dependent enhancement of GABA-evoked currents with a calculated EC₅₀ of 64±3 μM and an E_max of 68±2% (of the maximum response to GABA).
4. The actions of etomidate at Rdl_M314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 μM R-(+)-etomidate and S-(−)-etomidate enhancing the current induced by GABA (EC₁₀) to 52±6% and 12±1% of the GABA maximum respectively.
5. The effects of this mutation were selective for etomidate as the GABA-modulatory actions of 1 mM pentobarbitone at wild type Rdl (49±4% of the GABA maximum) and Rdl_M314N receptors (53±2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5α-pregnan-3α-ol-20-one (Rdl=25±4% of the GABA maximum) was not altered by this mutation (Rdl_M314N=18±3% of the GABA maximum).
6. Etomidate acting at β₁ (S290)-containing mammalian GABA_A receptors is known to produce only a modest GABA-modulatory effect. Similarly, etomidate acting at Rdl_M314S receptors produced an enhancement of GABA but the magnitude of the effect was reduced compared to Rdl_M314N receptors.
7. Etomidate acting at human α₆β₃γ_2L receptors is known to produce a large enhancement of GABA-evoked currents and at higher concentrations this anaesthetic directly activates the GABA_A receptor complex. Mutation of the human β₃ subunit asparagine to methionine (β_{3 N289M} found in the equivalent position in Rdl completely inhibited both the GABA-modulatory and GABA-mimetic action of etomidate (10–300 μM) acting at α₆β_{3 N289M}γ_2L receptors.
8. It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modification of their function by etomidate can be influenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction. Furthermore, this study provides a clear example of the specificity of anaesthetic action.

     

Influence of hormonal status on behavioral responses to an acute ethanol challenge during ethanol withdrawal in male and female rats

We previously reported significant sex differences in ethanol withdrawal (EW) recovery as well as in sensitivity to GABA_A receptor modulators during EW. The aim of the present study was to determine if hormonal status moderated behavioral responses to an acute ethanol challenge in EW animals comparing two different behaviors. An initial set of experiments explored motor-incoordinating effects of the acute ethanol injection during EW at either 1 day or 3 days of withdrawal. EW male, but not female, rats showed a decrease in coordination compared to controls that persisted through 3 days EW. Female rats displayed tolerance to the motor-incoordinating actions of the acute ethanol challenge at 1 day EW whereas tolerance was more evident in EW male rats at 3 days. In contrast, EW animals generally remained responsive to the anticonvulsant actions of ethanol, irrespective of hormonal status. While EW by itself did not significantly alter seizure latency, duration or severity, it increased seizure-induced mortality especially at 3 days EW. There was some evidence of tolerance to the anticonvulsant effect of the acute ethanol challenge at the lowest dose employed (0.62 g/kg), which varied by sex condition and time of EW. All sex conditions displayed marked sensitivity to the anticonvulsant effects of the ethanol challenge at the two higher doses studied. Overall, ovariectomized females showed the greatest response to the acute ethanol administration. These findings provide additional evidence of a divergence in behavioral responses during EW and suggest that multiple neuroadaptations moderate various responses to ethanol during EW, with minor contributions of hormonal status.     

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABAA receptors but with the opposite functionality

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA_A receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence.
2. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU-100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU-100079 with a trifluoroethyl substituent produced a positive allosteric response.
3. These functional characteristics were monitored with Cl⁻ currents measurements in cloned rat αxβ2γ2 subtypes of GABA_A receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA_A receptors with binding of [³⁵S]-TBPS, which is a high affinity ligand specific for GABA_A receptors with exquisite sensitivity to allosteric modulations.
4. This structure-functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

     

Alterations in brain monoamines and GABAA receptors in transgenic mice overexpressing TGFα

