Molecular and neuronal substrate for the selective attenuation of anxiety.

Benzodiazepine tranquilizers are used in the treatment of anxiety disorders. To identify the molecular and neuronal target mediating the anxiolytic action of benzodiazepines, we generated and analyzed two mouse lines in which the alpha2 or alpha3 GABAA (gamma-aminobutyric acid type A) receptors, respectively, were rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of diazepam was absent in mice with the alpha2(H101R) point mutation but present in mice with the alpha3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by alpha2 GABAA receptors, which are largely expressed in the limbic system, but not by alpha3 GABAA receptors, which predominate in the reticular activating system.

Cortical glutamatergic neurons mediate the motor sedative action of diazepam.

The neuronal circuits mediating the sedative action of diazepam are unknown. Although the motor-depressant action of diazepam is suppressed in alpha1(H101R) homozygous knockin mice expressing diazepam-insensitive alpha1-GABA(A) receptors, global alpha1-knockout mice show greater motor sedation with diazepam. To clarify this paradox, attributed to compensatory up-regulation of the alpha2 and alpha3 subunits, and to further identify the neuronal circuits supporting diazepam-induced sedation, we generated Emx1-cre-recombinase-mediated conditional mutant mice, selectively lacking the alpha1 subunit (forebrain-specific alpha1(-/-)) or expressing either a single wild-type (H) or a single point-mutated (R) alpha1 allele (forebrain-specific alpha1(-/H) and alpha1(-/R) mice, respectively) in forebrain glutamatergic neurons. In the rest of the brain, alpha1(-/R) mutants are heterozygous alpha1(H101R) mice. Forebrain-specific alpha1(-/-) mice showed enhanced diazepam-induced motor depression and increased expression of the alpha2 and alpha3 subunits in the neocortex and hippocampus, in comparison with their pseudo-wild-type littermates. Forebrain-specific alpha1(-/R) mice were less sensitive than alpha1(-/H) mice to the motor-depressing action of diazepam, but each of these conditional mutants had a similar behavioral response as their corresponding control littermates. Unexpectedly, expression of the alpha1 subunit was reduced in forebrain, notably in alpha1(-/R) mice, and the alpha3 subunit was up-regulated in neocortex, indicating that proper alpha1 subunit expression requires both alleles. In conclusion, conditional manipulation of GABA(A) receptor alpha1 subunit expression can induce compensatory changes in the affected areas. Specifically, alterations in GABA(A) receptor expression restricted to forebrain glutamatergic neurons reproduce the behavioral effects seen after a global alteration, thereby implicating these neurons in the motor-sedative effect of diazepam.

Decreased GABAA-receptor clustering results in enhanced anxiety and a bias for threat cues.

Patients with panic disorders show a deficit of GABAA receptors in the hippocampus, parahippocampus and orbitofrontal cortex. Synaptic clustering of GABAA receptors in mice heterozygous for the gamma2 subunit was reduced, mainly in hippocampus and cerebral cortex. The gamma2 +/- mice showed enhanced behavioral inhibition toward natural aversive stimuli and heightened responsiveness in trace fear conditioning and ambiguous cue discrimination learning. Implicit and spatial memory as well as long-term potentiation in hippocampus were unchanged. Thus gamma2 +/- mice represent a model of anxiety characterized by harm avoidance behavior and an explicit memory bias for threat cues, resulting in heightened sensitivity to negative associations. This model implicates GABAA-receptor dysfunction in patients as a causal predisposition to anxiety disorders.

Medical Cannabis in Patients with Chronic Pain: Effect on Pain Relief, Pain Disability, and Psychological aspects. A Prospective Non randomized Single Arm Clinical Trial.

BACKGROUND: There is an increasing interest in the medical use of cannabis, particularly in the treatment of chronic pain. OBJECTIVES: The aim is to evaluate the effects of cannabis use and the associated benefits reported by patients with various chronic pain diagnoses. MATERIAL AND METHODS: A total of 338 patients with different chro- nic pain conditions were treated with a Cannabis Flos 19% decoction for 12 months, in addition to their pharmacological therapy. Baseline levels for pain medications, pain intensity, pain disability, anxiety and depression were recorded at 1, 3, 6 and 12 months. RESULTS: Pain intensity records a statistically significant reduction from Baseline to 12 months follow up (X² 61.375; P<0,001); the im- provements from Baseline to 12 months follow up are also recorded in pain disability (X² 39.423; P<0,001) and in anxiety and depression symptoms (X²30.362; P<0,001; X²27.786; P<0,001). CONCLUSIONS: Our study suggest that Cannabis therapy, as an adjun- ct a traditional analgesic therapy, can be an efficacious tool to make more effective the management of chronic pain and its consequences on functional and psychological dimension. Further randomized, controlled trials are needed to confirm our conclusions.

