Anna Dondzillo

Light scattering properties vary across different regions of the adult mouse brain.

Recently developed optogenetic tools provide powerful approaches to optically excite or inhibit neural activity. In a typical in-vivo experiment, light is delivered to deep nuclei via an implanted optical fiber. Light intensity attenuates with increasing distance from the fiber tip, determining the volume of tissue in which optogenetic proteins can successfully be activated. However, whether and how this volume of effective light intensity varies as a function of brain region or wavelength has not been systematically studied. The goal of this study was to measure and compare how light scatters in different areas of the mouse brain. We delivered different wavelengths of light via optical fibers to acute slices of mouse brainstem, midbrain and forebrain tissue. We measured light intensity as a function of distance from the fiber tip, and used the data to model the spread of light in specific regions of the mouse brain. We found substantial differences in effective attenuation coefficients among different brain areas, which lead to substantial differences in light intensity demands for optogenetic experiments. The use of light of different wavelengths additionally changes how light illuminates a given brain area. We created a brain atlas of effective attenuation coefficients of the adult mouse brain, and integrated our data into an application that can be used to estimate light scattering as well as required light intensity for optogenetic manipulation within a given volume of tissue.

Inhibitory projections from the ventral nucleus of the trapezoid body to the medial nucleus of the trapezoid body in the mouse

Neurons in the medial nucleus of the trapezoid body (MNTB) receive prominent excitatory input through the calyx of Held, a giant synapse that produces large and fast excitatory currents. MNTB neurons also receive inhibitory glycinergic inputs that are also large and fast, and match the calyceal excitation in terms of synaptic strength. GABAergic inputs provide additional inhibition to MNTB neurons. Inhibitory inputs to MNTB modify spiking of MNTB neurons both in-vitro and in-vivo, underscoring their importance. Surprisingly, the origin of the inhibitory inputs to MNTB has not been shown conclusively. We performed retrograde tracing, anterograde tracing, immunohistochemical experiments, and electrophysiological recordings to address this question. The results support the ventral nucleus of the trapezoid body (VNTB) as at least one major source of glycinergic input to MNTB. VNTB fibers enter the ipsilateral MNTB, travel along MNTB principal neurons and produce several bouton-like presynaptic terminals. Further, the contribution of GABA to the total inhibition declines during development, resulting in only a very minor fraction of GABAergic inhibition in adulthood, which is matched in time by a reduction in expression of a GABA synthetic enzyme in VNTB principal neurons.

Glycinergic inhibition to the medial nucleus of the trapezoid body shows prominent facilitation and can sustain high levels of ongoing activity.

Neurons in the medial nucleus of the trapezoid body (MNTB) are well known for their prominent excitatory inputs, mediated by the calyx of Held. Less attention has been paid to the prominent inhibitory inputs that MNTB neurons also receive. Because of their auditory nature, both excitatory and inhibitory synapses are highly active in vivo. These high levels of activity are known to reduce excitatory synaptic currents considerably, such that in vivo synaptic currents produced by the calyx are smaller than typically measured in standard brain slice experiments. The goal of this study was to investigate the properties of the inhibitory inputs in the Mongolian gerbil (Meriones unguiculatus) under activity levels that correspond to those in the intact brain to facilitate a direct comparison between the two inputs. Our results suggest that inhibitory inputs to MNTB are largely mediated by a fast and phasic glycinergic component, and to a lesser degree by a GABAergic component. The glycinergic component can sustain prolonged high levels of activity. Even when challenged with stimulus patterns consisting of thousands of stimuli over tens of minutes, glycinergic inputs to MNTB maintain large conductances and fast decays and even facilitate substantially when the stimulation frequency is increased. The inhibition is mediated by a relatively small number of independent input fibers. The data presented here suggest that inhibitory inputs to MNTB sustain high levels of activity and need to be considered for a full understanding of mechanisms underlying processing of auditory information in MNTB.

Recurrent Inhibition to the Medial Nucleus of the Trapezoid Body in the Mongolian Gerbil (Meriones Unguiculatus).

