Recent findings indicate a high level of specialization at the level of microcircuits and cell populations within brain structures with regards to the control of fear and anxiety. The hippocampus, ...however, has been treated as a unitary structure in anxiety and fear research despite mounting evidence that different hippocampal subregions have specialized roles in other cognitive domains. Using novel cell-type- and region-specific conditional knockouts of the GABAA receptor α2 subunit, we demonstrate that inhibition of the principal neurons of the dentate gyrus or CA3 via α2-containing GABAA receptors (α2GABAARs) is required to suppress anxiety, while the inhibition of CA1 pyramidal neurons is required to suppress fear responses. We further show that the diazepam-modulation of hippocampal theta activity shows certain parallels with our behavioral findings, suggesting a possible mechanism for the observed behavioral effects. Thus, our findings demonstrate a double dissociation in the regulation of anxiety versus fear by hippocampal microcircuitry.
GABAergic transmission regulates adult neurogenesis by exerting negative feedback on cell proliferation and enabling dendrite formation and outgrowth. Further, GABAergic synapses target ...differentiating dentate gyrus granule cells prior to formation of glutamatergic connections. GABAA receptors (GABAARs) mediating tonic (extrasynaptic) and phasic (synaptic) transmission are molecularly and functionally distinct, but their specific role in regulating adult neurogenesis is unknown. Using global and single‐cell targeted gene deletion of subunits contributing to the assembly of GABAARs mediating tonic (α4, δ) or phasic (α2) GABAergic transmission, we demonstrate here in the dentate gyrus of adult mice that GABAARs containing α4, but not δ, subunits mediate GABAergic effects on cell proliferation, initial migration and early dendritic development. In contrast, α2‐GABAARs cell‐autonomously signal to control positioning of newborn neurons and regulate late maturation of their dendritic tree. In particular, we observed pruning of distal dendrites in immature granule cells lacking the α2 subunit. This alteration could be prevented by pharmacological inhibition of thrombospondin signaling with chronic gabapentin treatment, shown previously to reduce glutamatergic synaptogenesis. These observations point to homeostatic regulation of inhibitory and excitatory inputs onto newborn granule cells under the control of α2‐GABAARs. Taken together, the availability of distinct GABAAR subtypes provides a molecular mechanism endowing spatiotemporal specificity to GABAergic control of neuronal maturation in adult brain.
Impairment of brain functional connectivity (FC) is thought to be an early event occurring in diseases with cerebral amyloidosis, such as Alzheimer's disease. Regions sustaining altered functional ...networks have been shown to colocalize with regions marked with amyloid plaques burden suggesting a strong link between FC and amyloidosis. Whether the decline in FC precedes amyloid plaque deposition or is a consequence thereof is currently unknown. The sequence of events during early stages of the disease is difficult to capture in humans due to the difficulties in providing an early diagnosis and also in view of the heterogeneity among patients. Transgenic mouse lines overexpressing amyloid precursor proteins develop cerebral amyloidosis and constitute an attractive model system for studying the relationship between plaque and functional changes. In this study, ArcAβ transgenic and wild-type mice were imaged using resting-state fMRI methods across their life-span in a cross-sectional design to analyze changes in FC in relation to the pathology. Transgenic mice show compromised development of FC during the first months of postnatal life compared with wild-type animals, resulting in functional impairments that affect in particular the sensory-motor cortex already in preplaque stage. These functional alterations were accompanied by structural changes as reflected by reduced fractional anisotropy values, as derived from diffusion tensor imaging. Our results suggest cerebral amyloidosis in mice is preceded by impairment of neuronal networks and white matter structures. FC analysis in mice is an attractive tool for studying the implications of impaired neuronal networks in models of cerebral amyloid pathology.
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 α2 or α3 GABAA(γ-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 α2(H101R) point mutation but present in mice with the α3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by α2 GABAAreceptors, which are largely expressed in the limbic system, but not by α3 GABAAreceptors, which predominate in the reticular activating system.
Magnetic resonance imaging (MRI) can be used to monitor pathological changes in Alzheimer's disease (AD). The objective of this longitudinal study was to assess the effects of progressive ...amyloid-related pathology on multiple MRI parameters in transgenic arcAβ mice, a mouse model of cerebral amyloidosis. Diffusion-weighted imaging (DWI), T1-mapping and quantitative susceptibility mapping (QSM), a novel MRI based technique, were applied to monitor structural alterations and changes in tissue composition imposed by the pathology over time. Vascular function and integrity was studied by assessing blood-brain barrier integrity with dynamic contrast-enhanced MRI and cerebral microbleed (CMB) load with susceptibility weighted imaging and QSM. A linear mixed effects model was built for each MRI parameter to incorporate effects within and between groups (i.e. genotype) and to account for changes unrelated to the disease pathology. Linear mixed effects modelling revealed a strong association of all investigated MRI parameters with age. DWI and QSM in addition revealed differences between arcAβ and wt mice over time. CMBs became apparent in arcAβ mice with 9 month of age; and the CMB load reflected disease stage. This study demonstrates the benefits of linear mixed effects modelling of longitudinal imaging data. Moreover, the diagnostic utility of QSM and assessment of CMB load should be exploited further in studies of AD.