This study investigated the possibility that overexpression of transforming growth factor α (TGFα) changes those neurotransmitter systems that have been associated with behaviors found to be altered in the transgenic TGFα CD-1 mice. The female TGFα mice showed elevated levels of norepinephrine (NE) in the hypothalamus and serotonin (5-HT) in the cortex and brain stem when compared with nontransgenic CD-1 females. The concentrations of monoamines were not altered in the male transgenic brain. The 5-hydroxyindoleacitic acid (5-HIAA)/5-HT ratio was significantly reduced in the brain stem of the male TGFα mice and frontal cortex in the female transgenics. The binding of the [³H]GBR 12935-labeled DA transporter was lower in the frontal cortex in the transgenic male TGFα mice than in the female TGFα mice. No gender difference in dopamine (DA) transporter binding was noted between the nontransgenic male and female mice. Serotonin and GABA_A receptors were measured only in males. No differences in the number of 5-HT_1A and 5-HT₂ receptors were found in the cortex or hippocampus. Maximal GABA stimulation of [³H]flunitrazepam binding in the forebrain hemispheres and cerebellar binding of an imidazobenzodiazepine, [³H]Ro 15-4513, were not different between transgenic and nontransgenic male mice. However, forebrain [³⁵S]TBPS binding in male TGFa mice was less affected by the blockade of the GABA agonist sites by the specific GABA_A antagonists SR 95531 and bicuculline than the binding of the controls, suggesting either altered endogenous GABA concentrations or a change in receptor populations. Taken together, the previously reported behavioral alterations in male TGFa mice, including increased levels of aggressive behavior, locomotor activity, voluntary alcohol consumption, and immobility in the swim test, or the altered behavioral responses to alcohol and monoamine uptake inhibitors, may be due to a reduced 5-HIAA/5-HT ratio, [³H]GBR 12935-labeled DA transporter binding, or altered regulation of [³⁵S]TBPS binding by endogenous GABA in the brain. Reduced aggressive behavior and shortened immobility in the swim test in the female TGFa mice, on the other hand, might reflect elevated levels of NE and 5-HT in the brain. It is possible that TGFα-induced increase in plasma estrogen levels in the transgenic mice is the common mechanism of action that causes gender-specific changes in certain neurotransmitter systems.     

Effects of diazepam and zolpidem on EEG beta frequencies are behavior-specific in rats

A pharmacological dissociation of the relation between electroencephalographic (EEG) activity and behavior has been described for the benzodiazepines. While a decrease in high frequency EEG activity is associated with a decrease in arousal in drug-free conditions, sedative benzodiazepines increase beta activity. Non-benzodiazepine GABA(A) receptor modulators can increase beta activity as well. To further study the relationship between rat behavior and EEG under GABA(A) receptor modulation, EEG effects of diazepam (2.5 mg/kg) and zolpidem (2.5 mg/kg) were studied during different behaviors. Both drugs modulate the GABA(A) receptor, albeit that zolpidem shows alpha(1) subunit selectivity while diazepam is non-selective. A detailed analysis of rat open field behavior was made with a distinction of 25 behavioral elements. The EEG was segmented according to each behavioral element and a corresponding power spectrum calculated. Both diazepam and zolpidem increased EEG beta frequencies, characteristic for the benzodiazepines. However, the beta and gamma increase was specific for active behavior and not for inactivity. Interestingly, diazepam and zolpidem seemed to amplify, rather than dissociate, the relation between behavior and the EEG. It is hypothesized that the large increase in beta-3/gamma activity caused by diazepam and zolpidem is a compensatory mechanism that allows for behavioral activation, despite pharmacologically induced sedation.     

