Benedikt Grothe

Functional properties of neurons derived from in vitro reprogrammed postnatal astroglia

With the exception of astroglia-like cells in the neurogenic niches of the telencephalic subependymal or hippocampal subgranular zone, astroglia in all other regions of the adult mouse brain do not normally generate neurons. Previous studies have shown, however, that early postnatal cortical astroglia in culture can be reprogrammed to adopt a neuronal fate after forced expression of Pax6, a transcription factor (TF) required for proper neuronal specification during embryonic corticogenesis. Here we show that also the proneural genes neurogenin-2 and Mash1 (mammalian achaete schute homolog 1) possess the ability to reprogram astroglial cells from early postnatal cerebral cortex. By means of time-lapse imaging of green fluorescent astroglia, we provide direct evidence that it is indeed cells with astroglial characteristics that give rise to neurons. Using patch-clamp recordings in culture, we show that astroglia-derived neurons acquire active conductances and are capable of firing action potentials, thus displaying hallmarks of true neurons. However, independent of the TF used for reprogramming, astroglia-derived neurons appear to mature more slowly compared with embryonic-born neurons and fail to generate a functional presynaptic output within the culturing period. However, when cocultured with embryonic cortical neurons, astroglia-derived neurons receive synaptic input, demonstrating that they are competent of establishing a functional postsynaptic compartment. Our data demonstrate that single TFs are capable of inducing a remarkable functional reprogramming of astroglia toward a truly neuronal identity.

The functional role of GABA and glycine in monaural and binaural processing in the inferior colliculus of horseshoe bats

SummaryThe functional role of GABA and glycine in monaural and binaural signal analysis was studied in single unit recordings from the central nucleus of the inferior colliculus (IC) of horseshoe bats (Rhinolophus rouxi) employing microiontophoresis of the putative neurotransmitters and their antagonists bicuculline and strychnine.Most neurons were inhibited by GABA (98%; N=107) and glycine (92%; N=118). Both neurotransmitters appear involved in several functional contexts, but to different degrees.Bicuculline-induced increases of discharge activity (99% of cells; N=191) were accompanied by changes of temporal response patterns in 35% of neurons distributed throughout the IC. Strychnine enhanced activity in only 53% of neurons (N=147); cells exhibiting response pattern changes were rare (9%) and confined to greater recording depths. In individual cells, the effects of both antagonists could markedly differ, suggesting a differential supply by GABAergic and glycinergic networks.Bicuculline changed the shape of the excitatory tuning curve by antagonizing lateral inhibition at neighboring frequencies and/or inhibition at high stimulation levels. Such effects were rarely observed with strychnine.Binaural response properties of single units were influenced either by antagonization of inhibition mediated by ipsilateral stimulation (bicuculline) or by changing the strength of the main excitatory input (bicuculline and strychnine).

Interaural Time Difference Processing in the Mammalian Medial Superior Olive: The Role of Glycinergic Inhibition

The dominant cue for localization of low-frequency sounds are microsecond differences in the time-of-arrival of sounds at the two ears [interaural time difference (ITD)]. In mammals, ITD sensitivity is established in the medial superior olive (MSO) by coincidence detection of excitatory inputs from both ears. Hence the relative delay of the binaural inputs is crucial for adjusting ITD sensitivity in MSO cells. How these delays are constructed is, however, still unknown. Specifically, the question of whether inhibitory inputs are involved in timing the net excitation in MSO cells, and if so how, is controversial. These inhibitory inputs derive from the nuclei of the trapezoid body, which have physiological and structural specializations for high-fidelity temporal transmission, raising the possibility that well timed inhibition is involved in tuning ITD sensitivity. Here, we present physiological and pharmacological data from in vivo extracellular MSO recordings in anesthetized gerbils. Reversible blockade of synaptic inhibition by iontophoretic application of the glycine antagonist strychnine increased firing rates and significantly shifted ITD sensitivity of MSO neurons. This indicates that glycinergic inhibition plays a major role in tuning the delays of binaural excitation. We also tonically applied glycine, which lowered firing rates but also shifted ITD sensitivity in a way analogous to strychnine. Hence tonic glycine application experimentally decoupled the effect of inhibition from the timing of its inputs. We conclude that, for proper ITD processing, not only is inhibition necessary, but it must also be precisely timed.