GABA(A) receptor subtypes: dissecting their pharmacological functions.

The enhancement of GABA-mediated synaptic transmission underlies the pharmacotherapy of various neurological and psychiatric disorders. GABA(A) receptors are pluripotent drug targets that display an extraordinary structural heterogeneity: they are assembled from a repertoire of at least 18 subunits (alpha1-6, beta1-3, gamma1-3, delta, epsilon, theta, rho1-3). However, differentiating defined GABA(A) receptor subtypes on the basis of function has had to await recent progress in the genetic dissection of receptor subtypes in vivo. Evidence that the various actions of allosteric modulators of GABA(A) receptors, in particular the benzodiazepines, can be attributed to specific GABA(A) receptor subtypes will be discussed. Such discoveries could open up new avenues for drug development.

GABA(A)-receptor subtypes: a new pharmacology.

The GABA(A) receptor is a pluripotent drug target mediating anxiolytic, sedative, anticonvulsant, muscle relaxant and amnesic activity. These drug actions have now been attributed to defined receptor subtypes. Thus, precise guidelines are available for the development of novel drugs with more selective action and less side effects than those currently in clinical use.

Pathophysiology and pharmacology of GABA(A) receptors.

By controlling spike timing and sculpting neuronal rhythms, inhibitory interneurons play a key role in brain function. GABAergic interneurons are highly diverse. The respective GABA(A) receptor subtypes, therefore, provide new opportunities not only for understanding GABA-dependent pathophysiologies but also for targeting of selective neuronal circuits by drugs. The pharmacological relevance of GABA(A) receptor subtypes is increasingly being recognized. A new central nervous system pharmacology is on the horizon. The development of anxiolytic drugs devoid of sedation and of agents that enhance hippocampus-dependent learning and memory has become a novel and highly selective therapeutic opportunity.

Contribution of the alpha1-GABA(A) receptor subtype to the pharmacological actions of benzodiazepine site inverse agonists.

A histidine-to-arginine point-mutation at position 101 in the alpha1-subunit of gamma-aminobutyric acid (GABA)(A) receptors has been shown to switch the in vitro efficacy of Ro 15-4513 from inverse agonism to agonism. In order to assess the consequences of this pharmacological switch in vivo, the motor and proconvulsant effects of Ro 15-4513 were analyzed in knock-in mice containing point-mutated alpha1(H101R)-GABA(A) receptors. Furthermore the influence of the alpha1(H101R) substitution on the efficacy of the beta-carboline inverse agonist DMCM was examined both in vitro and in vivo. Ro 15-4513 (10 mg/kg) increased baseline locomotion and potentiated the convulsant effect of pentylenetetrazole in wild type mice. In alpha1(H101R) mice, Ro 15-4513 decreased locomotion and, at a higher dose (30 mg/kg) it displayed an anticonvulsant action. In vitro, DMCM acted as an inverse agonist at recombinant alpha1beta2gamma2 receptors whereas it potentiated GABA-evoked chloride currents at alpha1(H101R)beta2gamma2 receptors. DMCM was inactive as a convulsant in alpha1(H101R) mice. In keeping with the major contribution of these receptors to the sedative and anticonvulsant properties of benzodiazepine site agonists, the present findings identify the alpha1-GABA(A) receptors as the molecular targets for the allosteric modulation by benzodiazepine site ligands in either direction with regard to the behavioral outputs, sedation/motor stimulation and anticonvulsion/proconvulsion.

Mechanism of action of the hypnotic zolpidem in vivo.