Unraveling the identity of benzodiazepine binding sites in rat hipppocampus and olfactory bulb

The goals of the work reported here were (i) to identify distinct GABA(A)/benzodiazepine receptors in the rat hippocampus and olfactory bulb using receptor binding assays, and (ii) to determine the affinities and selectivities of benzodiazepine receptor ligands from structurally diverse chemical families at each site identified. These studies were aided by the use of software AFFINITY ANALYSIS SYSTEM, developed in our laboratory for analysis of receptor binding data that allows the determination of receptor heterogeneity using non-selective radioligands. Saturation binding assays using [3H]RO15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1, 5-a]-[1,4]benzodiazepine-3-carboxylate) revealed two binding sites in each of these two tissues. The higher affinity site corresponds to alpha(5) subunit-containing GABA(A) receptor and the lower affinity site to a combination of alpha(1), alpha(2), and alpha(3) subunit-containing receptors. These results should be useful in the challenging task of identifying the various functional GABA(A) receptors in the central nervous system, and in providing a link between receptor affinities and in vivo activities of the GABA(A)/benzodiazepine receptor ligands studied.     

Ipsapirone and 8-OH-DPAT reduce ethanol preference in rats: involvement of presynaptic 5-HT1A receptors

The selective serotonin(5-HT)_1A receptor agonists 8-OH-DPAT and ipsapirone were tested in selectively inbred Wistar rats, with high preference [70–90%: defined as the ratio of ethanol (EtOH) to total fluid intake] for EtOH (10% v/v) over water in a two-bottle free choice situation. Rats were injected shortly before the overnight test session (8:00p.m.–8:00a.m.). EtOH and water consumption were determined in 20-min intervals; food consumption after the session. 8-OH-DPAT (ED₅₀: 2.4 mg/kg, SC) and ipsapirone (ED₅₀: 12.5 mg/kg, SC) reduced EtOH preference in a dose-dependent manner. In addition, 8-OH-DPAT increased total fluid intake, whereas ipsapirone enhanced total food intake. The EtOH preference reduction was time-dependent and reached a maximum within the second 4 h after application of 8-OH-DPAT (–73%) and ipsapirone (–72%). The preference reducing effect of ipsapirone (20 mg/kg, PO) was completely blocked by the nonselective 5-HT_1A antagonist spiperone (0.05 mg/kg, SC). Local application of 8-OH-DPAT (10 µg, 0.5 µl) into the dorsal raphe nucleus (DRN, a brain area rich in somatodendritic 5-HT_1A autoreceptors), reduced the EtOH preference significantly as compared to the saline injection in the same animal (–12%, 8:00–12:00p.m.). Only marginal effects on ingestion behavior were observed after micro-injection into the nucleus accumbens. Reduction of brain 5-HT levels by pretreatment with the 5-HT synthesis inhibitor pCPA (2×150 mg/kg, IP) resulted in a short lasting, marked reduction (–54%) and a long lasting, small attenuation of the EtOH preference. Total food consumption was strongly decreased but returned soon to normal; total fluid intake was only slightly decreased. The EtOH preference reducing effect of ipsapirone (5 and 20 mg/kg, SC) was attenuated in pCPA-pretreated rats. The present data suggest that 5-HT_1A receptor ligands reduce EtOH preference via stimulation of 5-HT_1A receptors in the DRN. The possibility of additional mechanism(s) is discussed.     

Activation of peripheral mitochondrial benzodiazepine receptors in the hippocampus stimulates allopregnanolone synthesis and produces anxiolytic-like effects in the rat

