Humanized Foxp2 specifically affects cortico-basal ganglia circuits.

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution and influence aspects of speech and language. Recently it was shown that when these substitutions are introduced into the endogenous Foxp2 gene of mice, they increase dendrite length and long-term depression (LTD) in medium spiny neurons of the striatum. Here we investigated if these effects are found in other brain regions. We found that neurons in the cerebral cortex, the thalamus and the striatum have increased dendrite lengths in the humanized mice whereas neurons in the amygdala and the cerebellum do not. In agreement with previous work we found increased LTD in medium spiny neurons, but did not detect alterations of synaptic plasticity in Purkinje cells. We conclude that although Foxp2 is expressed in many brain regions and has multiple roles during mammalian development, the evolutionary changes that occurred in the protein in human ancestors specifically affect brain regions that are connected via cortico-basal ganglia circuits.

Altered receptor subtypes in the forebrain of GABA(A) receptor delta subunit-deficient mice: recruitment of gamma 2 subunits.

A GABA(A) receptor delta subunit-deficient mouse line was created by homologous recombination in embryonic stem cells to investigate the role of the subunit in the brain GABA(A) receptors. High-affinity [(3)H]muscimol binding to GABA sites as studied by ligand autoradiography was reduced in various brain regions of delta(-/-) animals. [(3)H]Ro 15-4513 binding to benzodiazepine sites was increased in delta(-/-) animals, partly due to an increment of diazepam-insensitive receptors, indicating an augmented forebrain assembly of gamma 2 subunits with alpha 4 subunits. In the western blots of forebrain membranes of delta(-/-) animals, the level of gamma 2 subunit was increased and that of alpha 4 decreased, while the level of alpha1 subunits remained unchanged. In the delta(-/-) forebrains, the remaining alpha 4 subunits were associated more often with gamma 2 subunits, since there was an increase in the alpha 4 subunit level immunoprecipitated by the gamma 2 subunit antibody. The pharmacological properties of t-butylbicyclophosphoro[(35)S]thionate binding to the integral ion-channel sites were slightly altered in the forebrain and cerebellum, consistent with elevated levels of alpha 4 gamma 2 and alpha 6 gamma 2 subunit-containing receptors, respectively.The altered pharmacology of forebrain GABA(A) receptors and the decrease of the alpha 4 subunit level in delta subunit-deficient mice suggest that the delta subunit preferentially assembles with the alpha 4 subunit. The delta subunit seems to interfere with the co-assembly of alpha 4 and gamma 2 subunits and, therefore, in its absence, the gamma 2 subunit is recruited into a larger population of alpha 4 subunit-containing functional receptors. These results support the idea of subunit competition during the assembly of native GABA(A) receptors.

Pharmacological heterogeneity of gamma-aminobutyric acid receptors during development suggests distinct classes of rat cerebellar granule cells in situ.

The gamma-aminobutyric acid receptor (GABA(A)R) represents a ligand-gated Cl(-)-channel assembling as heteropentamere from 19 known subunits. Cerebellar granule cells contain a unique subset, namely the alpha1-, alpha6-, beta2-, gamma2- and delta-subunits. We studied their GABAergic pharmacology in situ using whole-cell patch-clamp recordings in brain slices and a modified Y-tube application system. The distribution of the EC50s for GABA in young (P8-P14) and medium aged animals (P15-P28) could be fitted with the sum of two Gaussian distributions with means of 60 and 185 microM and 27 and 214 microM, respectively. In older animals (P29-P48) the observed homogeneous range of sensitivities fitted a single Gaussian distribution (11 microM). In young animals (< or =P14) GABA-responses were largely insensitive towards 300 microM of the alpha6-specific inhibitor furosemide (82% of control response). The sensitivity increased in older animals at the EC5-20 of GABA (31% of control responses), supporting an increased expression of alpha6-subunits as molecular basis for the observed developmental changes. Approximately 50% of cells in the age range P15-P48 were potentiated by 1 microM diazepam and by 3 microM methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), suggesting the concurrent presence of alpha1- and alpha6-containing receptors, whereas the remaining of cells were neither potentiated by diazepam nor did they show the alpha6-typical DMCM potentiation, though they were potentiated by loreclezole. These properties indicate unknown pharmacological characteristics of cerebellar receptor-subunit combinations in approximately 50% of granule cells in situ.

Coupling between agonist and chloride ionophore sites of the GABA(A) receptor: agonist/antagonist efficacy of 4-PIOL.

