Assessment of the AAV-mediated expression of channelrhodopsin-2 and halorhodopsin in brainstem neurons mediating auditory signaling.

The physiology and circuitry associated with dorsal cochlear nucleus neurons (DCN) have been well described. The ability to remotely manipulate neuronal activity in these neurons would represent a step forward in the ability to understand the specific function of DCN neurons in hearing. Although, optogenetics has been used to study the function of pathways in other systems for several years, in the auditory system only neurons in the auditory cortex have been studied using this technique. Adeno-associated viral vectors with either channelrhodopsin-2 fused with GFP (ChR2-GFP) or halorhodopsin fused with mCherry (HaloR-mCherry), capable of expressing light sensitive cation channels or chloride pumps, respectively, were delivered into the dorsal cochlear nucleus (DCN). One to 18 months later, expression of ChR2 and HaloR was observed throughout the DCN. Rhodopsin distribution within the DCN was determined to be within several cell types identified based on morphology and location within the DCN. Expression of ChR2-GFP and HaloR-mCherry was found at both the injection site as well as in regions receiving projections from the site. Wavelength appropriate optical stimulation in vivo resulted in neuronal activity that was significantly increased over pre-stimulation levels with no return to baseline levels during the time of the light exposure. We also examined the effects of optically driven neuronal activity on subsequent tone driven responses in the DCN. In the DCN 75% of the 16 electrode sites showed decreased neuronal activity in response to a tone immediately following light stimulation while six percent were decreased following tone stimulation and 19% of the electrode sites showed no change. This is in contrast to tone driven neuronal activity prior to the light exposure in which the majority of electrode sites showed increased neuronal activity. Our results indicate that expression and activation of rhodopsin within neurons involved in auditory processing does not appear to have deleterious effects on hearing even 18 months following expression. In addition, virally targeted rhodopsins may be useful as tract tracers to delineate as well as modulate the activity of pathways and specific neurons. In the future rhodopsins can be targeted to specific subpopulations of auditory neurons. Ultimately, photostimulation may provide a physiologically relevant method for modulating the function of auditory neurons and affecting hearing outcomes. This article is part of a Special Issue entitled Optogenetics (7th BRES).

[Asthma control with the salmeterol-fluticasone-combination disc compared to standard treatment]. / Asthmakontrolle mit dem Salmeterol/Fluticason Kombinationsdiskus in hoher und mittlerer Dosis im Vergleich zur Standardtherapie.

BACKGROUND: The present study aimed to investigate whether a fixed combination of salmeterol and fluticasone (SFC) from a single inhaler provides sufficient asthma control comparable to that achieved with standard treatment (inhaled steroid in a dose of 1,000 mcg BDP- (beclomethasone dipropionate) equivalent plus a LABA and/or theophylline and/or montelukast). PATIENTS AND METHODS: In a prospective, randomised study patients with moderate or severe asthma were either switched to a twice daily inhalation of 50 mcg salmeterol plus 500 mcg fluticasone from the Viani forte 50/500 mcg Diskus (n = 142 patients), or they were maintained on standard treatment (n = 89 patients). If adequate asthma control was achieved after 8 weeks, the dose of the inhaled steroid was reduced by 50 % during weeks 9 to 16. RESULTS: After 8 weeks, 81 % of the patients who had been switched to SFC and 80 % of patients on standard treatment achieved sufficient asthma control. After reducing the ICS dose by 50 %, asthma control remained appropriate in 90 % of SFC-patients, but only in 75 % of patients receiving standard treatment (p = 0.031). In addition, asthma symptoms and use of rescue medication were significantly more stable in SFC patients (p < 0.05). CONCLUSIONS: With the salmeterol fluticasone combination product, patients with moderate asthma can achieve a control of their asthma, which is as good as that after standard treatment. In most SFC patients the fluticasone dosage can be reduced by 50 % without losing asthma control.

Voltage-gated Ca2+ conductances in acutely isolated guinea pig dorsal cochlear nucleus neurons.

