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Therapeutic ultrasound is a widely used application in clinics. The response to ultrasound is tissue specific and the parameters of ultrasonic treatments are decisive. Here, the responses of primary enteric (ENS) and central nervous system (CNS) neural stem cells were investigated to different frequency – energy density – combination treatments between 510 kHz–3 W s/cm2 and 4.36 MHz–25 W s/cm2. Responses to ultrasonic treatments were both frequency and energy density dependent. Ultrasound enhanced the expansion and showed enlarged neurospheres for both ENS and CNS neural stem cell cultures. Differentiation was not impaired in regard to neuronal and glial cell number as well as neurite and glial fiber outgrowth. Ultrasound is a promising tool to expand neural stem cells in vitro neither influencing neurogenesis nor gliogenesis by applying specific frequency – energy density – combinations. Keywords: Enteric nervous system, Ultrasound, Neural stem cells

Childhood stress and trauma are associated with substance use disorders in adulthood, but the neurological changes that confer increased vulnerability are largely unknown. In this study, maternal separation (MS) stress, restricted to the pre-weaning period, was used as a model to study mechanisms of protracted effects of childhood stress/traumatic experiences on binge drinking and impulsivity. Using an operant self-administration model of binge drinking and a delay discounting assay to measure impulsive-like behavior, we report that early life stress due to MS facilitated acquisition of binge drinking and impulsivity during adulthood in rats. Previous studies have shown heightened levels of corticotropin releasing factor (CRF) after MS, and here, we add that MS increased expression levels of GABA(A) α2 subunit in central stress circuits. To investigate the precise role of these circuits in regulating impulsivity and binge drinking, the CRF1 receptor antagonist antalarmin and the novel GABA(A) α2 subunit ligand 3-PBC were infused into the central amygdala (CeA) and medial prefrontal cortex (mPFC). Antalarmin and 3-PBC at each site markedly reduced impulsivity and produced profound reductions on binge-motivated alcohol drinking, without altering responding for sucrose. Furthermore, whole-cell patch-clamp studies showed that low concentrations of 3-PBC directly reversed the effect of relatively high concentrations of ethanol on α2β3γ2 GABA(A) receptors, by a benzodiazepine site-independent mechanism. Together, our data provide strong evidence that maternal separation, i.e. early life stress, is a risk factor for binge drinking, and is linked to impulsivity, another key risk factor for excessive alcohol drinking. We further show that pharmacological manipulation of CRF and GABA receptor signaling is effective to reverse binge drinking and impulsive-like behavior in MS rats. These results provide novel insights into the role of the brain stress systems in the development of impulsivity and excessive alcohol consumption.

Multielectrode array (MEA) technology is used as an extracellular recording method to investigate the electrical activity of complex excitable cell networks and tissue slices. In this context, an array of single-contactable, sensing electrodes record the electrical field potentials induced by the ion flux across the cell membranes. Therefore, MEAs are an ideal tool to investigate network activities for prolonged periods. Still, MEA technology suffers from low-signal-to-noise ratios caused by a dissatisfied cell-electrode contact, thus revealing only limited measurements. To improve cell recording, a tight cell-electrode contact is inalienable. This can be achieved by three-dimensional micro- or nanostructures on top of the sensing electrodes. Beside the fact of increasing the electrochemically active surface area, thus leading to a decrease in the overall chip impedance, the nano-sized structures can also trigger an engulfment process of the cells. By minimizing the gap between cell and electrode, a high seal resistance can be achieved which in return results in recording of higher signal amplitudes. In this work, electrochemical concepts are used for fabrication as well as for characterizing the chips and to investigate the cell-electrode interaction. The developed process allows the fabrication of half-structured MEAs with different three-dimensional nanostructure shapes, geometries and pitch lengths and is mainly based on thermal nanoimprint lithography (NIL), gold electroplating and microstructuring techniques. During thermal NIL a customized, nanostructured master mold is pressed into a polymer resist of a wafer substrate, thus creating a negative copy of the master´s nanostructures. The subsequent electroplating step can be adjusted in a way, that it fills or overgrows the NIL templates, thereby creating different shapes. Photolithography, chemical vapor deposition and etching steps are used to fabricate the overall MEA geometry. Cyclic voltammetry as well as electrochemical impedance spectroscopy have been applied to characterize the effect of different nanostructure geometries on charge transfer, double-layer capacitance and impedance of the MEA electrodes. Compared to unstructured electrodes, charge transfer and double-layer capacitance of the nanostructured electrodes are increased, whereas the impedance of the nanostructured electrodes are decreased. These results can directly be related to an increase of the electrochemically active surface area and vary with the investigated nanostructure layout. To examine the effect of the nanostructures on cell-electrode coupling in detail and to quantify cell adhesion, impedance sensing at a fixed frequency of 1 kHz has been performed using the impedance-stable human embryonic kidney cell line HEK 293. Impedance values have been measured with and without cells. Due to the chip layout where non- and nanostructured electrodes have been included on the chip, the relative change of the impedance values could directly be correlated with cell-electrode adhesion. By this, a significant increase or decrease of cell-electrode coupling depending on the nanostructure layout could be demonstrated. For verification, action potential recordings with neural stem cells of the subventricular zone of postnatal mice have been performed, showing a distinct increase of the recorded signal amplitudes of the nanostructured electrodes compared to unstructured electrodes of the same chip. The latter might be correlated to an engulfment process of the cells. Figure: Left: Impedance measurement to quantify cell-electrode adhesion. Right: SEM picture of attached SVZ cells on top of overgrown pillar structures (Inset: Magnification, scale bar: 2 µm). Figure 1