Eight gamma-aminobutyric acid (GABA) mimetics were tested on their ability to differentiate native GABA(A) receptor subtypes present in various rat brain regions. In rat brain cryostat sections, little regional variations by the agonistic actions of muscimol, thiomuscimol, 4,5,6,7-tetrahydroisoazolo(5,4-c)pyridin-3-ol, piperidine-4-sulphonic acid, taurine and beta-alanine on [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABA(A) receptor channels were found. They were very similar to those found for GABA itself and indicated no direct correlation with single subunit distributions for any of these compounds. Only the low-efficacy GABA mimetic 5-(4-piperidyl)isoxazol-3-ol (4-PIOL) acted like a weak partial agonist or antagonist depending on the brain area. As the cerebellar granule cell layer was relatively insensitive to both modes of action, we tested 4-PIOL in recombinant alpha1beta2gamma2 (widespread major subtype) and alpha6beta2gamma2 (cerebellar granule cell restricted) receptors where it had different effects on GABA-modulated [35S]TBPS binding and on electrophysiological responses. 4-PIOL may thus serve as a potential lead for receptor subtype selective compounds.

Ionic currents of Drosophila embryonic neurons derived from selectively cultured CNS midline precursors.

In order to investigate the electrogenesis of defined cell populations, we applied an in vitro system that allows the selective culturing of individual Drosophila CNS precursors under different conditions. CNS midline (ML) precursors prepared from gastrula stage embryos gave rise to progeny cells with neuronal and glial morphology that expressed specific markers. Using whole-cell patch-clamp recordings, a detailed description of ionic currents present in this defined cell population is provided. Most ionic currents of cultured ML neurons were similar to other cultured Drosophila neurons, even though their embryonic origin is different. They displayed at least two voltage-gated potassium currents, a voltage-gated sodium, two voltage-gated calcium currents, and responded to the neurotransmitters ACh and GABA. They showed homogeneity in action potential firing properties, generating only a single spike even upon sustained depolarization. Interestingly, although the expression of the voltage-gated potassium currents appeared to be highly cell autonomous, for all other currents significant changes were observed in the presence of fiber contacts.

Assembly of functional alpha6beta3gamma2delta GABA(A) receptors in vitro.

Transgenic mice deficient in the alpha6 subunit of the GABA(A) receptor show reduced levels of the delta subunit protein and an altered GABA(A) receptor pharmacology, suggesting selective assembly mechanisms. Delta reduced the binding of [3H]Ro15-4513 or t-butylbicyclophosphoro[35S]thionate and, to a lesser extent, [3H]muscimol to recombinant alpha1beta1gamma2(delta), alpha4beta1gamma2(delta) and alpha6beta1gamma2(delta) receptors, paralleled by diminished GABA-evoked maximal currents in electrophysiological recordings for the latter one. The delta subunit gave rise to a lower EC50 for GABA and a slowed desensitization indicating its assembly in alpha6beta2delta, alpha6beta1gamma2delta and alpha6beta2gamma2delta receptors. The data show that the delta subunits assemble in various functional GABA(A) receptor subtypes in vitro to reduce GABA-evoked maximal currents and ligand binding, but increase the potency for GABA.

Modulation of the light response by cAMP in Drosophila photoreceptors.

Phototransduction in Drosophila is mediated by a G-protein-coupled phospholipase C transduction cascade in which each absorbed photon generates a discrete electrical event, the quantum bump. In whole-cell voltage-clamp recordings, cAMP, as well as its nonhydrolyzable and membrane-permeant analogs 8-bromo-cAMP (8-Br-cAMP) and dibutyryl-cAMP, slowed down the macroscopic light response by increasing quantum bump latency, without changes in bump amplitude or duration. In contrast, cGMP or 8-Br-cGMP had no effect on light response amplitude or kinetics. None of the cyclic nucleotides activated any channels in the plasma membrane. The effects of cAMP were mimicked by application of the non-specific phosphodiesterase inhibitor IBMX and the adenylyl cyclase activator forskolin; zaprinast, a specific cGMP-phosphodiesterase inhibitor, was ineffective. Bump latency was also increased by targeted expression of either an activated G(s) alpha subunit, which increased endogenous adenylyl cyclase activity, or an activated catalytic protein kinase A (PKA) subunit. The action of IBMX was blocked by pretreatment with the PKA inhibitor H-89. The effects of cAMP were abolished in mutants of the ninaC gene, suggesting this nonconventional myosin as a possible target for PKA-mediated phosphorylation. Dopamine (10 microM) and octopamine (100 microM) mimicked the effects of cAMP. These results indicate the existence of a G-protein-coupled adenylyl cyclase pathway in Drosophila photoreceptors, which modulates the phospholipase C-based phototransduction cascade.

Electrophysiological evidence for an inverse benzodiazepine receptor agonist in panic disorder.

Inverse agonists of the GABA(A) receptor clearly decrease the amplitudes of the spontaneous EEG in the beta-frequency range. Therefore, we tested the hypothesis that panic patients exhibit a reduction of the EEG's spectral power in the beta-frequency band. Ten unmedicated patients with panic disorder and agoraphobia according to DSM-III-R criteria and 10 matched controls were investigated under baseline conditions, after hyperventilation and 30 min after hyperventilation. EEG recordings from the position Pz and Cz were performed under eyes closed conditions. At baseline conditions the patients suffering from panic disorder depicted a reduced beta-power reaching statistically significance for lead position Pz. Immediately after hyperventilation for both channels we observed a decreased beta-power. After hyperventilation we observed the same situation as under baseline conditions. Taken together, our results point to the view that in panic disorder an endogenous inverse agonist of the GABA(A)-benzodiazepine receptor could be hypothesized.

