Somatosensory and sensorimotor consequences associated with the heterozygous disruption of the autism candidate gene, Gabrb3.

Autism spectrum disorder (ASD) is diagnosed based on three core features: impaired social interactions, deficits in communication and repetitive or restricted behavioral patterns. Against this backdrop, abnormal sensory processing receives little attention despite its prevalence and the impact it exerts on the core diagnostic features. Understanding the source of these sensory abnormalities is paramount to developing intervention strategies aimed at maximizing the coping ability of those with ASD. Consequently, we chose to examine whether sensory abnormalities were present in mice heterozygous for the Gabrb3 gene, a gene strongly associated with ASD. Mice were assessed for tactile and heat sensitivity, sensorimotor competence (accelerating rotarod task) and sensorimotor gating by prepulse inhibition of the acoustic startle reflex (PPI). All heterozygotes exhibited an increase in seizure susceptibility and similar reductions in Gabrb3 expression in the dorsal root ganglia, spinal cord, whole brain and amygdala. Interestingly, significant differences were noted between heterozygous variants in regards to tactile sensitivity, heat sensitivity, sensorimotor competence and PPI along with differences in Gabrb3 expression in the reticular thalamic nucleus and the bed nucleus of stria terminalis. These differences were influenced by the heterozygotes' gender and whether the Gabrb3 gene was of paternal or maternal origin. These results are not adequately explained by simple haploinsufficiency of Gabrb3, therefore, additional mechanisms are likely to be involved. In addition, this is the first report of the occurrence of tactile and heat hypersensitivity in an ASD mouse model, two features often associated with ASD.

Selective influence on contextual memory: physiochemical properties associated with selectivity of benzodiazepine ligands at GABAA receptors containing the alpha5 subunit.

Ligands that bind to the benzodiazepine binding site on the GABA A receptor can attenuate or potentiate cognition. To investigate this property, the chemical determinants favoring selective binding or selective activation of the alpha5beta2gamma2 and alpha1beta2gamma2 GABA A receptor isoforms were examined. A 3D-pharmacophore, developed from a diverse set of BDZR ligands, was used as an initial basis for multivariate discriminant, fragment, and 3D-quantitative structure-activity relationship analyses, which formed the criteria for selection of additional compounds for study. We found that the electrostatic potential near the ligands' terminal substituent correlated with its binding selectivity toward the alpha5beta2gamma2 versus alpha1beta2gamma2 isoform; while the fragment length and frontier molecular orbital energetics correlated with a compounds influence on electrophysiological activity. Compounds with promising alpha5 profiles were further assessed for their ability to attenuate scopolamine-induced contextual memory impairment in mice. Surprisingly, both weak inverse agonist and antagonists that display binding selectivity toward the alpha5beta2gamma2 isoform were able to attenuate contextual memory impairment.

Gabrb3 gene deficient mice exhibit impaired social and exploratory behaviors, deficits in non-selective attention and hypoplasia of cerebellar vermal lobules: a potential model of autism spectrum disorder.

OBJECTIVE: GABA(A) receptors play an important regulatory role in the developmental events leading to the formation of complex neuronal networks and to the behaviors they govern. The primary aim of this study was to assess whether gabrb3 gene deficient (gabrb3(-/-)) mice exhibit abnormal social behavior, a core deficit associated with autism spectrum disorder. METHODS: Social and exploratory behaviors along with non-selective attention were assessed in gabrb3(-/-), littermates (gabrb3(+/+)) and progenitor strains, C57BL/6J and 129/SvJ. In addition, semi-quantitative assessments of the size of cerebellar vermal lobules were performed on gabrb3(+/+) and gabrb3(-/-) mice. RESULTS: Relative to controls, gabrb3(-/-) mice exhibited significant deficits in activities related to social behavior including sociability, social novelty and nesting. In addition, gabrb3(-/-) mice also exhibited differences in exploratory behavior compared to controls, as well as reductions in the frequency and duration of rearing episodes, suggested as being an index of non-selective attention. Gabrb3(-/-) mice also displayed significant hypoplasia of the cerebellar vermis compared to gabrb3(+/+) mice. CONCLUSIONS: The observed behavioral deficits, especially regarding social behaviors, strengthens the face validity of the gabrb3 gene deficient mouse as being a model of autism spectrum disorder.