A chemically heterogeneous group of compounds acts at the benzodiazepine (BZ) recognition site of the diverse gamma-aminobutyric acid type A (GABA(A)) receptor complexes which can assemble from more than 16 known subunits. Most 1,4-BZs like diazepam recognize all GABA(A)/BZ receptors containing the alpha1-3 or alpha5 together with any beta and the gamma2 subunit. Other compounds differentiate less, e.g. Ro15-4513, that additionally recognizes alpha4- and a6-containing receptors, or differentiate more, e.g. zolpidem, that recognizes preferentially alpha1-containing receptors. Here we describe the functional properties of 1-(4-chloro-phenyl)-4-piperidin-1-yl-1,5-dihydro-imidazol-2-on (ELB139) in the presence and absence of the BZ receptor antagonist flumazenil (Ro15-1788) on recombinant alphaibeta2gamma2 (i=1-5) receptor subtypes expressed in HEK 293 cells. The properties were measured with the whole-cell variation of the patch-clamp technique and compared to those of diazepam. Like the latter, ELB139 did not potentiate GABA-induced currents in alpha4-containing receptors, but it displays functional subtype specificity between alpha1, alpha2, alpha3, and alpha5beta2gamma2 receptors with highest potency in alpha3-containing receptors but highest efficacy in alpha1- or alpha2-containing receptors, respectively. ELB139 acted as a partial agonist on these receptor subtypes reaching 40-50% of the efficacy of diazepam.

The enteric nervous system (ENS) orchestrates a broad range of important gastrointestinal functions such as intestinal motility and gastric secretion. The ENS can be affected by environmental factors, diet and disease. Changes due to these alterations are often hard to evaluate in detail when whole gut samples are used. Analyses based on pure ENS tissue can more effectively reflect the ongoing changes during pathological processes. Here, we present an optimized approach for the isolation of pure myenteric plexus (MP) from adult mouse and human. To do so, muscle tissue was individually digested with a purified collagenase. After incubation and a gentle mechanical disruption step, MP networks could be collected with anatomical integrity. These tissues could be stored and used either for immediate genomic, proteomic or in vitro approaches and enteric neurospheres could be generated and differentiated. In a pilot experiment, the influence of bacterial lipopolysaccharide on human MP was analyzed using 2-dimensional gel electrophoresis. The method also allows investigation of factors that are secreted by myenteric tissue in vitro. The isolation of pure MP in large amounts allows new analytical approaches that can provide a new perspective in evaluating changes of the ENS in experimental models, human disease and aging.

[35S]TBPS binding to the GABAA receptor ionophore binding site is anion dependent. Using autoradiography on rat brain sections, we show that permeabilities of anions through the receptor channel correlate with their efficiencies to promote basal [35S]TBPS binding. Phosphate made an exception as it induced more binding than expected from its permeability. Well-permeable anions (chloride, nitrate, formate) allowed [35S]TBPS binding to be effectively displaced by 1 mM GABA, whereas low-permeable anions (acetate, phosphate, propionate) markedly prevented this GABA effect, especially in the thalamus, the transition from the high to the low GABA effect being between formate and acetate. In the presence of phosphate, GABA enhanced [3H]flunitrazepam binding to benzodiazepine site of recombinant alpha1beta2gamma2 receptors with the same efficacy but lower potency as compared to the presence of chloride, whereas [35S]TBPS binding was abnormally modulated by GABA. These results suggest that inorganic phosphate affects coupling between agonist and ionophore sites in GABAA receptors.

