Eckhard Friauf

A Shared Vesicular Carrier Allows Synaptic Corelease of GABA and Glycine

The type of vesicular transporter expressed by a neuron is thought to determine its neurotransmitter phenotype. We show that inactivation of the vesicular inhibitory amino acid transporter (Viaat, VGAT) leads to embryonic lethality, an abdominal defect known as omphalocele, and a cleft palate. Loss of Viaat causes a drastic reduction of neurotransmitter release in both GABAergic and glycinergic neurons, indicating that glycinergic neurons do not express a separate vesicular glycine transporter. This loss of GABAergic and glycinergic synaptic transmission does not impair the development of inhibitory synapses or the expression of KCC2, the K+ -Cl- cotransporter known to be essential for the establishment of inhibitory neurotransmission. In the absence of Viaat, GABA-synthesizing enzymes are partially lost from presynaptic terminals. Since GABA and glycine compete for vesicular uptake, these data point to a close association of Viaat with GABA-synthesizing enzymes as a key factor in specifying GABAergic neuronal phenotypes.

Expression and Function of Chloride Transporters during Development of Inhibitory Neurotransmission in the Auditory Brainstem

Glycine and GABA, the dominant inhibitory neurotransmitters in the CNS, assume a depolarizing role in early development, leading to increased cytoplasmic Ca2+ levels and action potentials. The effect is thought to be of some significance for maturation. The depolarization is caused by Cl− efflux, and chloride transporters contribute to the phenomenon by raising the intracellular Cl− concentration ([Cl−]i) above equilibrium, thereby generating an outward-directed electrochemical gradient for Cl−. In mature neurons, the [Cl−]i is reduced below equilibrium, thus rendering glycine activity hyperpolarizing. Here, we investigated the temporal expression of the K-Cl cotransporter KCC2 and the Na-K-Cl cotransporter NKCC1 in the lateral superior olive (LSO) of rats and mice. The two cation cotransporters normally extrude and accumulate Cl−, respectively. As evidenced by several methods, KCC2 mRNA was present in LSO neurons during both the depolarizing and hyperpolarizing periods. Western blots confirmed a constant level of KCC2 in the brainstem, and immunohistochemistry showed that the protein is diffusely distributed within neonatal LSO neurons, becoming integrated into the plasma membrane only with increasing age. The glycine reversal potential in KCC2 knock-out mice differed significantly from that determined in wild-type controls at postnatal day 12 (P12) but not at P3, demonstrating that KCC2 is not active in neonates, despite its early presence. NKCC1 mRNA was not detected during the depolarizing phase in the LSO, implying that this transporter does not contribute to the high [Cl−]i. Our results reveal major differences in the development of [Cl−]i regulation mechanisms seen in brainstem versus forebrain regions.

Development and influence of inhibition in the lateral superior olivary nucleus.

While studies of neuronal development and plasticity have focused on excitatory pathways, the inhibitory projection from the MNTB to the LSO provides a favorable model for studies of synaptic inhibition. This review covers recent studies from our laboratories indicating that inhibitory connections are quite dynamic during development. These findings suggest that there are two phases inhibitory transmission. During an initial depolarizing phase is growth and branching of pre- and postsynaptic elements in the LSO. During a second hyperpolarizing phase there is refinement of inhibitory afferent arborizations and the LSO dendrites that they innervate.

Shift from depolarizing to hyperpolarizing glycine action occurs at different perinatal ages in superior olivary complex nuclei.

The inhibitory transmitters glycine and GABA undergo a developmental shift from depolarizing to hyperpolarizing action (D/H‐shift). To analyse this shift in functionally related nuclei of the rat superior olivary complex (SOC), we employed voltage‐sensitive dye recordings in auditory brainstem slices. Complementarily, we analysed single neurons in gramicidin perforated‐patch recordings. Our results show a differential timing of the D/H‐shift in the four SOC nuclei analysed. In the medial superior olive (MSO), the shift occurred at postnatal day (P) 5–9. In the superior paraolivary nucleus (SPN), it occurred between embryonic day (E) 18 and P1. No D/H‐shift was observed in the medial nucleus of the trapezoid body (MNTB) until P10. This is in line with the finding that most of the patched MNTB neurons displayed glycine‐induced depolarizations between P0–9. While no regional differences regarding the D/H‐shift were found within the MSO, SPN, and MNTB, we observed such differences in the lateral superior olive (LSO). All LSO regions showed a D/H‐shift at P4–5. However, in the high‐frequency regions, hyperpolarizations were large already at P6, yet amplitudes of this size were not present until P8 in the low‐frequency regions, suggesting a delayed development in the latter regions. Our physiological results demonstrate that D/H‐shifts in SOC nuclei are staggered in time and occur over a period of almost two weeks. Membrane‐associated immunoreactivity of the Cl– outward transporter KCC2 was found in every SOC nucleus already at times when glycine was still depolarizing. This implies that the mere presence of KCC2 does not correlate with functional Cl– outward transport.