RATIONALE AND OBJECTIVES: Stimulation of the mitochondrial benzodiazepine receptor (MBR) in the brain activates the synthesis of neurosteroids that can act as positive modulators of the GABA(A) receptor complex. Allopregnanolone is a potent anxiolytic, anticonvulsant, sedative and hypnotic GABAergic neurosteroid. The anxiolytic-like effects of FGIN 1-27, an MBR agonist, were determined after microinjection into the dorsal hippocampus. METHODS: Behavior in the elevated plus-maze was assessed in adult male rats after bilateral injections of 0, 1.25, 2.5, or 5 microg FGIN 1-27. The behavioral effects of FGIN 1-27 were also determined in animals receiving intrahippocampal co-administration of 20 ng picrotoxin, 5 microg flumazenil, or 200 ng PK 11195. The effects of FGIN 1-27 on behavior in the elevated plus-maze and shock-probe burying test were measured in animals pretreated systemically with 10 mg/kg 4-MA, a 5alpha-reductase inhibitor. Hippocampal and blood plasma levels of allopregnanolone were measured in separate groups of animals pretreated with 4-MA and receiving an intrahippocampal injection of FGIN 1-27. RESULTS: Intrahippocampal injections of FGIN 1-27 produced anxiolytic-like effects in the plus-maze and in the shock-probe burying test. Hippocampal and blood levels of allopregnanolone were also increased by FGIN 1-27. The anxiolytic-like effects of FGIN 1-27 were attenuated by PK 11195 and were blocked by picrotoxin and 4-MA pretreatment, but remained unaffected by flumazenil pretreatment. The neurosteroidogenic effect of FGIN 1-27 was also eliminated by 4-MA. CONCLUSION: Activation of the MBR in the hippocampus leads to the synthesis of allopregnanolone, an anxiolytic neurosteroid that potentiates GABA(A) receptor function.     

Effects of ethanol and GABA<Subscript>B</Subscript> drugs on working memory in C57BL/6J and DBA/2J mice

Rationale It has been suggested that GABA_B receptors may be part of a neural substrate mediating some of the effects of ethanol.Objective The purpose of this experiment was to investigate, in mice, the effects of ethanol on working memory in a delayed matching-to position (DMTP) task, and additionally to determine if these effects were modulated by GABA_B receptors.Methods Female C57BL/6J and DBA/2J mice were trained in the DMTP task, and after asymptotic levels of performance accuracy were achieved, injections (IP) of ethanol, baclofen, or phaclofen were administered. Baclofen or phaclofen were then co-administered with ethanol. Each test was repeated twice.Results Ethanol caused deficits in working memory at 2.0 g/kg and higher. The highest dose (2.5 g/kg) produced additional non-specific effects, indicative of sedation. Baclofen increased performance accuracy (2.5 mg/kg), while decreasing the total number of trials completed. When combined with ethanol (1.5 g/kg), baclofen increased memory deficits at the highest dose (7.5 mg/kg). Phaclofen increased performance accuracy at 10 and 30 mg/kg but had no effect on the total number of trials completed. When combined with ethanol (2.5 g/kg), phaclofen did not significantly alter ethanol-induced deficits in performance.Conclusions Analyses of performance accuracy, total trials completed and variables indexing bias and motor impairment indicated that GABA_B drugs modulate working memory in a behaviorally specific manner. Overall, these receptors may be part of a neural substrate that modulates some of the effects of ethanol.     

Effects of chronic selective serotonin reuptake inhibitors on 8-OH-DPAT-induced facilitation of ejaculation in rats: comparison of fluvoxamine and paroxetine

Rationale Chronic treatment with selective serotonin reuptake inhibitors (SSRIs) can delay ejaculation in humans, but the extent of this effect differs between SSRIs. The involvement of 5-HT_1A receptors is likely, since 5-HT_1A receptor agonists accelerate ejaculation and chronic SSRI treatment is thought to desensitize 5-HT_1A receptors.Objectives This study was conducted to examine the effects of chronic pretreatment with the SSRIs fluvoxamine and paroxetine on the facilitation of ejaculation induced by the 5-HT_1A receptor agonist 8-OH-DPAT.Methods Sexually experienced Wistar rats with normal ejaculatory behavior were treated for 22 days with vehicle, fluvoxamine (30 mg/kg/day), or paroxetine (10 or 20 mg/kg/day, p.o.). On day 22, rats received a challenge with saline or 8-OH-DPAT (0.4 mg/kg, s.c.). Sexual behavior was tested on days 1, 8, 15, and 22 of the SSRI-treatment.Results Treatment with both doses of paroxetine, but not fluvoxamine, delayed ejaculation. 8-OH-DPAT strongly accelerated ejaculation under vehicle conditions. Pretreatment with paroxetine reduced the effects of 8-OH-DPAT on ejaculation in a dose-dependent manner and more strongly than fluvoxamine.Conclusions SSRIs affect 5-HT_1A receptors involved in ejaculation. The degree to which this occurs, with paroxetine exerting a stronger effect than fluvoxamine, might determine the extent of SSRI-induced delayed ejaculation.     