Nicotinic acetylcholine receptors (nAChR) are the archetypes of drug receptors. In 1905, John Newport Langley introduced the concept of a receptive substance on the surface of skeletal muscle that mediated the action of a drug, such as nicotine and curare (a neurotoxin made from a plant) (Langley et al., 1905). He also proposed that these receptive substances were different in different species and tissues, and they undergo conformational changes in response to the respective drug. Today nAChRs are considered prototypes of receptors that function as integral signal transducers. They have the response element and the ion channel domain within the same molecular entity as the ligand-binding domain that is activated by acetylcholine. In contrast, muscarinic acetylcholine receptors (mAChR) are prototypic G protein-coupled receptors. They also sense molecules outside the cell, but they require the G proteins to induce cellular responses by coupling to intracellular signalling pathways. More important than their historical role, nicotinic receptors continue to be at the forefront of science, as they are drug targets for muscle and nerve diseases, such as Alzheimer’s and Parkinson’s diseases, Schizophrenia and Myasthenia gravis. Therefore, this receptor family serves as an excellent example for demonstrating the suitability of site-directed mutagenesis for investigating receptor function and exploring drug action.

We have used the selective metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) to investigate in the CA1 hippocampal subregion in vitro whether coactivation of N-methyl-D-aspartate (NMDA) and metabotropic glutamate receptors is necessary for the induction of long-term potentiation (LTP) when LTP is induced by theta burst stimulation (TBS). When MCPG (500 microM) was bath applied 14-30 min prior to a triple high-frequency tetanization (100 Hz, 1 s) and washed out immediately afterwards the potentiation of the extracellularly recorded field potentials decayed gradually to baseline (P0.05) over 2-3 h. However, when MCPG was applied in the same manner before a triple TBS (10 bursts at 5 Hz, 100 Hz within the bursts) the resulting potentiation remained stable for at least 4 h. MCPG had no effect on baseline synaptic transmission or post-tetanic potentiation. These results demonstrate a clear difference in the mechanisms underlying these two different forms of LTP.

The potent sedative-hypnotic zolpidem and the convulsant methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) act primarily by binding to the benzodiazepine site of the main inhibitory neurotransmitter receptor, the pentameric γ-aminobutyric acid type A receptor (GABA(A)). This binding depends critically on the wild-type F77 residue of the GABA(A) receptor γ2 subunit. Mice with γ2 subunit F77I point mutation (γ2I77 mouse line) lose the high-affinity nanomolar binding of these ligands as well as their most robust behavioral actions at low doses. Interestingly, the γ2I77 mice offer a tool to study the actions of these substances mediated via other possible binding sites of the GABA(A) receptor. In ligand autoradiographic experiments, we discovered in γ2I77 mouse brain sections a significant amount of residual non-γ2 subunit-dependent benzodiazepine site binding enriched to the striatum and septum. Zolpidem only weakly affected this residual binding at micromolar concentrations, and only a high zolpidem dose (≥ 40 mg/kg) caused sedation and deficits in motor coordination in γ2I77 mice. DMCM had an agonistic action through a secondary, low-affinity non-benzodiazepine binding site of the GABA(A) receptor in the forebrain of γ2I77 mice, and this drug also fully displaced the residual benzodiazepine-site labeling. In behavioral tests, a high dose (20mg/kg) of DMCM was sedative and modulated fear learning. DMCM, but not zolpidem, acted as an agonist in recombinant GABA(A) α1/6β3 receptors studied using ligand binding and electrophysiological assays. Our results highlight the less well-known actions of high doses of DMCM and zolpidem that are not mediated via the γ2 subunit-containing benzodiazepine site of the GABA(A) receptor.

Drugs used to treat various disorders target GABA A receptors. To develop alpha subunit selective compounds, we synthesized 5-(4-piperidyl)-3-isoxazolol (4-PIOL) derivatives. The 3-isoxazolol moiety was substituted by 1,3,5-oxadiazol-2-one, 1,3,5-oxadiazol-2-thione, and substituted 1,2,4-triazol-3-ol heterocycles with modifications to the basic piperidine substituent as well as substituents without basic nitrogen. Compounds were screened by [(3)H]muscimol binding and in patch-clamp experiments with heterologously expressed GABA A alpha ibeta 3gamma 2 receptors (i = 1-6). The effects of 5-aminomethyl-3 H-[1,3,4]oxadiazol-2-one 5d were comparable to GABA for all alpha subunit isoforms. 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-one 5a and 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-thione 6a were weak agonists at alpha 2-, alpha 3-, and alpha 5-containing receptors. When coapplied with GABA, they were antagonistic in alpha 2-, alpha 4-, and alpha 6-containing receptors and potentiated alpha 3-containing receptors. 6a protected GABA binding site cysteine-substitution mutants alpha 1F64C and alpha 1S68C from reacting with methanethiosulfonate-ethylsulfonate. 6a specifically covalently modified the alpha 1R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing alpha subtype selective GABA mimetic drugs.