The diversity of GABAA receptors. Pharmacological and electrophysiological properties of GABAA channel subtypes.

The amino acid gamma-aminobutyric-acid (GABA) prevails in the CNS as an inhibitory neurotransmitter that mediates most of its effects through fast GABA-gated Cl(-)-channels (GABAAR). Molecular biology uncovered the complex subunit architecture of this receptor channel, in which a pentameric assembly derived from five of at least 17 mammalian subunits, grouped in the six classes alpha, beta, gamma, delta, sigma and epsilon, permits a vast number of putative receptor isoforms. The subunit composition of a particular receptor determines the specific effects of allosterical modulators of the GABAARs like benzodiazepines (BZs), barbiturates, steroids, some convulsants, polyvalent cations, and ethanol. To understand the physiology and diversity of GABAARs, the native isoforms have to be identified by their localization in the brain and by their pharmacology. In heterologous expression systems, channels require the presence of alpha, beta, and gamma subunits in order to mimic the full repertoire of native receptor responses to drugs, with the BZ pharmacology being determined by the particular alpha and gamma subunit variants. Little is known about the functional properties of the beta, delta, and epsilon subunit classes and only a few receptor subtype-specific substances like loreclezole and furosemide are known that enable the identification of defined receptor subtypes. We will summarize the pharmacology of putative receptor isoforms and emphasize the characteristics of functional channels. Knowledge of the complex pharmacology of GABAARs might eventually enable site-directed drug design to further our understanding of GABA-related disorders and of the complex interaction of excitatory and inhibitory mechanisms in neuronal processing.

The main determinant of furosemide inhibition on GABA(A) receptors is located close to the first transmembrane domain.

Inhibitory GABA(A) receptors are regulated by numerous allosteric modulators, the most receptor-subtype specific of which is furosemide. It recognises receptors of the subunit composition alpha6beta2/3gamma2, restricted to cerebellar granule cells. To locate furosemide's site of action we constructed chimeras of the furosemide-sensitive alpha6 and the furosemide-insensitive alpha1 subunit, and expressed and studied them together with the beta3 and gamma2 subunits in Xenopus oocytes by the two-electrode voltage clamp technique. The inhibition of GABA-induced currents by furosemide mainly depended on a short domain proximal to the first transmembrane region of the alpha6 subunit.

Ultrastructural changes of the microvillar cytoskeleton in the photoreceptor of the crayfish Orconectes limosus related to different adaptation conditions.

In the retina of crayfish microvilli of seven of the eight photoreceptor cells build highly organized structures, the rhabdoms. Cytoskeletal elements inside the microvilli were investigated in conventional and slightly extracted electron microscopical preparations. In conventional preparations the ultrastructure of these cytoskeletal elements depended on the adaptational state of the animal. They appeared as central filament-like structures inside each microvillus when dark-adapted retinae were prepared and fixed at night in the absence of calcium. Changes of these conditions (light, daytime, or calcium concentration) impaired the detectability of these central filaments; in light-adapted eyes prepared at midday they were rarely seen. Nevertheless, single microvillar filaments were present in light-adapted retinae after mild cell permeabilization with the saponin beta-escin. They appeared as a regular structure in each microvillus, often attached to the membrane. Their fine structure was consistent with the ultrastructure of single actin filaments as indicated by fast-Fourier-analysis and further supported by the presence of anti-actin immunoreactivity in electron microscopical and immunocytochemical preparations. These results indicate that microvillar filaments are not necessarily destroyed by light as previously described; we suggest that their appearance inside the microvillus might be altered by the properties of associated, maybe sidearm-like proteins.

Serotonin modulates the voltage dependence of delayed rectifier and Shaker potassium channels in Drosophila photoreceptors.

We describe the in situ modulation of potassium channels in a semi-intact preparation of the Drosophila retina. In whole-cell recordings of photoreceptors, rapidly inactivating Shaker channels are characterized by a conspicuously negative voltage operating range; together with a delayed rectifier, these channels are specifically modulated by the putative efferent neurotransmitter serotonin. Contrary to most potassium channel modulations, serotonin induced a reversible positive shift in the voltage operating range, of +30 mV for the Shaker channels and +10-14 mV for the delayed rectifier. The maximal current amplitudes were unaffected. Modulation was not affected by the subunit-specific Shaker mutations ShE62 and T(1;Y)W32 or a null mutation of the putative modulatory subunit eag. The modulation of both channels was mimicked by intracellularly applied GTP gamma S.