Plug-and-play adaptive optics for commercial laser scanning fluorescence microscopes based on an adaptive lens.

In this Letter, we present a solution for simple implementation of adaptive optics in any existing laser scanning fluorescence microscope. Adaptive optics are implemented by the introduction of a multiactuator adaptive lens between the microscope body and the objective lens. Correction is performed with a sensorless method by optimizing the quality of the images presented on screen by the microscope software. We present the results acquired on both a commercial linear excitation confocal microscope and a custom-made multiphoton excitation microscope.

A novel QTL underlying early-onset, low-frequency hearing loss in BXD recombinant inbred strains.

The DBA/2J inbred strain of mice has been used extensively in hearing research as it suffers from early-onset, progressive hearing loss. Initially, it mostly affects high frequencies, but already at 2-3 months hearing loss becomes broad. In search for hearing loss genes other than Cadherin 23 (otocadherin) and fascin-2, which make a large contribution to the high-frequency deficits, we used a large set of the genetic reference population of BXD recombinant inbred strains. For frequencies 4, 8, 16 and 32 kHz, auditory brainstem response hearing thresholds were longitudinally determined from 2-3 up to 12 weeks of age. Apart from a significant, broad quantitative trait locus (QTL) for high-frequency hearing loss on chromosome 11 containing the fascin-2 gene, we found a novel, small QTL for low-frequency hearing loss on chromosome 18, from hereon called ahl9. Real-time quantitative polymerase chain reaction of organs of Corti, isolated from a subset of strains, showed that a limited number of genes at the QTL were expressed in the organ of Corti. Of those genes, several showed significant expression differences based on the parental line contributing to the allele. Our results may aid in the future identification of genes involved in low-frequency, early-onset hearing loss.

In vivo dynamic clamp study of I(h) in the mouse inferior colliculus.

Approximately half of the cells in the mouse inferior colliculus have the hyperpolarization-activated mixed cation current I(h), yet little is known about its functional relevance in vivo. We therefore studied its contribution to the processing of sound information in single cells by making in vivo whole cell recordings from the inferior colliculus (IC) of young-adult anesthetized C57Bl/6 mice. Following pharmacological block of the endogenous channels, a dynamic clamp approach allowed us to study the responses to current injections or auditory stimuli in the presence and absence of I(h) within the same neuron, thus avoiding network or developmental effects. The presence of I(h) changed basic cellular properties, including depolarizing the resting membrane potential and decreasing resting membrane resistance. Sound-evoked excitatory postsynaptic potentials were smaller but at the same time reached a more positive membrane potential when I(h) was present. With I(h), a subset of cells showed rebound spiking following hyperpolarizing current injection. Its presence also changed more complex cellular properties. It decreased temporal summation in response to both hyperpolarizing and depolarizing repetitive current stimuli, and resulted in small changes in the cycle-averaged membrane potential during sinusoidal amplitude modulated (SAM) tones. Furthermore, I(h) minimally decreased the response to a tone following a depolarization, an effect that may make a small contribution to forward masking. Our results thus suggest that previously observed differences in IC cells are a mixture of direct effects of I(h) and indirect effects due to the change in membrane potential or effects due to the co-expression with other channels.

Intracellular responses of neurons in the mouse inferior colliculus to sinusoidal amplitude-modulated tones.

Changes in the temporal envelope are important defining features of natural acoustic signals. Many cells in the inferior colliculus (IC) respond preferentially to certain modulation frequencies, but how they accomplish this is not yet clear. We therefore made whole cell patch-clamp recordings in the IC of anesthetized mice while presenting sinusoidal amplitude-modulated (SAM) tones. The relation between the number of evoked spikes and modulation frequency was used to construct rate modulation transfer functions (rMTFs). We observed different types of rate tuning, including band-pass (16%), band-reject (13%), high-pass (6%), and low-pass (6%) tuning. In the high-pass rMTF neurons and some of the low-pass rMTF neurons, the tuning characteristics appeared to be already present in the inputs. In both band-pass and band-reject rMTF neurons, the nonlinear relation between membrane potential and spike probability ensured preferential spiking during only a small part of the modulation period. Band-pass rMTF neurons had rapidly rising excitatory postsynaptic potentials, allowing good phase-locking to brief tones and intermediate modulation frequencies. At low modulation frequencies, adaptation of their spike threshold contributed to the onset response. In contrast, band-reject rMTF neurons responded with small excitatory or inhibitory postsynaptic potentials to brief tones. In these cells, a power law could describe the supralinear relation between average membrane potential and spike rate. Differences in timing of synaptic input and presence or absence of spike adaptation therefore define band-pass and band-reject rate tuning to SAM tones in the mouse IC.