New insight into the role of the beta3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout.

BACKGROUND: The beta3 subunit of the gamma-aminobutyric acid type A receptor (GABAA-R) has been reported to be important for palate formation, anesthetic action, and normal nervous system function. This subunit has also been implicated in the pathogenesis of Angelman syndrome and autism spectrum disorder. To further investigate involvement of this subunit, we previously produced mice with a global knockout of beta3. However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model. To overcome many of these limitations, a mouse line with a conditionally inactivated beta3 gene was engineered. RESULTS: Gene targeting and embryonic stem cell technologies were used to create mice in which exon 3 of the beta3 subunit was flanked by loxP sites (i.e., floxed). Crossing the floxed beta3 mice to a cre general deleter mouse line reproduced the phenotype of the previously described global knockout. Pan-neuronal knockout of beta3 was achieved by crossing floxed beta3 mice to Synapsin I-cre transgenic mice. Palate development was normal in pan-neuronal beta3 knockouts but ~61% died as neonates. Survivors were overtly normal, fertile, and were less sensitive to etomidate. Forebrain selective knockout of beta3 was achieved using alpha CamKII-cre transgenic mice. Palate development was normal in forebrain selective beta3 knockout mice. These knockouts survived the neonatal period, but ~30% died between 15-25 days of age. Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese. CONCLUSION: Conditional inactivation of the beta3 gene revealed novel insight into the function of this GABAA-R subunit. The floxed beta3 knockout mice described here will be very useful for conditional knockout studies to further investigate the role of the beta3 subunit in development, ethanol and anesthetic action, normal physiology, and pathophysiologic processes.

Gabrb3 gene deficient mice exhibit increased risk assessment behavior, hypotonia and expansion of the plexus of locus coeruleus dendrites.

Gabrb3 gene deficient (gabrb3(-/-)) mice, control littermates (gabrb3(+/+)) and their progenitor strains C57Bl/6J and 129/SvJ were assessed for changes in the morphology of the main noradrenergic nuclei, the locus coeruleus (LC) and LC-associated behaviors including anxiety and muscle tone. While the area defined by the cell bodies of the LC was found not to differ between gabrb3(-/-) mice and controls, the pericoerulear dendritic zone of the LC was found to be significantly enlarged in gabrb3(-/-) mice. Relative to controls, gabrb3(-/-) mice were also found to be hypotonic, as was indicated by poor performance on the wire hanging task. Gabrb3(-/-) mice also exhibited a significant increase in stretch-attend posturing, a form of risk assessment behavior associated with anxiety. However, in the plus maze, a commonly used behavioral test for assessing anxiety, no significant difference was observed between gabrb3(-/-) and control mice. Lastly, relative to controls, gabrb3(-/-) mice exhibited significantly less marble burying behavior, a method commonly used to assess obsessive-compulsive behavior. However, the poor marble burying performance of the gabrb3(-/-) mice could be associated with the hypotonic condition exhibited by these mice. In conclusion, the results of this study indicate that the gabrb3 gene contributes to LC noradrenergic dendrite development with the disruption of this gene in mice resulting in an enlarged plexus of LC dendrites with a concurrent reduction in muscle tone and marble burying behavior, an increase in risk assessment behavior but no change in the plus maze parameters that are commonly used for assessing anxiety.

Transcutaneous blood gas CO2 monitoring of induced ventilatory depression in mice.