Localization of rat glycine receptor α1 and α2 subunit transcripts in the developing auditory brainstem

Glycine receptors (GlyRs), aside from GABAA receptors, mediate fast postsynaptic inhibition in the mammalian nervous system. Spatial and temporal expression of the genes encoding ligand‐binding α subunits (α1–α4) and the structural β subunit leads to the formation of various GlyR isoforms. Currently, the idea of the GlyRs containing the “adult” α1 subunit replacing those with the “neonatal” α2 subunit during early postnatal development predominates. Here, we describe the patterns of expression of the GlyR α1 and α2 subunit genes in the rat auditory brainstem between postnatal day (P) 0 and P20, by using both nonradioactive and radioactive in situ hybridization. We show that the α1 subunit mRNA appears throughout the auditory brainstem during the first 8 postnatal days, which resembles the time of onset described within the spinal cord. In the rostral auditory nuclei (nuclei of the lateral lemniscus and inferior colliculus), the α1 subunit transcript appears later (P8) than in the caudal nuclei (cochlear nuclear complex and superior olivary complex; P0). Surprisingly, we found that low levels of the α2 subunit transcript are present in the auditory brainstem at birth and persist throughout the period analyzed. However, α2 subunit mRNA is present at high levels in other neonatal brainstem structures, such as cranial motor nuclei. Therefore, we conclude that the changes in GlyR composition in the auditory brainstem deviate from the classic α2 to α1 subunit switch observed in spinal cord. Our data suggest that genes for other GlyR subunits (e.g., α3 and α4) may be expressed during early development in the auditory brainstem. J. Comp. Neurol. 438:336–352, 2001.

Cav1.3 Calcium Channels Are Required for Normal Development of the Auditory Brainstem

Within the Cav1 family of voltage-gated calcium channels, Cav1.2 and Cav1.3 channels are the predominant subtypes in the brain. Whereas specific functions for each subtype were described in the adult brain, their role in brain development is poorly understood. Here we assess the role of Cav1.3 subunits in the activity-dependent development of the auditory brainstem. We used Cav1.3-deficient (Cav1.3−/−) mice because these mice lack cochlea-driven activity that deprives the auditory centers from peripheral input. We found a drastically reduced volume in all auditory brainstem centers (range 25–59%, total 35%), which was manifest before hearing onset. A reduction was not obvious outside the auditory system. The lateral superior olive (LSO) was strikingly malformed in Cav1.3−/− mice and had fewer neurons (1/3 less). The remaining LSO neurons displayed normal dendritic trees and received functional glutamatergic input, yet they fired action potentials predominantly with a multiple pattern upon depolarization, in contrast to the single firing pattern prevalent in controls. The latter finding appears to be due to a reduction of dendrototoxin-sensitive potassium conductances, presumably mediated through the Kv1.2 subtype. Fura2 imaging provided evidence for functional Cav1.3 channels in the LSO of wild-type mice. Our results imply that Cav1.3 channels are indispensable for the development of the central auditory system. We propose that the unique LSO phenotype in Cav1.3−/− mice, which hitherto was not described in other hereditary deafness models, is caused by the synergistic contribution of two factors: on-site loss of Cav1.3 channels in the neurons plus lack of peripheral input.

Synaptic Refinement of an Inhibitory Topographic Map in the Auditory Brainstem Requires Functional CaV1.3 Calcium Channels

Synaptic refinement via the elimination of inappropriate synapses and strengthening of appropriate ones is crucially important for the establishment of specific, topographic neural circuits. The mechanisms driving these processes are poorly understood, particularly concerning inhibitory projections. Here, we address the refinement of an inhibitory topographic projection in the auditory brainstem in functional and anatomical mapping studies involving patch-clamp recordings in combination with minimal and maximal stimulation, caged glutamate photolysis, and single axon tracing. We demonstrate a crucial dependency of the refinement on CaV1.3 calcium channels: CaV1.3−/− mice displayed virtually no elimination of projections up to hearing onset. Furthermore, strengthening was strongly impaired, in line with a reduced number of axonal boutons. The mediolateral topography was less precise and the shift from a mixed GABA/glycinergic to a purely glycinergic transmission before hearing onset did not occur. Together, our findings provide evidence for a CaV1.3-dependent mechanism through which both inhibitory circuit formation and determination of the neurotransmitter phenotype are achieved.