Maturation of glycinergic inhibition in the gerbil medial superior olive after hearing onset

The neurones of the medial superior olive (MSO) are the most temporally sensitive neurones in the brain. They respond to the arrival time difference of sound at the two ears with a microsecond resolution; these interaural time differences are used to localize low‐frequency sounds. In addition to the excitatory inputs from each ear, the MSO neurones also receive binaural glycinergic projections, which have a critical role in sound localization processing. Recently, it was shown that the glycinergic input to the MSO undergoes an experience‐dependent structural reorganization after hearing onset. To explore the maturation of inhibition during the development of sound localization on a cellular level, glycinergic currents and potentials were measured in gerbil MSO principal cells from postnatal (P) day P12–P25 by whole‐cell patch‐clamp recordings. The synaptic glycinergic currents accelerated to rapid decay kinetics (∼2 ms) and rise times (∼0.4 ms) after hearing onset, reaching maturity around P17. Since the kinetics of miniature glycinergic currents did not change with age, it is likely that a higher degree of transmitter release synchrony is the underlying mechanism influencing the acceleration of the kinetics. During the same period, the synaptic glycinergic potentials accelerated four‐fold, largely as a result of a prominent decrease in input resistance. In accordance with a reorganization of the glycinergic inputs, the evoked peak conductances decreased more than two‐fold, together with a three‐fold reduction in the frequency of miniature events after hearing onset. These age‐dependent changes were absent in animals that had been reared in omni‐directional noise, indicating that an experience‐dependent pruning of synaptic inputs is important for the maturation of functional inhibition in the MSO. Taken together, these striking developmental adjustments of the glycinergic inhibition in the MSO most probably reflect an adaptation to improve the encoding of auditory cues with great temporal precision and fidelity during the maturation of sound localization behaviour.

Medial Superior Olivary Neurons Receive Surprisingly Few Excitatory and Inhibitory Inputs with Balanced Strength and Short-Term Dynamics

Neurons in the medial superior olive (MSO) process microsecond interaural time differences, the major cue for localizing low-frequency sounds, by comparing the relative arrival time of binaural, glutamatergic excitatory inputs. This coincidence detection mechanism is additionally shaped by highly specialized glycinergic inhibition. Traditionally, it is assumed that the binaural inputs are conveyed by many independent fibers, but such an anatomical arrangement may decrease temporal precision. Short-term depression on the other hand might enhance temporal fidelity during ongoing activity. For the first time we show that binaural coincidence detection in MSO neurons may require surprisingly few but strong inputs, challenging long-held assumptions about mammalian coincidence detection. This study exclusively uses adult gerbils for in vitro electrophysiology, single-cell electroporation and immunohistochemistry to characterize the size and short-term plasticity of inputs to the MSO. We find that the excitatory and inhibitory inputs to the MSO are well balanced both in strength and short-term dynamics, redefining this fastest of all mammalian coincidence detector circuits.

Retrograde GABA Signaling Adjusts Sound Localization by Balancing Excitation and Inhibition in the Brainstem

Central processing of acoustic cues is critically dependent on the balance between excitation and inhibition. This balance is particularly important for auditory neurons in the lateral superior olive, because these compare excitatory inputs from one ear and inhibitory inputs from the other ear to compute sound source location. By applying GABA(B) receptor antagonists during sound stimulation in vivo, it was revealed that these neurons adjust their binaural sensitivity through GABA(B) receptors. Using an in vitro approach, we then demonstrate that these neurons release GABA during spiking activity. Consequently, GABA differentially regulates transmitter release from the excitatory and inhibitory terminals via feedback to presynaptic GABA(B) receptors. Modulation of the synaptic input strength, by putative retrograde release of neurotransmitter, may enable these auditory neurons to rapidly adjust the balance between excitation and inhibition, and thus their binaural sensitivity, which could play an important role as an adaptation to various listening situations.

Tuning of Ranvier node and internode properties in myelinated axons to adjust action potential timing

Action potential timing is fundamental to information processing; however, its determinants are not fully understood. Here we report unexpected structural specializations in the Ranvier nodes and internodes of auditory brainstem axons involved in sound localization. Myelination properties deviated significantly from the traditionally assumed structure. Axons responding best to low-frequency sounds had a larger diameter than high-frequency axons but, surprisingly, shorter internodes. Simulations predicted that this geometry helps to adjust the conduction velocity and timing of action potentials within the circuit. Electrophysiological recordings in vitro and in vivo confirmed higher conduction velocities in low-frequency axons. Moreover, internode length decreased and Ranvier node diameter increased progressively along the distal axon segments, which simulations show was essential to ensure precisely timed depolarization of the giant calyx of Held presynaptic terminal. Thus, individual anatomical parameters of myelinated axons can be tuned to optimize pathways involved in temporal processing.