We assessed a simple, noninvasive method of monitoring transcutaneous partial pressure of CO2 (Ptcco2) in mice to determine whether it would provide an accurate and reproducible method to assess ventilatory depression in mice. To this end, Ptcco2 and Paco2 (partial pressure of arterial CO2) measurements were performed on isoflurane-anesthetized male C57Bl/6 mice breathing differing percentages of CO2 or fentanyl, a known ventilatory depressive drug. All doses of fentanyl produced a sharp increase in Ptcco2 values within 20 min with difference in Ptcco2 values between saline and all fentanyl groups being statistically significant (P < 0.0001). A good correlation between Paco2 and Ptcco2 values was established (r2 = 0.91). A Bland-Altman analysis likewise found that Ptcco2 measurements in the mice reliably and accurately reflected their Paco2 values. Therefore, under controlled conditions, Ptcco2 measurements were found to reliably reflect Paco2 values in mice. Consequently, the Ptcco2 method can be used as a means to rapidly and quantitatively assess the ventilatory depressive properties of a wide spectrum of drugs, under varying conditions in numerous mouse models.

Pharmacological profiles of opioid ligands at kappa opioid receptors.

BACKGROUND: The aim of the present study was to describe the activity of a set of opioid drugs, including partial agonists, in a human embryonic kidney cell system stably expressing only the mouse kappa-opioid receptors. Receptor activation was assessed by measuring the inhibition of cyclic adenosine mono phosphate (cAMP) production stimulated by 5 microM forskolin. Intrinsic activities and potencies of these ligands were determined relative to the endogenous ligand dynorphin and the kappa agonist with the highest intrinsic activity that was identified in this study, fentanyl. RESULTS: Among the ligands studied naltrexone, WIN 44,441 and dezocine, were classified as antagonists, while the remaining ligands were agonists. Intrinsic activity of agonists was assessed by determining the extent of inhibition of forskolin-stimulated cAMP production. The absolute levels of inhibition of cAMP production by each ligand was used to describe the rank order of intrinsic activity of the agonists; fentanyl = lofentanil > or = hydromorphone = morphine = nalorphine > or = etorphine > or = xorphanol > or = metazocine > or = SKF 10047 = cyclazocine > or = butorphanol > nalbuphine. The rank order of affinity of these ligands was; cyclazocine > naltrexone > or = SKF 10047 > or = xorphanol > or = WIN 44,441 > nalorphine > butorphanol > nalbuphine > or = lofentanil > dezocine > or = metazocine > or = morphine > hydromorphone > fentanyl. CONCLUSION: These results elucidate the relative activities of a set of opioid ligands at kappa-opioid receptor and can serve as the initial step in a systematic study leading to understanding of the mode of action of these opioid ligands at this receptor.

Pharmacologic evidence for abnormal thalamocortical functioning in GABA receptor beta3 subunit-deficient mice, a model of Angelman syndrome.

PURPOSE: gamma-Aminobutyric acid receptor (GABA(A)r) subunit beta3-deficient mice model Angelman syndrome by displaying impaired learning, abnormal EEG with interictal spikes and slowing, myoclonus, and convulsions. The beta3-subunit deficiency causes a failure of intrathalamic reticular nucleus inhibition, leading to abnormally synchronized thalamocortical oscillations. We postulated that this pathophysiology underlies the abnormal cortical EEG and triggers interictal spikes and seizures, but extrathalamic regions also contribute to interictal spikes and seizures, so that the EEG slowing should reveal an absence-like response profile, whereas spikes and seizures have dual responsiveness to absence and partial-seizure drugs. METHODS: Recording electrodes were implanted over the parietal cortices of wild-type, heterozygotes, and homozygous null mice. In each experiment, EEG was recorded for 45 min, either drug or vehicle administered, and EEG recorded for another 3 h. Each EEG was scored for slow-wave activity, interictal spikes, and seizures by a reader blinded to treatments. RESULTS: Interictal spiking and percentage of time in EEG slowing in heterozygotes were increased by the proabsence drug baclofen (GABA(B)-receptor agonist), whereas CGP 35348 (GABA(B)-receptor antagonist) had the opposite effect. The antiabsence drug ethosuximide markedly suppressed EEG slowing and interictal spiking in heterozygote and null mice. Broad-spectrum clonazepam and valproate were more effective on interictal spiking than on EEG slowing, and fosphenytoin suppressed only interictal spiking. CONCLUSIONS: The results suggest that this model of Angelman syndrome, although not expressing typical absence seizures, is characterized by hypersynchronous thalamocortical oscillations that possess absence-like pharmacologic responsiveness and promote EEG slowing, interictal spikes, and convulsive seizures.