Effects of isolation-rearing on voluntary consumption of ethanol, sucrose and saccharin solutions in Fawn Hooded and Wistar rats

These experiments examined the hypothesis that isolation-rearing and strain influence hedonic mechanisms. In experiment 1, voluntary consumption of ethanol and water was monitored in the home cage of Fawn Hooded (FH) and Wistar rats. FH rats were found to consume more ethanol at low concentrations than Wistar rats, independent of rearing condition, and isolation-reared rats were found to consume more of high ethanol concentrations, independent of strain. In experiment 2, isolation-reared rats were found to consume more sucrose, independent of concentration, than socially reared rats. In experiment 3, Fawn Hooded rats were found to be more sensitive to low concentration solutions of saccharin, and to consume less of the high concentration solutions, while isolation-rearing was found to enhance consumption of high concentrations. Thus, hedonic processes are independently modulated by strain and rearing conditions, although the effects of isolation-rearing appear to be exacerbated in Fawn Hooded rats. Received: 19 August 1997 / Final version: 26 November 1997     

5-HT1A receptors are involved in the cannabidiol-induced attenuation of behavioural and cardiovascular responses to acute restraint stress in rats

Background and purpose

Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa which induces anxiolytic- and antipsychotic-like effects in rodents. These effects could be mediated by facilitation of the endocannabinoid system or by the activation of 5-HT_1A receptors. As either of these mechanisms could promote adaptation to inescapable stress, the aim of the present work was to test the hypothesis that CBD would attenuate the autonomic and behavioural consequences of restraint stress (RS). We also investigated if the responses to CBD depended on activation of 5-HT_1A receptors.

Experimental approach

Male Wistar rats received i.p. injections of vehicle or CBD (1, 10 or 20 mg kg⁻¹) and 30 min later were submitted to 60 min of restraint where their cardiovascular responses were recorded. The protocol of the second experiment was similar to the first one except that animals received i.p. injections of the 5-HT_1A receptor antagonist WAY100635 (0.1 mg kg⁻¹) before CBD treatment and exposure to restraint. 24 h later they were also tested in the elevated plus-maze (EPM), an animal model of anxiety.

Key results

Exposure to RS increased blood pressure and heart rate and induced an anxiogenic response in the EPM 24 h later. These effects were attenuated by CBD. WAY100635 by itself did not change the cardiovascular and anxiogenic response to RS, but blocked the effects of CBD.

Conclusion and implications

The results suggest that CBD can attenuate acute autonomic responses to stress and its delayed emotional consequences by facilitating 5-HT_1A receptor-mediated neurotransmission.     

Pharmacological profile of 5-hydroxytryptamine-induced inhibition on the pressor effect elicited by sympathetic stimulation in long-term diabetic pithed rats