Inhibiting the Inhibition: A Neuronal Network for Sound Localization in Reverberant Environments

The precedence effect describes the phenomenon whereby echoes are spatially fused to the location of an initial sound by selectively suppressing the directional information of lagging sounds (echo suppression). Echo suppression is a prerequisite for faithful sound localization in natural environments but can break down depending on the behavioral context. To date, the neural mechanisms that suppress echo directional information without suppressing the perception of echoes themselves are not understood. We performed in vivo recordings in Mongolian gerbils of neurons of the dorsal nucleus of the lateral lemniscus (DNLL), a GABAergic brainstem nucleus that targets the auditory midbrain, and show that these DNLL neurons exhibit inhibition that persists tens of milliseconds beyond the stimulus offset, so-called persistent inhibition (PI). Using in vitro recordings, we demonstrate that PI stems from GABAergic projections from the opposite DNLL. Furthermore, these recordings show that PI is attributable to intrinsic features of this GABAergic innervation. Implementation of these physiological findings into a neuronal model of the auditory brainstem demonstrates that, on a circuit level, PI creates an enhancement of responsiveness to lagging sounds in auditory midbrain cells. Moreover, the model revealed that such response enhancement is a sufficient cue for an ideal observer to identify echoes and to exhibit echo suppression, which agrees closely with the percepts of human subjects.

The long adventurous journey of rhombic lip cells in jawed vertebrates: a comparative developmental analysis.

This review summarizes vertebrate rhombic lip and early cerebellar development covering classic approaches up to modern developmental genetics which identifies the relevant differential gene expression domains and their progeny. Most of this information is derived from amniotes. However, progress in anamniotes, particularly in the zebrafish, has recently been made. The current picture suggests that rhombic lip and cerebellar development in jawed vertebrates (gnathostomes) share many characteristics. Regarding cerebellar development, these include a ptf1a expressing ventral cerebellar proliferation (VCP) giving rise to Purkinje cells and other inhibitory cerebellar cell types, and an atoh1 expressing upper rhombic lip giving rise to an external granular layer (EGL, i.e., excitatory granule cells) and an early ventral migration into the anterior rhombencephalon (cholinergic nuclei). As for the lower rhombic lip (LRL), gnathostome commonalities likely include the formation of precerebellar nuclei (mossy fiber origins) and partially primary auditory nuclei (likely convergently evolved) from the atoh1 expressing dorsal zone. The fate of the ptf1a expressing ventral LRL zone which gives rise to (excitatory cells of) the inferior olive (climbing fiber origin) and (inhibitory cells of ) cochlear nuclei in amniotes, has not been determined in anamniotes. Special for the zebrafish in comparison to amniotes is the predominant origin of anamniote excitatory deep cerebellar nuclei homologs (i.e., eurydendroid cells) from ptf1a expressing VCP cells, the sequential activity of various atoh1 paralogs and the incomplete coverage of the subpial cerebellar plate with proliferative EGL cells. Nevertheless, the conclusion that a rhombic lip and its major derivatives evolved with gnathostome vertebrates only and are thus not an ancestral craniate character complex is supported by the absence of a cerebellum (and likely absence of its afferent and efferent nuclei) in jawless fishes

Experience-Dependent Refinement of the Inhibitory Axons Projecting to the Medial Superior Olive

Neurons in the medial superior olive (MSO) analyze interaural time differences (ITDs) by comparing the arrival times of the two excitatory inputs from each ear using a coincidence detection mechanism. They also receive a prominent inhibitory, glycinergic projection from the ipsilateral medial nucleus of the trapezoid body (MNTB), which contributes to the fine‐tuning of ITD analysis. Here, we investigated developmental changes of the axonal arborisation pattern of single Microruby‐labeled MNTB neurons projecting to the MSO region. During the first 2 weeks after hearing onset, the axonal arborisation of MNTB neurons was significantly refined resulting in a narrowed projection area across the tonotopic axis of the MSO and a redistribution of the axonal endsegments to a mostly somatic location. Rearing the animals in omnidirectional noise prevented the structural changes of single MNTB projections. These results indicate that the functional elimination of inhibitory inputs on MSO neurons after hearing onset, as described previously, is paralleled by a structural, site‐specific refinement of the inputs and is dependent on the normal acoustic experience of the animal.