Although it is known that voltage-gated Ca2+ conductances (VGCCs) contribute to the responses of dorsal cochlear nucleus (DCN) neurons, little is known about the properties of VGCCs in the DCN. In this study, the whole cell voltage-clamp technique was used to examine the pharmacology and voltage dependence of VGCCs in unidentified DCN neurons acutely isolated from guinea pig brain stem. The majority of cells responded to depolarization with sustained inward currents that were enhanced when Ca2+ was replaced by Ba2+, were blocked partially by Ni2+ (100 microM), and were blocked almost completely by Cd2+ (50 microM). Experiments using nifedipine (10 microM), omegaAga IVA (100 nM) and omegaCTX GVIA (500 nM) demonstrated that a variety of VGCC subtypes contributed to the Ba2+ current in most cells, including the L, N, and P/Q types and antagonist-insensitive R type. Although a large depolarization from rest was required to activate VGCCs in DCN neurons, VGCC activation was rapid at depolarized levels, having time constants <1 ms at 22 degrees C. No fast low-threshold inactivation was observed, and a slow high-threshold inactivation was observed at voltages more positive than -20 mV, indicating that Ba2+ currents were carried by high-voltage activated VGCCs. The VGCC subtypes contributing to the overall Ba2+ current had similar voltage-dependent properties, with the exception of the antagonist-insensitive R-type component, which had a slower activation and a more pronounced inactivation than the other components. These data suggest that a variety of VGCCs is present in DCN neurons, and these conductances generate a rapid Ca2+ influx in response to depolarizing stimuli.

Evidence for functional metabotropic glutamate receptors in the dorsal cochlear nucleus.

The parallel fibers (PFs) of the dorsal cochlear nucleus (DCN) molecular layer use glutamate as a neurotransmitter. Although metabotropic glutamate receptors (mGluRs) have been identified on cells postsynaptic to the PFs, little is known about the effects of mGluR activation in PF synaptic transmission in the DCN. To investigate these effects, PF-evoked field potentials were recorded from the DCN in guinea pig brain stem slice preparations. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated components of the field response were reversibly depressed by bathing the slice in the mGluR agonists (+/-)-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD]. A similar depression was produced by the mGluR1/5 agonist (RS)-3,5-dihydroxyphenylglycine, but not by the mGluR2/3 agonist (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine or by the mGluR4/6/7/8 agonist L(+)-2-amino-4-phosphonobutyric acid. In addition to the AMPA component, an N-methyl-D-aspartate (NMDA) receptor-dependent component of the field potentials could be identified when the slices were bathed in a low magnesium solution. Under these conditions, the ACPD-induced depression of the AMPA component did not completely recover, whereas the depression of the NMDA component usually recovered and potentiated in some slices. Intracellular recordings of PF-evoked responses were obtained to ascertain which neuronal populations were affected by mGluR activation. Activation of mGluRs produced a reversible depression of PF-evoked responses in cartwheel cells that was not accompanied by any changes in paired-pulse facilitation. The PF-evoked responses recorded from pyramidal cells were unaffected by mGluR activation. Both cell types exhibited a reversible depolarization during (1S,3R)-ACPD application. Subsequent experiments explored the involvement of protein kinases in mediating the effects of mGluRs. The protein kinase C (PKC) activator phorbol-12,13-diacetate partially inhibited the mGluR-mediated depression of the field response; however, the PKC inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide or the protein kinase A inhibitor N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide had little effect on the actions of (1S,3R)-ACPD. These results demonstrate that functional mGluRs are present at PF synapses and are capable of modulating PF synaptic transmission in the DCN.

[Cost of asthma therapy in relation to severity. An empirical study]. / Kosten der Asthmatherapie nach Schweregrad. Eine empirische Untersuchung.