Zolpidem is a widely used hypnotic agent acting at the GABA(A) receptor benzodiazepine site. On recombinant receptors, zolpidem displays a high affinity to alpha 1-GABA(A) receptors, an intermediate affinity to alpha(2)- and alpha(3)-GABA(A) receptors and fails to bind to alpha(5)-GABA(A) receptors. However, it is not known which receptor subtype is essential for mediating the sedative-hypnotic action in vivo. Studying alpha1(H101R) mice, which possess zolpidem-insensitive alpha(1)-GABA(A) receptors, we show that the sedative action of zolpidem is exclusively mediated by alpha(1)-GABA(A) receptors. Similarly, the activity of zolpidem against pentylenetetrazole-induced tonic convulsions is also completely mediated by alpha(1)-GABA(A) receptors. These results establish that the sedative-hypnotic and anticonvulsant activities of zolpidem are due to its action on alpha(1)-GABA(A) receptors and not on alpha(2)- or alpha(3)-GABA(A) receptors.

Specific GABA(A) circuits in brain development and therapy.

GABAergic interneurons are highly diverse and operate with a corresponding diversity of GABA(A) receptor subtypes in controlling behaviour. In this article, we review the significance of GABA(A) receptor heterogeneity for neural circuit development and central nervous system pharmacology. GABA(A) receptor subtypes were identified as selective targets for behavioural actions of benzodiazepines and of selected intravenous anesthetic agents using point mutations which render a specific receptor subtype insensitive to the action of the respective drugs and also by novel subtype-selective ligands. The pharmacological separation of anxiolysis and sedation guides the development of novel anxiolytics, while inverse agonism at extrasynaptic GABA(A) receptors involved in learning and memory is currently being evaluated as a novel therapeutic principle for symptomatic memory enhancement.

Activation of the GABA(A)-receptor delta-subunit gene promoter following pentylenetetrazole-induced seizures in transgenic mice.

An impairment of GABA(A)-receptor-mediated inhibitory neurotransmission has been implicated in the development of epileptic seizures. To determine whether seizures affect GABA(A)-receptor gene transcription in vivo, a transgenic mouse line carrying a lacZ-fusion gene driven by GABA(A)-receptor delta-subunit promoter and upstream sequences was subjected to pentylenetetrazol (PTZ)-induced seizures. After injection of a single convulsive dose of PTZ, the activity of the delta-subunit promoter, as monitored by beta-galactosidase immunohistochemistry, was increased selectively in neurons of layers II-IV of neocortex. In contrast, mice kindled by repeated administration of initially subconvulsive doses of PTZ did not show a change in transgene expression, even shortly after the last PTZ-induced seizure. These results show that transient changes in transcription of the GABA(A)-receptor delta-subunit gene occur after acute seizures, but not after kindling. The limited responsiveness of the GABA(A)-receptor delta-subunit promoter after repeated stimulation may reflect an inappropriate adaptation of cellular responses to recurrent excitation, thereby contributing to the development of seizure disorders.

Flumazenil induces benzodiazepine partial agonist-like effects in BALB/c but not C57BL/6 mice.

RATIONALE: Some anxiety disorders may be treated in a different way than normal anxiety. OBJECTIVE: This study was aimed at investigating the action of the benzodiazepine receptor antagonist flumazenil, compared to that of the benzodiazepine receptor full agonist chlordiazepoxide, in an animal model of generalised anxiety disorder (the BALB/c mouse). METHODS: Flumazenil (0.0001, 0.001, 0. 01, 0.1 and 1 mg/kg) or chlordiazepoxide (5 mg/kg) were administered to BALB/c or C57BL/6 mice subjected to the light/dark test, the elevated plus maze or a passive avoidance step-through paradigm. RESULTS: Chlordiazepoxide and flumazenil (at all doses tested in the elevated plus maze and at the doses of 0.001 and 0.01 mg/kg in the light/dark test) induced a strong anxiolytic effect in BALB/c mice. Flumazenil did not induce anxiolysis in C57BL/6 mice, whatever the behavioral test or the dose used. However, chlordiazepoxide elicited anxiolysis in this strain in both procedures. In the passive avoidance test, chlordiazepoxide was amnesic in both strains but flumazenil had no effect. CONCLUSION: Flumazenil induces partial agonist-like effects in BALB/c and not in C57BL/6 mice, suggesting a possible benzodiazepine receptor set point shift toward the agonistic direction in some pathological anxiety states such as generalised anxiety disorder.