Membrane properties and firing patterns of inferior colliculus neurons: an in vivo patch-clamp study in rodents.

The inferior colliculus (IC) is a large auditory nucleus in the midbrain, which is a nearly obligatory relay center for ascending auditory projections. We made in vivo whole cell patch-clamp recordings of IC cells in young-adult anesthetized C57/Bl6 mice and Wistar rats to characterize their membrane properties and spontaneous inputs. We observed spikelets in both rat (18%) and mouse (13%) IC neurons, suggesting that IC neurons may be connected by electrical synapses. In many cells, spontaneous postsynaptic potentials were sufficiently large to contribute to spike irregularity. Cells differed considerably in the number of simultaneous spontaneous postsynaptic potentials that would be needed to trigger an action potential. Depolarizing and hyperpolarizing current injections showed six different types of firing patterns: buildup, accelerating, burst-onset, burst-sustained, sustained, and accommodating. Their relative frequencies were similar in both species. In mice, about half of the cells showed a clear depolarizing sag, suggesting that they have the hyperpolarization-activated current I(h). This sag was observed more often in burst and in accommodating cells than in buildup, accelerating, or sustained neurons. Cells with I(h) had a significantly more depolarized resting membrane potential. They were more likely to fire rebound spikes and generally showed long-lasting afterhyperpolarizations following long depolarizations. We therefore suggest a separate functional role for I(h).

Comparison of responses of neurons in the mouse inferior colliculus to current injections, tones of different durations, and sinusoidal amplitude-modulated tones.

We made in vivo whole cell patch-clamp recordings from the inferior colliculus of young-adult, anesthetized C57/Bl6 mice to compare the responses to constant-current injections with the responses to tones of different duration or to sinusoidal amplitude-modulated (SAM) tones. We observed that voltage-dependent ion channels contributed in several ways to the response to tones. A sustained response to long tones was observed only in cells showing little accommodation during current injection. Cells showing burst-onset firing during current injection showed a small response to SAM tones, whereas burst-sustained cells showed a good response to SAM tones. The hyperpolarization-activated nonselective cation channel I(h) had a special role in shaping the responses: I(h) was associated with an increased excitability, with chopper and pauser responses, and with an afterhyperpolarization following tones. Synaptic properties were more important in determining the responses to tones of different durations. A short-latency inhibitory response appeared to contribute to the long-pass response in some cells and short-pass and band-pass neurons were characterized by their slow recovery from synaptic adaptation. Cells that recovered slowly from synaptic adaptation showed a relatively small response to SAM tones. Our results show an important role for both intrinsic membrane properties -- most notably the presence of I(h) and the extent of accommodation -- and synaptic adaptation in shaping the response to tones in the inferior colliculus.

Two modes of vesicle recycling in the rat calyx of Held.

Vesicle recycling was studied in the rat calyx of Held, a giant brainstem terminal involved in sound localization. Stimulation of brain slices containing the calyx-type synapse with a high extracellular potassium ion concentration in the presence of horseradish peroxidase resulted within several minutes in a reduction of the number of neurotransmitter vesicles and in the appearance of labeled endosome-like structures. After returning to normal solution, the endosome-like structures disappeared over a period of several minutes, whereas simultaneously the number of labeled vesicles increased. A comparison with afferent stimulation suggested that the endosome-like structures normally do not participate in the vesicle cycle. Afferent stimulation at 5 Hz resulted in sustained synaptic transmission, without vesicle depletion but with an estimated endocytotic activity of <0.2 synaptic vesicles per active zone per second. At 20 Hz, the presynaptic action potentials generally failed during prolonged stimulation. In identified synapses, the number of vesicles labeled by photoconversion after stimulation at 5 Hz in the presence of the styryl dye RH414 was much lower than the number of vesicles that were released, as determined by measuring EPSCs. No more than approximately 5% of the vesicles were labeled after 20 min stimulation at 5 Hz, whereas this stimulation protocol was sufficient to largely destain a terminal after previous loading. The results support a scheme for recycling in which two different modes coexist. At physiological demands, a pool of approximately 5% of all vesicles provides sufficient vesicles for release. During intense stimulation, such as occurs in the presence of high extracellular K+, the synapse resorts to bulk endocytosis, a very slow mode of recycling.