Tumor hypoxia and the hypoxia-inducible factors (HIFs) play a central role in the development of cancer. To study the relationship between tumor growth, tumor hypoxia, the stabilization of HIF-1 a, ...and HIF transcriptional activity, we have established an in vivo imaging tool that allows longitudinal and noninvasive monitoring of these processes in a mouse C51 allograft tumor model. We used positron emission tomography (PET) with the hypoxia-sensitive tracer ¹⁸F-fluoromisonidazole (FMISO) to measure tumor hypoxia over 14 days. Stabilization of HIF-1α and HIF transcriptional activity were assessed by bioluminescence imaging using the reporter constructs HIF-1α-luciferase and hypoxia response element-luciferase, respectively, stably expressed in C51 cells. Interestingly, we did not observe any major change in the level of tumor hypoxia throughout the observation period whereas HIF-1α levels and HIF activity showed drastic temporal variations. When comparing the readouts as a function of time we found a good correlation between HIF-1α levels and HIF activity. In contrast, there was no significant correlation between the ¹⁸F-FMISO PET and HIF readouts. The tool developed in this work allows for the longitudinal study of tumor hypoxia and HIF-1α in cancer in an individual animal and will be of value when monitoring the efficacy of therapeutical interventions targeting the HIF pathway.
Diazepam is used clinically for its myorelaxant, anxiolytic, sedative, and anticonvulsant properties. Although the anxiolytic action is mediated by alpha2 gamma-aminobutyric acid A (GABA(A)) ...receptors, the sedative action and in part the anticonvulsant action are mediated by alpha1 GABA(A) receptors. To identify the GABA(A) receptor subtypes mediating the action of diazepam on muscle tone, we have assessed the myorelaxant properties of diazepam in alpha2(H101R) and alpha3(H126R) knock-in mice harboring diazepam-insensitive alpha2 or alpha3 GABA(A) receptors, respectively. Whereas in alpha2(H101R) mice the myorelaxant action of diazepam was almost completely abolished at doses up to 10 mg/kg, the same dose induced myorelaxation in both wild-type and alpha3(H126R) mice. It was only at a very high dose (30 mg/kg diazepam) that alpha2(H101R) mice showed partial myorelaxation and alpha3(H126R) mice were partially protected from myorelaxation compared with wild-type mice. Thus, the myorelaxant activity of diazepam seems to be mediated primarily by alpha2 GABA(A) receptors and at high concentrations also by alpha3 GABA(A) receptors.
Intratumoral hypoxia changes the metabolism of gliomas, leading to a more aggressive phenotype with increased resistance to radio- and chemotherapy. Hypoxia triggers a signaling cascade with ...hypoxia-inducible factor (HIF) as a key regulator. We monitored activation of the HIF pathway longitudinally in murine glioma tumors. GL261 cells, stably transfected with a luciferase reporter driven under the control of a promoter comprising the HIF target gene motive hypoxia response element, were implanted either subcutaneously or orthotopically. In vivo experiments were carried out using bioluminescence imaging. Tumors were subsequently analyzed using immunofluorescence staining for hypoxia, endothelial cells, tumor perfusion, and glucose transporter expression. Transient upregulation of the HIF signaling was observed in both subcutaneous and orthotopic gliomas. Immunofluorescence staining confirmed hypoxic regions in subcutaneous and, to a lesser extent, intracranial tumors. Subcutaneous tumors showed substantial necrosis, which might contribute to the decreased bioluminescence output observed toward the end of the experiment. Orthotopic tumors were less hypoxic than subcutaneous ones and did not develop extensive necrotic areas. Although this may be the result of the overall smaller size of orthotopic tumors, it might also reflect differences in the local environment, such as the better intrinsic vascularization of brain tissue compared to the subcutaneous tissue compartment.
Benzodiazepines are in wide clinical use for their sedative and tranquilizing actions, the former being mediated via α1‐containing GABAA receptors. The signal transduction pathways elicited beyond ...the receptor are only poorly understood. Changes of transcript levels in cerebral cortex induced by acute diazepam administration were therefore compared by microarray analysis between wild‐type and point mutated α1(H101R) mice, in which the α1 GABAA receptor subunit had been rendered insensitive to diazepam. In wild‐type animals, diazepam reduced the expression levels of the α subunit of the calcium/calmodulin‐dependent protein kinase II, as well as brain‐derived neurotrophic factor, MAP kinase phosphatase, transcription factor GIF, c‐fos and nerve growth factor induced gene‐A. None of these transcripts was changed in the α1(H101R) mice after treatment with diazepam. Thus, the sedative action of diazepam is correlated with a selective down‐regulation of transcripts involved in the regulation of neuronal plasticity and neurotrophic responses. Most transcript changes were transient except for the decrease of the CaMKIIα transcript which persisted even 40 h after the single dose of diazepam. This long‐term alteration is likely to contribute to the resetting of the neuronal responsiveness, which may be involved in rebound phenomena and, under chronic treatment, in the development of tolerance and dependence.
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