The behavioral and functional significance of the extrasynaptic inhibitory GABA(A) receptors in the brain is still poorly known. We used a transgenic mouse line expressing the GABA(A) receptor alpha6 subunit gene in the forebrain under the Thy-1.2 promoter (Thy1alpha6) mice ectopically expressing alpha6 subunits especially in the hippocampus to study how extrasynaptically enriched alphabeta(gamma2)-type receptors alter animal behavior and receptor responses. In these mice extrasynaptic alpha6beta receptors make up about 10% of the hippocampal GABA(A) receptors resulting in imbalance between synaptic and extrasynaptic inhibition. The synthetic GABA-site competitive agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; 3 mg/kg) induced remarkable anxiolytic-like response in the light : dark exploration and elevated plus-maze tests in Thy1alpha6 mice, while being almost inactive in wild-type mice. The transgenic mice also lost quicker and for longer time their righting reflex after 25 mg/kg gaboxadol than wild-type mice. In hippocampal sections of Thy1alpha6 mice, the alpha6beta receptors could be visualized autoradiographically by interactions between gaboxadol and GABA via [(35)S]TBPS binding to the GABA(A) receptor ionophore. Gaboxadol inhibition of the binding could be partially prevented by GABA. Electrophysiology of recombinant GABA(A) receptors revealed that GABA was a partial agonist at alpha6beta3 and alpha6beta3delta receptors, but a full agonist at alpha6beta3gamma2 receptors when compared with gaboxadol. The results suggest strong behavioral effects via selective pharmacological activation of enriched extrasynaptic alphabeta GABA(A) receptors, and the mouse model represents an example of the functional consequences of altered balance between extrasynaptic and synaptic inhibition.

In rodent models, γ-aminobutyric acid A (GABAA) receptors with the α6 and δ subunits, expressed in the cerebellar and cochlear nucleus granule cells, have been linked to ethanol sensitivity and voluntary ethanol drinking. Here, we review the findings. When considering both in vivo contributions and data on cloned receptors, the evidence for direct participation of the α6-containing receptors to increased ethanol sensitivity is poor. The α6 subunit-knockout mouse lines do not have any changed sensitivity to ethanol, although these mice do display increased benzodiazepine sensitivity. However, in general the compensations occurring in knockout mice (regardless of which particular gene is knocked out) tend to fog interpretations of drug actions at the systems level. For example, the α6 knockout mice have increased TASK-1 channel expression in their cerebellar granule cells, which could influence sensitivity to ethanol in the opposite direction to that obtained with the α6 knockouts. Indeed, TASK-1 knockout mice are more impaired than wild types in motor skills when given ethanol; this might explain why GABAA receptor α6 knockout mice have unchanged ethanol sensitivities. As an alternative to studying knockout mice, we examined the claimed δ subunit-dependent/γ2 subunit-independent ethanol/[3H]Ro 15-4513 binding sites on GABAA receptors. We looked at [3H]Ro 15-4513 binding in HEK 293 cell membrane homogenates containing rat recombinant α6/4β3δ receptors and in mouse brain sections. Specific high-affinity [3H]Ro 15-4513 binding could not be detected under any conditions to the recombinant receptors or to the cerebellar sections of γ2(F77I) knockin mice, nor was this binding to brain sections of wild-type C57BL/6 inhibited by 1–100 mM ethanol. Since ethanol may act on many receptor and channel protein targets in neuronal membranes, we consider the α6 (and α4) subunit-containing GABAA receptors unlikely to be directly responsible for any major part of ethanol's actions. Therefore, we finish the review by discussing more generally alcohol and GABAA receptors and by suggesting potential future directions for this research., This study and review was supported by the Finnish Foundation for Alcohol Studies, the Academy of Finland and the Sigrid Juselius Foundation.

GABA(A) receptors, mediators of fast inhibitory neurotransmission, are heteropentameric assemblies from a large array of subunits. Differences in the sensitivity of receptor subtypes to endogenous GABA may permit subunit-dependent finely tuned responsiveness to the same GABAergic inputs. Using both radioligand binding and electrophysiology combined with mutagenesis, we identified a domain of four amino acids within the alpha subunits that mediates the distinct sensitivities to GABA allowing their selective switch between alphabeta3gamma2 combinations. Replacing this domain in alpha3 by the corresponding segments of alpha1-alpha5 resulted in mutant receptors displaying the GABA EC(50) values of the respective wild-type receptors. Vice versa, the alpha3 motif forced the low sensitivity to GABA of alpha3 upon alpha1beta3gamma2, alpha4beta3gamma2, and alpha5beta3gamma2. Binding of the GABA agonist [(3)H]muscimol was not affected by the exchange of the motif between alpha1 and alpha3 subunits. Thus, the equilibrium binding pocket is maintained upon replacement of the four amino acids. Taken together our data suggest that the identified motifs contribute to a structure involved in the transduction of the binding signal rather than to the binding itself.

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.