Mechanisms of the behavioral effects of interleukin 1. Role of prostaglandins and CRF.

Sickness behavior induced by IL-1 can be assessed quantitatively by measuring disruption of schedule-controlled behavior and loss of interest in social activities displayed by rats or mice injected peripherally or centrally with this monokine. These effects are mediated via the release of prostaglandins since they are blocked by the prostaglandin synthesis inhibitors indomethacin and piroxicam. They do not depend, however, on the central release of CRF, since they are not altered by central administration of either CRF or the specific antagonist of CRF receptors.

Behavioural effects of peripherally injected interleukin-1: role of prostaglandins.

To investigate the possible mediation by prostaglandins of changes in behaviour induced by peripheral injection of interleukin-1 (IL-1), two types of behavioural tests were chosen, social exploration in mice and schedule-controlled behaviour in rats. Mice treated with 1 and 2.5 micrograms recombinant human IL-1 beta showed a time- and dose-dependent decrease in exploration of a juvenile conspecific. This effect was completely blocked by pretreatment with 10 mg/kg indomethacin or 10 mg/kg piroxicam, but not with 50 mg/kg aspirin. The disruption of operant responding induced by 5 micrograms IL-1 in rats was also suppressed by pretreatment with 5 mg/kg indomethacin or 10 mg/kg piroxicam. These results indicate that prostaglandins mediate the behavioural effects of peripherally injected IL-1.

Interleukin-1 induces conditioned taste aversion in rats: a possible explanation for its pituitary-adrenal stimulating activity.

To investigate the possible aversive stimulus properties of peripherally administered interleukin 1 (IL-1), rats received two pairings of ingestion of a saccharin solution with various doses of recombinant rat interleukin 1 in a conditioned taste aversion paradigm, using 20 mg/kg lipopolysaccharide endotoxin as a positive control. Rats treated with 1 and 10 micrograms IL-1 showed a dose-dependent reduced preference for saccharin together with dose-dependent impairments in weight gain. Since these effects were obtained within the range of doses that has been previously reported to stimulate the release of ACTH, it is proposed that this last action of IL-1 is likely to be secondary to the aversive effects of IL-1.

Treatment of neck pain with type A botulinum toxin evaluated by Neck Pain Questionnaire (NPQ).

AIMS: To investigate the effects of a treatment with botulinum toxin in patients suffering of cervical pain can be defined as a painful condition of the upper region of the spinal column affecting neck and shoulders. It is one of the most frequent complaints and estimates suggest it affects 7 out of 10 people, with varying degrees of intensity. Although there is a number of causes, most often muscular contraction is involved as a probable consequence of a variety of irritative phenomena. MATERIALS AND METHODS: In our study 26 patients suffering from cervical pain were treated (21 women and 5 men, mean age 60.8 years, range 37-88). Three scales were used to evaluate pain: the Visual Analogue Scale (VAS), the Verbal Scale (VS) and the Neck ad Pain Questionnaire (NPQ). Scoring on all three of these scales was carried out at time 0 (T0) prior to treatment and at time 1 (T1) after 4 months. Patients were treated with 500 MU of Type A Dysport Toxin (Ipsen SpA), made up with 2.5 ml of sodium chloride solution at 0.9%, giving 20 units of toxin in 1 ml. An average of 180 units of toxin were administered to each patient, equivalent to 0.9 ml per point/unit (range 40- 360 units). Patients were given from 1 to 4 units at each session with an average of 2 units. The most common muscles injected were splenius cervicis, the sternocleidomastioid, trapetius, paravertebral and levator scapulae. of patients with cervical pain treated with botulinum toxin were as follows: the mean score on the Visual analogue scale (VAS) at T0 was 6.31, while at T1 it fell to 2.65, i.e. a decrease of 3.66 scores, equivalent to 58%. On the verbal scale (VS), the mean score at T0 was 2, which dropped to 0.8 at T1, a decrease of 1.2 scores, equivalent to 60%. By the Neck Pain Questionnaire (NPQ) the mean T0 score was 12.76, a figure which fell to 7.62 at T1, a drop of 5.14 points, equivalent to 40.28%. The statistical analysis shows that between T0 and T1 the decrease in the VAS, VA and NPQ scores was highly significant (p< 0.01). CONCLUSIONS: The administration of botulinum toxin appears to be a useful therapy for the treatment of muscular-skeletal pain. Technically, the therapeutic use and efficacy of botulinum toxin principally consists in the practical possibility of delivering the toxin principally remains in the target structures. If performed by qualified personnel, the treatment with botulinum toxin can be carried out in day surgery and it appears to be safe, with few risks and a minimum of side effects. To conclude, the main goals in the treatment of muscular-skeletal pain with botulinum toxin are in the breaking of the vicious circle of contraction pain -contraction, in stopping the pain from becoming chronic and achieving a valid alternative therapy.