We analysed the type and/or subtype of 5-hydroxytryptamine (5-HT) receptors involved in the inhibitory mechanisms of 5-HT on the pressor responses induced by stimulation of sympathetic vasopressor outflow in long-term diabetic pithed rats. Diabetes was induced in male Wistar rats by a single subcutaneous injection of alloxan. Eight weeks later, rats were anaesthetized, pre-treated with atropine, and pithed. The effect of 5-HT on the pressor responses elicited by stimulation of the sympathetic outflow was analysed in eight-week alloxan-induced diabetic pithed rats. 5-HT (20 μg/kg/min) reduced the pressor action obtained by electrical stimulation of the sympathetic outflow. However, there was no effect on exogenous noradrenaline-induced pressor responses. 5-CT (5 μg/kg/min), 8-OH-DPAT (5 μg/kg/min), and α-methyl-5-HT (5 μg/kg/min), selective 5-HT₁, 5-HT_1A and 5-HT₂ receptor agonists, respectively, reproduced the 5-HT inhibitory action. Nevertheless, infusion of 5 μg/kg/min of 1-phenylbiguanide, CGS-12066B, L-694,247, BW273C86 or MK212 (5-HT₃, 5-HT_1B, 5-HT_1D, 5-HT_2B and 5-HT_2C receptor agonists, respectively) had no effect on the pressor responses elicited by stimulation of the sympathetic outflow. Methiothepin (100 μg/kg) and a cocktail of WAY-100,635 (100 μg/kg) and spiperone (125 μg/kg) blocked the 5-HT inhibitory effect on the pressor action obtained by sympathetic stimulation. Moreover, WAY-100, 635 abolished the 8-OH-DPAT inhibitory effect and spiperone blocked α-methyl-5-HT action. In conclusion, this study revealed that long-term experimental diabetes induces changes in the receptor type/subtype involved in the 5-HT inhibitory action on the sympathetic pressor responses produced by electrical stimulation. This is mainly mediated by pre-junctional 5-HT_1A and 5-HT_2A receptors.     

Anxiolytic-Like Effects of Chrysanthemum indicum Aqueous Extract in Mice: Possible Involvement of GABAA Receptors and 5-HT1A Receptors

Chrysanthemum indicum Linne is an ancient herbal medicine used to treat bone and muscle deterioration, ocular infl ammation, headache, and anxiety in Korea, China, and Japan. Furthermore, tea derived from Chrysanthemum indicum Linne has been used to treat anxiety by facilitating relaxation and curing insomnia. However, no reports exist on the anxiolytic-like effects of Chrysanthemum indicum Linne water extract (CWE) in mice. In the present study, we investigated the anxiolytic-like effects of CWE using the elevated plus-maze (EPM) test in mice. CWE, at a dose of 500 mg/kg (p.o.), signifi cantly increased the time spent in the open arms of the EPM compared to a vehicle-injected control group. Moreover, the effect of CWE (500 mg/kg) was blocked by bicuculline (a selective GABA_A receptor antagonist) and WAY 100635 (a selective 5-HT_1A receptor antagonist). Taken together, these fi ndings suggest that the anxiolytic-like effects of CWE might be mediated by the GABA_A receptor and the 5-HT_1A receptor.     

Discriminative stimulus effects of ethanol and 3α-hydroxy-5α-pregnan-20-one in relation to menstrual cycle phase in cynomolgus monkeys (Macaca fascicularis)

 The present study was designed to characterize the discriminative stimulus effects of ethanol and the neurosteroid 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) in nonhuman primates as a function of menstrual cycle phase. Female cynomolgus monkeys (Macaca fascicularis) were trained in a two-lever procedure to discriminate 1.0 g/kg ethanol (IG, 30 min pretreatment) from water using food reinforcement. A cumulative dosing procedure was used to assess changes in the potency of ethanol and an endogenous anxiolytic steroid in the follicular versus the luteal phase of the menstrual cycle. Plasma progesterone and allopregnanolone levels were determined within 24 h of testing to verify phase of menstrual cycle. The monkeys were more sensitive to the discriminative stimulus effects of ethanol and the ethanol-like effects of the endogenous neuroactive steroid allopregnanolone during the luteal phase of the menstrual cycle. These findings suggest that changes in the endogenous levels of ovarian-derived progesterone and allopregnanolone alter sensitivity to the discriminative stimulus effects of ethanol. Received: 13 March 1996 / Final version: 26 September 1996