ADF/Cofilin Controls Synaptic Actin Dynamics and Regulates Synaptic Vesicle Mobilization and Exocytosis

Actin is a regulator of synaptic vesicle mobilization and exocytosis, but little is known about the mechanisms that regulate actin at presynaptic terminals. Genetic data on LIMK1, a negative regulator of actin-depolymerizing proteins of the ADF/cofilin family, suggest a role for ADF/cofilin in presynaptic function. However, synapse physiology is fully preserved upon genetic ablation of ADF in mice, and n-cofilin mutant mice display defects in postsynaptic plasticity, but not in presynaptic function. One explanation for this phenomenon is overlapping functions of ADF and n-cofilin in presynaptic physiology. Here, we tested this hypothesis and genetically removed ADF together with n-cofilin from synapses. In double mutants for ADF and n-cofilin, synaptic actin dynamics was impaired and more severely affected than in single mutants. The resulting cytoskeletal defects heavily affected the organization, mobilization, and exocytosis of synaptic vesicles in hippocampal CA3-CA1 synapses. Our data for the first time identify overlapping functions for ADF and n-cofilin in presynaptic physiology and vesicle trafficking. We conclude that n-cofilin is a limiting factor in postsynaptic plasticity, a function which cannot be substituted by ADF. On the presynaptic side, the presence of either ADF or n-cofilin is sufficient to control actin remodeling during vesicle release.

Retrocochlear function of the peripheral deafness gene Cacna1d

Hearing impairment represents the most common sensory deficit in humans. Genetic mutations contribute significantly to this disorder. Mostly, only malfunction of the ear is considered. Here, we assessed the role of the peripheral deafness gene Cacna1d, encoding the L-type channel Ca(v)1.3, in downstream processing of acoustic information. To this end, we generated a mouse conditional Cacna1d-eGFP(flex) allele. Upon pairing with Egr2::Cre mice, Ca(v)1.3 was ablated in the auditory brainstem, leaving the inner ear intact. Structural assessment of the superior olivary complex (SOC), an essential auditory brainstem center, revealed a dramatic volume reduction (43-47%) of major nuclei in young adult Egr2::Cre;Cacna1d-eGFP(flex) mice. This volume decline was mainly caused by a reduced cell number (decline by 46-56%). Abnormal formation of the lateral superior olive was already present at P4, demonstrating an essential perinatal role of Ca(v)1.3 in the SOC. Measurements of auditory brainstem responses demonstrated a decreased amplitude in the auditory nerve between 50 and 75 dB stimulation in Egr2::Cre;Cacna1d-eGFP(flex) knockout mice and increased amplitudes in central auditory processing centers. Immunohistochemical studies linked the amplitude changes in the central auditory system to reduced expression of K(v)1.2. No changes were observed for K(v)1.1, KCC2, a determinant of inhibitory neurotransmission, and choline acetyltransferase, a marker of efferent olivocochlear neurons. Together, these analyses identify a crucial retrocochlear role of Ca(v)1.3 and demonstrate that mutations in deafness genes can affect sensory cells and neurons alike. As a corollary, hearing aids have to address central auditory processing deficits as well.

Attention-Deficit/Hyperactivity Disorder-like Phenotype in a Mouse Model with Impaired Actin Dynamics.

BACKGROUND Actin depolymerizing proteins of the actin depolymerizing factor (ADF)/cofilin family are essential for actin dynamics, which is critical for synaptic function. Two ADF/cofilin family members, ADF and n-cofilin, are highly abundant in the brain, where they are present in excitatory synapses. Previous studies demonstrated the relevance of n-cofilin for postsynaptic plasticity, associative learning, and anxiety. These studies also suggested overlapping functions for ADF and n-cofilin. METHODS We performed pharmacobehavioral, electrophysiologic, and electron microscopic studies on ADF and n-cofilin single mutants and double mutants (named ACC mice) to characterize the importance of ADF/cofilin activity for synapse physiology and mouse behavior. RESULTS The ACC mice, but not single mutants, exhibited hyperlocomotion, impulsivity, and impaired working memory. Hyperlocomotion and impulsive behavior were reversed by methylphenidate, a psychostimulant commonly used for the treatment of attention-deficit/hyperactivity disorder (ADHD). Also, ACC mice displayed a disturbed morphology of striatal excitatory synapses, accompanied by strongly increased glutamate release. Blockade of dopamine or glutamate transmission resulted in normal locomotion. CONCLUSIONS Our study reveals that ADHD can result from a disturbed balance between excitation and inhibition in striatal circuits, providing novel insights into the mechanisms underlying this neurobehavioral disorder. Our results link actin dynamics to ADHD, suggesting that mutations in actin regulatory proteins may contribute to the etiology of ADHD in humans.