BACKGROUND: The aim of asthma therapy, i.e. the permanent elimination of the patient's symptoms, is as a rule, achievable over the long-term only with the aid of anti-inflammatory drugs. As well as medical, this approach also has considerable economic implications. The comparatively low compliance among asthmatics makes treatment in this context all the more difficult. An alternative that presents itself is the use of combination preparations, a mixture of a long-term prophylactic and a therapeutic agent. PATIENTS AND METHODS: With the aid of standardised questionnaires, data were acquired from 216 patients and assigned to subgroups in accordance with the degree of severity of the asthma. The patients were treated in the offices of a total of 23 GPs and internists selected at random from a complete list of all relevant practices in Germany. The use of resources, i.e. all diagnostic and therapeutic measures, was recorded retrospectively for a period of 1 year. In this way, all those resources of relevance to the health insurance carriers used during the observation period were identified. In addition to direct costs, so-called indirect costs were also estimated, i.e. in the present study the productivity loss to the economy due to illness-related absence from work. RESULTS: The annual cost of treating adult asthmatics was calculated to be DM 3,339 for level 1 severity, DM 5,260 for level 2 severity and DM 12,016 for level 3 severity. As the illness progresses in particular the direct cost of inpatient care and the indirect costs rise disproportionately. The yearly expenditure for women sufferers is about DM 800 more than for male sufferers. The direct cost of asthma treatment in children amounts to DM 2,950 for level 1, DM 3,225 for level 2, and DM 4,811 for level 3, severity. Here, drug-related costs in particular, rise significantly as the disease progresses. CONCLUSION: One of the results of the present study is the fact that for asthma sufferers in general, there is a positive correlation between average total costs and degree of severity. It may thus be postulated that preventive medical treatment of asthma that slows the progression of the illness, together with appropriate patient instruction, would have a positive effect on the total expenditure per patient. If, for example, the appropriate use of drugs in combination with patient instruction improved the compliance of asthmatics, lower treatment costs and a better quality of life for the patient could be expected.

N-methyl-D-aspartate receptors at parallel fiber synapses in the dorsal cochlear nucleus.

1. N-methyl-D-aspartate (NMDA) binding and NMDA-receptors immunolocalization experiments have revealed an enhanced expression of these receptors in the outer two layers of the dorsal cochlear nucleus (DCN). The distribution of the receptors is congruent with the distribution of synapses produced by the granule cell-parallel fiber system. To determine the functional distribution and contribution of NMDA receptors at parallel fiber synapses, synaptic responses to parallel fiber stimulation were studied in in vitro brain slice preparations of the guinea pig and rat dorsal cochlear nucleus. 2. The field potential response to parallel fiber stimulation in guinea pigs includes three postsynaptic components. The short latency components (the P3(2) and N2(2)) are blocked by general excitatory receptor antagonists, including the non-NMDA-receptor blockers 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but are insensitive to NMDA-receptor antagonists. 3. A slower component (P4(2)) is revealed when the slices are washed with a low magnesium solution to eliminate the magnesium block of currents through NMDA receptors. This slow component is reduced by D- or DL-2-amino-5-phosphonovaleric acid (D-APV, DL-APV) and 3-[(+/-)-2-carboxypiperazine-4-yl] propyl-1-phosphonate, but is not blocked by DNQX or CNQX. Eliminating the voltage dependence of the NMDA receptors also results in a complex oscillatory response in some slices. This response exhibits the same pharmacological sensitivity as the slow potential. The pharmacologic sensitivity to NMDA-receptor antagonists suggest that the slow component (P4(2)) and the associated oscillatory response are mediated through activation of NMDA receptors. 4. Current source-density analysis of the parallel fiber-evoked field potentials was carried out to determine the relative spatial distributions of the fast and slow synaptic currents. Both synaptic components were associated with a superficial current sink and a deeper current source, localized within the superficial 250 microM of the nucleus. The slow (APV-sensitive) current was slightly shifted in depth relative to the fast (DNQX-sensitive) current in three of five slices with the maximum current sink and source occurring approximately 16 microns further from the surface of the DCN. These data suggest that either the NMDA receptors are not present at all of the synapses that generate the fast non-NMDA currents or that postsynaptic cells with different dendritic distributions have different densities of NMDA receptors. 5. The types of cells in layers 1 and 2 exhibiting NMDA-receptor-mediated synaptic potentials were investigated. Intracellular recordings with sharp electrodes in guinea pig slices showed that eliminating the voltage dependence of the NMDA receptors in low magnesium revealed a slow excitatory postsynaptic potential (EPSP) in both simple and complex spiking cells. The late phase of the EPSP could be reduced by APV in both cell types. These results could be explained by NMDA receptors on the postsynaptic cells or by NMDA receptors on excitatory interneurons. Attempts to demonstrate an appropriate voltage dependence of the parallel fiber synaptic response in normal magnesium medium under current clamp were confounded by the intrinsic voltage-dependent conductances of the cells. 6. To determine whether NMDA receptors were present on postsynaptic cells, the direct sensitivity of DCN cells to NMDA application was examined during intracellular recording. Both simple spiking and complex spiking cells responded to NMDA with depolarization. The response to NMDA persisted when non-NMDA receptors were blocked with CNQX or DNQX. However in all cells tested, the response to NMDA was blocked by APV. These experiments further support the postsynaptic localization of NMDA receptors on both simple and complex spiking cells. (ABSTRACT TRUNCATED)

Hemodynamic and neuroendocrine response to acute administration of the phosphodiesterase inhibitor BM14.478 in patients with congestive heart failure.