Role of α1- and α2-GABA(A) receptors in mediating the respiratory changes associated with benzodiazepine sedation.

BACKGROUND AND PURPOSE: The molecular substrates underlying the respiratory changes associated with benzodiazepine sedation are unknown. We examined the effects of different doses of diazepam and alprazolam on resting breathing in wild-type (WT) mice and clarified the contribution of α1- and α2-GABA(A) receptors, which mediate the sedative and muscle relaxant action of diazepam, respectively, to these drug effects using point-mutated mice possessing either α1H101R- or α2H101R-GABA(A) receptors insensitive to benzodiazepine. EXPERIMENTAL APPROACH: Room air breathing was monitored using whole-body plethysmography. Different groups of WT mice were injected i.p. with diazepam (1-100 mg·kg(-1) ), alprazolam (0.3, 1 or 3 mg·kg(-1) ) or vehicle. α1H101R and α2H101R mice received 1 or 10 mg·kg(-1) diazepam or 0.3 or 3 mg·kg(-1) alprazolam. Respiratory frequency, tidal volume, time of expiration and time of inspiration before and 20 min after drug injection were analysed. KEY RESULTS: Diazepam (10 mg·kg(-1) ) decreased the time of expiration, thereby increasing the resting respiratory frequency, in WT and α2H101R mice, but not in α1H101R mice. The time of inspiration was shortened in WT and α1H101R mice, but not in α2H101R mice. Alprazolam (1-3 mg·kg(-1) ) stimulated the respiratory frequency by shortening expiration and inspiration duration in WT mice. This tachypnoeic effect was partially conserved in α1H101R mice while absent in α2H101R mice. CONCLUSIONS AND IMPLICATIONS: These results identify a specific role for α1-GABA(A) receptors and α2-GABA(A) receptors in mediating the shortening by benzodiazepines of the expiratory and inspiratory phase of resting breathing respectively.

A reduction in hippocampal GABAA receptor alpha5 subunits disrupts the memory for location of objects in mice.

The memory for location of objects, which binds information about objects to discrete positions or spatial contexts of occurrence, is a form of episodic memory particularly sensitive to hippocampal damage. Its early decline is symptomatic for elderly dementia. Substances that selectively reduce alpha5-GABA(A) receptor function are currently developed as potential cognition enhancers for Alzheimer's syndrome and other dementia, consistent with genetic studies implicating these receptors that are highly expressed in hippocampus in learning performance. Here we explored the consequences of reduced GABA(A)alpha5-subunit contents, as occurring in alpha5(H105R) knock-in mice, on the memory for location of objects. This required the behavioral characterization of alpha5(H105R) and wild-type animals in various tasks examining learning and memory retrieval strategies for objects, locations, contexts and their combinations. In mutants, decreased amounts of alpha5-subunits and retained long-term potentiation in hippocampus were confirmed. They exhibited hyperactivity with conserved circadian rhythm in familiar actimeters, and normal exploration and emotional reactivity in novel places, allocentric spatial guidance, and motor pattern learning acquisition, inhibition and flexibility in T- and eight-arm mazes. Processing of object, position and context memories and object-guided response learning were spared. Genotype difference in object-in-place memory retrieval and in encoding and response learning strategies for object-location combinations manifested as a bias favoring object-based recognition and guidance strategies over spatial processing of objects in the mutants. These findings identify in alpha5(H105R) mice a behavioral-cognitive phenotype affecting basal locomotion and the memory for location of objects indicative of hippocampal dysfunction resulting from moderately decreased alpha5-subunit contents.