Activation of alpha2 adrenergic receptors suppresses fear conditioning: expression of c-Fos and phosphorylated CREB in mouse amygdala.

alpha(2) adrenergic agonists such as dexmedetomidine generally suppress noradrenergic transmission and have sedative, analgesic, and antihypertensive properties. Considering the importance of the neurotransmitter norepinephrine in forming memories for fearful events, we have investigated the acute and chronic effects of dexmedetomidine on discrete cue and contextual fear conditioning in mice. When administered before training, dexmedetomidine (10-20 microg/kg, i.p.) selectively suppressed discrete cue fear conditioning without affecting contextual memory. This behavioral change was associated with a decrease in memory retrieval-induced expression of c-Fos and P-CREB in the lateral, basolateral, and central nuclei of the amygdala. Dexmedetomidine's action on discrete cue memory did not occur in alpha(2A) adrenoceptor knockout (KO) mice. When dexmedetomidine was administered after training, it suppressed contextual memory, an effect that did not occur in alpha(2A) adrenoceptor KO mice. We conclude that dexmedetomidine, acting at alpha(2A) adrenoceptors, must be present during the encoding process to decrease discrete cue fear memory; however, its ability to suppress contextual memory is likely the result of blocking the consolidation process. The ability of alpha(2) agonists to suppress fear memory may be a valuable property clinically in order to suppress the formation of memories during stressful situations.

Augmentation of the noradrenergic system in alpha-2 adrenergic receptor deficient mice: anatomical changes associated with enhanced fear memory.

We have investigated sensitivity to the conditioned fear procedure of mice is influenced by the genetic deletion of alpha2A adrenoceptors (ARs). We observed a heightened freezing response in the discrete cue memory test in alpha2A AR knockout (alpha2A AR KO) mice and in D79N mice, a transgenic mouse strain with functionally impaired alpha2A ARs. No significant differences in contextual memory were observed between control and alpha2A AR KO or D79N mice suggesting a minimal role for the noradrenergic system in contextual memory. We speculated that the increased freezing response of the alpha2A AR KO and D79N mice in the discrete cue setting was due to increased release of norepinephrine evoked by the unconditioned footshock stimulus. In alpha2A AR KO mice we measured a doubling in the number of noradrenergic neurons in the locus coeruleus (LC) and a large increase in the cell volume of tyrosine hydroxylase positive neurons, likely due to selective preservation of large, multipolar neurons in the subcoeruleus. Hyperplasia of the noradrenergic neurons in the nucleus tractus solitarius, A5 and A7, was also observed. Alpha2A AR KO mice exhibit greater c-Fos expression in the LC compared to wild type mice suggesting that the LC neurons in the alpha2A AR KO mice were spontaneously more active. This study suggests that alpha2A ARs are involved in the development of the central noradrenergic system and raises the possibility that alterations in alpha2A AR expression may contribute to variations in fear and stress responses.

Sleep states and sleep electroencephalographic spectral power in mice lacking the beta 3 subunit of the GABA(A) receptor.

Mice lacking the GABA(A) receptor beta(3) subunit exhibit a profound disruption in thalamic circuitry. We have studied sleep in these mice under baseline conditions and following treatment with the benzodiazepine midazolam. Under baseline conditions, NREM sleep time did not differ between beta(3) subunit knockout mice and wild type mice, while REM sleep time was significantly lower in knockout mice than in wild type mice during the light portion of a 24-h light-dark cycle. In constant dark conditions, circadian rhythmicity remained intact in mutant mice for a period of at least 9 days. EEG delta power (1-4 Hz) was significantly greater in the knockout than in wild type mice during NREM sleep but not during other states. A transient increase in EEG power in the 12-16 Hz range that occurred in wild type mice just prior to the transition from NREM to REM sleep was present but significantly blunted in the knockout. Midazolam decreased NREM delta power and REM time in wild type mice. The former but not the latter response to midazolam was intact in the knockout. These results further support a role for GABAergic transmission in regulating REM sleep and EEG spectral phenomena associated with NREM sleep.