The benzimidazol analogue BM14.478 is a phosphodiesterase inhibitor with both vasodilator and positive inotropic properties. Hemodynamic parameters and plasma hormone levels of 8 patients (1 female, 7 male) with chronic congestive heart failure NYHA Classes II-IV (1 patient with coronary artery disease, 7 patients with primary dilated cardiomyopathy) were assessed before and until 6 h after the intravenous application of 1.0 mg BM14.478. There was a significant decrease of mean pulmonary artery pressure (28 +/- 11 vs. 23 +/- 11 mmHg; p less than 0.05), mean right atrial pressure (8.6 +/- 5.2 vs. 5.0 +/- 4.7 mmHg; p less than 0.02), and systemic vascular resistance (1651 +/- 484 vs. 1206 +/- 252 dynes.s.cm-5; p less than 0.05) as early as 10 min after injection of BM14.478. Pulmonary vascular resistance also was reduced (128 +/- 86 vs. 61 +/- 39 dynes.s.cm-5, 30 min after injection; p less than 0.02). Simultaneously there was a significant increase of cardiac index (2.3 +/- 0.7 vs. 3.1 +/- 0.8 l.min-1.m-2, 10 min after injection; p less than 0.02), and stroke volume index (28.8 +/- 11.7 vs. 33.9 +/- 8.5 ml.min-1.m-2; 30 min after injection; p less than 0.05). Although mean heart rate did not change significantly, some patients reacted with a transient increase. There was also a slight but insignificant increase of the double product. No serious side effects were observed. The hemodynamic improvement was followed by a delayed reduction of plasma levels of epinephrine (51 +/- 20 vs. 41 +/- 21 pg/ml; p less than 0.02; 30 min after injection) and atrial natriuretic peptide (229 +/- 283 vs. 121 +/- 168 pg/ml; p less than 0.05; 1 h after injection). Mean levels of plasma norepinephrine, however, did not change significantly and individual responses showed large variations, which could not be predicted by the behavior of the hemodynamic parameters. Three of eight patients (2 of these with elevated baseline filling pressures) even showed a marked increase of plasma norepinephrine levels after BM14.478. Response of plasma renin activity and plasma vasopressin levels to BM14.478 also was heterogeneous. According to the results of this study, acute administration of the phosphodiesterase inhibitor BM14.478 has an immediate beneficial hemodynamic effect in patients with severe congestive heart failure by reducing both preload and afterload, and by increasing cardiac index and stroke volume. However, this improvement of hemodynamic parameters is not necessarily accompanied by a favorable short-term response of plasma hormones, and therefore does not allow any conclusions on survival of these patients.

[Idiopathic lung fibrosis]. / Die idiopathische Lungenfibrose.

In a 39-year-old patient with chronic progressive idiopathic pulmonary fibrosis, the genetic aspects, course and therapeutic possibilities of the disease are discussed. In February, 1987, the English-born patient, Anthony V., attended for initial examination on account of progressive dyspnoea, on which occasion radiology and pulmonary function analysis revealed advanced pulmonary fibrosis. The patient's family history revealed a familial genesis, since both his father (?) and his sister had died of this disease. A comparative of the patient's chest films with original chest films of his sister revealed almost identical findings. Within the previous twelve months, follow-up examinations done on A.V. revealed an increase in the restrictive component (reduction of vital capacity from 2,400 ml to 1,500 ml), development of partial respiratory failure at rest, and global respiratory failure in response to mild ergometric exercise despite intermittent high-dose steroid administrations superimposed on long-term, low-dose steroid therapy. The unfavourable evolution observed over the past 12 months is underscored by an increase in mean pulmonary arterial pressure from 18 mmHg initially to a present 34 mmHg at rest, and 46 mmHg under submaximal ergometric loading. The only option still left to the patient is the possibility of a lung transplantation, which - probably initially unilateral - is scheduled to be carried out in the near future at the Chest Surgery Department of the Medical University at Hannover.