Principal neurons in the medial nucleus of the trapezoid body (MNTB) receive strong and temporally precise excitatory input from globular bushy cells in the cochlear nucleus through the calyx of Held. The extremely large synaptic currents produced by the calyx have sometimes led to the view of the MNTB as a simple relay synapse which converts incoming excitation to outgoing inhibition. However, electrophysiological and anatomical studies have shown the additional presence of inhibitory glycinergic currents that are large enough to suppress action potentials in MNTB neurons at least in some cases. The source(s) of glycinergic inhibition to MNTB are not fully understood. One major extrinsic source of glycinergic inhibitory input to MNTB is the ventral nucleus of the trapezoid body. However, it has been suggested that MNTB neurons receive additional inhibitory inputs via intrinsic connections (collaterals of glycinergic projections of MNTB neurons). While several authors have postulated their presence, these collaterals have never been examined in detail. Here we test the hypothesis that collaterals of MNTB principal cells provide glycinergic inhibition to the MNTB. We injected dye into single principal neurons in the MNTB, traced their projections, and immunohistochemically identified their synapses. We found that collaterals terminate within the MNTB and provide an additional source of inhibition to other principal cells, creating an inhibitory microcircuit within the MNTB. Only about a quarter to a third of MNTB neurons receive such collateral inputs. This microcircuit could produce side band inhibition and enhance frequency tuning of MNTB neurons, consistent with physiological observations.

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. Neurons typically receive multiple excitatory and inhibitory afferent and/or efferent inputs that integrate with each other, and the ultimate measured response properties of the neuron are driven by the neural integrations of these inputs. To study information processing in neural systems, it is necessary to understand the various inputs to a neuron or neural system, and the specific properties of these inputs. A powerful and technically relatively simple method to assess the functional role of certain inputs that a given neuron is receiving is to dynamically and reversibly suppress or eliminate these inputs, and measure the changes in the neurons output caused by this manipulation. This can be accomplished by pharmacologically altering the neurons immediate environment with piggy-back multibarrel electrodes. These electrodes consist of a single barrel recording electrode and a multibarrel drug electrode that can carry up to 4 different synaptic agonists or antagonists. The pharmacological agents can be applied iontophoretically at desired times during the experiment, allowing for time-controlled delivery and reversible reconfiguration of synaptic inputs. As such, pharmacological manipulation of the microenvironment represents a powerful and unparalleled method to test specific hypotheses about neural circuit function. Here we describe how piggy-back electrodes are manufactured, and how they are used during in vivo experiments. The piggy-back system allows an investigator to combine a single barrel recording electrode of any arbitrary property (resistance, tip size, shape etc) with a multibarrel drug electrode. This is a major advantage over standard multi-electrodes, where all barrels have more or less similar shapes and properties. Multibarrel electrodes were first introduced over 40 years ago 1-3, and have undergone a number of design improvements 2,3 until the piggy-back type was introduced in the 1980s 4,5. Here we present a set of important improvements in the laboratory production of piggy-back electrodes that allow for deep brain penetration in intact in vivo animal preparations due to a relatively thin electrode shaft that causes minimal damage. Furthermore these electrodes are characterized by low noise recordings, and have low resistance drug barrels for very effective iontophoresis of the desired pharmacological agents.

Challenges in Cell-Based Therapies for the Treatment of Hearing Loss

Hearing loss in mammals is an irreversible process caused by degeneration of the hair cells of the inner ear. Current therapies for hearing loss include hearing aids and cochlear implants that provide substantial benefits to most patients, but also have several shortcomings. There is great interest in the development of regenerative therapies to treat deafness in the future. Cell-based therapies, based either on adult, multipotent stem, or other types of pluripotent cells, offer promise for generating differentiated cell types to replace lost or damaged hair cells of the inner ear. In this review, we focus on the methods proposed and avenues for research that seem the most promising for stem cell-based auditory sensory cell regeneration, from work collected over the past 15 years.

A recording chamber for small volume slice electrophysiology.

Electrophysiological recordings from brain slices are typically performed in small recording chambers that allow for the superfusion of the tissue with artificial extracellular solution (ECS), while the chamber holding the tissue is mounted in the optical path of a microscope to image neurons in the tissue. ECS itself is inexpensive, and thus superfusion rates and volumes of ECS consumed during an experiment using standard ECS are not critical. However, some experiments require the addition of expensive pharmacological agents or other chemical compounds to the ECS, creating a need to build superfusion systems that operate on small volumes while still delivering appropriate amounts of oxygen and other nutrients to the tissue. We developed a closed circulation tissue chamber for slice recordings that operates with small volumes of bath solution in the range of 1.0 to 2.6 ml and a constant oxygen/carbon dioxide delivery to the solution in the bath. In our chamber, the ECS is oxygenated and recirculated directly in the recording chamber, eliminating the need for tubes and external bottles/containers to recirculate and bubble ECS and greatly reducing the total ECS volume required for superfusion. At the same time, the efficiency of tissue oxygenation and health of the section are comparable to standard superfusion methods. We also determined that the small volume of ECS contains a sufficient amount of nutrients to support the health of a standard brain slice for several hours without concern for either depletion of nutrients or accumulation of waste products.