Detlev Grabs

The SH3 Domain of Amphiphysin Binds the Proline-rich Domain of Dynamin at a Single Site That Defines a New SH3 Binding Consensus Sequence

Amphiphysin is an SH3 domain-containing neuronal protein that is highly concentrated in nerve terminals where it interacts via its SH3 domain with dynamin I, a GTPase implicated in synaptic vesicle endocytosis. We show here that the SH3 domain of amphiphysin, but not a mutant SH3 domain, bound with high affinity to a single site in the long proline-rich region of human dynamin I, that this site was distinct from the binding sites for other SH3 domains, and that the mutation of two adjacent amino acids in dynamin I was sufficient to abolish binding. The dynamin I sequence critically required for amphiphysin binding (PSRPNR) fits in the novel SH3 binding consensus identified for the SH3 domain of amphiphysin via a combinatorial peptide library approach: PXRPXR(H)R(H). Our data demonstrate that the long proline-rich stretch present in dynamin I contained multiple SH3 domain binding sites that recognize interacting proteins with high specificity.

Differential Expression of Synaptophysin and Synaptoporin During Pre- and Postnatal Development of the Rat Hippocampal Network

The closely related synaptic vesicle membrane proteins synaptophysin and synaptoporin are abundant in the hippocampal formation of the adult rat. But the prenatal hippocampal formation contains only synaptophysin, which is first detected at embryonic day 17 (E17) in perikarya and axons of the pyramidal neurons. At E21 synaptophysin immunoreactivity extends into the apical dendrites of these cells and in newly formed terminals contacting these dendrites. The transient presence of synaptophysin in axons and dendrites suggests a functional involvement of synaptophysin in fibre outgrowth of developing pyramidal neurons. Synaptoporin expression parallels the formation of dentate granule cell synaptic contacts with pyramidal neurons: the amount of hippocampal synaptoporin, determined in immunoblots and by synaptoporin immunostaining of developing mossy fibre terminals, increases during the first postnatal week. Moreover, in the adult, synaptoporin is found exclusively in the mossy fibre terminals present in the hilar region of the dentate gyrus and the regio inferior of the cornu ammonis. In contrast, synaptophysin is present in all synaptic fields of the hippocampal formation, including the mossy fibre terminals, where it colocalizes with synaptoporin in the same boutons. Our data indicate that granule neuron terminals differ from all other terminals of the hippocampal formation by the presence of both synaptoporin and synaptophysin. This difference, observed in the earliest synaptic contacts in the postnatal hippocampus and persisting into adult life, suggests distinct functions of synaptoporin in these nerve terminals.

Rab3 Proteins and SNAP‐25, Essential Components of the Exocytosis Machinery in Conventional Synapses, are Absent from Ribbon Synapses of the Mouse Retina

GTP‐binding rab proteins, present in synaptic vesicles and endocrine secretory granules, have been shown to be involved in the control of regulated exocytosis. We found rab3 proteins in immunoblots of diverse areas of the mouse central nervous system (spinal cord, olfactory bulb, hippocampus, cerebellum and neocortex). Immunohistochemical observations at light‐ and electron‐microscopical levels in the hippocampus and other areas revealed rab3 proteins in virtually all synaptic fields and terminals of the areas investigated. In the retina, rab3A immunoreactivity was confined to the inner and outer plexiform layers. Ultrastructural examination revealed that rab3A was present in conventional terminals in the inner plexiform layer and in horizontal cell processes of the outer plexiform layer. In contrast ribbon synapses, which play a key role in transferring information from the photoreceptor cells to the central nervous system, were immunonegative. We also tested whether other proteins of the rab3 family are present in ribbon synapses. However, using an antibody recognizing rab3B and rab3C in addition to rab3A, we found no immunoreactivity in these synapses. Interestingly, we observed also no immunoreactivity for synaptosomal‐associated protein 25 (SNAP‐25) in ribbon synapses, but conventional synapses and horizontal cell processes were heavily stained. Our data show that the known rab3 and SNAP‐25 isoforms, which are components of the secretory apparatus of conventional synapses, are absent from ribbon synapses of the retina. Our observations suggest different mechanisms of transmitter exocytosis in conventional and ribbon terminals.

Synaptophysin and synaptoporin expression in the developing rat olfactory system

The expressions of two closely related synaptic vesicle antigens synaptophysin and synaptoporin were examined in the olfactory system of the adult rat and during pre- and postnatal development. In the adult, immunocytochemistry showed that the continuously regenerating olfactory receptor neurons (primary neurons) produce both synaptophysin and synaptoporin which were localized in the cell bodies of the receptor neurons in the olfactory epithelium, their dendrites, axonal processes in the olfactory nerve and their terminals in the olfactory bulb glomeruli. Furthermore, ultrastructural analysis revealed synaptophysin- and synaptoporin-immunoreactivities associated with synaptic vesicles in most olfactory receptor axonal terminals impinging on dendrites of the mitral and tufted neurons (secondary neurons in the olfactory bulb circuitry) in the olfactory glomeruli. In like manner, tufted neurons, granule and periglomerular neurons (interneurons in the olfactory bulb circuitry) express both synaptophysin and synaptoporin. In contrast, mitral neurons expressed only the synaptophysin antigen which was likewise associated with mitral axonal terminals in their target the olfactory cortex. The patterns of synaptophysin and synaptoporin expressions in mitral neurons (synaptophysin only) and tufted neurons (synaptophysin and synaptoporin) were similar in prenatal, postnatal and adult rats as revealed by immunocytochemistry and in situ hybridization. However, the biosynthesis of synaptophysin and synaptoporin by granule and periglomerular neurons, olfactory bulb interneurons, occurred mainly postnatally.

Selective ultrasound guided pectoral nerve targeting in breast augmentation: How to spare the brachial plexus cords?

Subpectoral breast augmentation surgery under regional anesthesia requires the selective neural blockade of the medial and lateral pectoral nerves to diminish postoperative pain syndromes. The purpose of this cadaver study is to demonstrate a reliable ultrasound guided approach to selectively target the pectoral nerves and their branches while sparing the brachial plexus cords. After evaluating the position and appearance of the pectoral nerves in 25 cadavers (50 sides), a portable ultrasound machine was used to guide the injection of 10 ml of 0.2% aqueous methylene blue solution in the pectoral region on both sides of three Thiels embalmed cadavers using a single entry point—triple injection technique. This technique uses a medial to lateral approach with the entry point just medial to the pectoral minor muscle and three subsequent infiltrations: (1) deep lateral part of the pectoralis minor muscle, (2) between the pectoralis minor and major muscles, and (3) between the pectoralis major muscle and its posterior fascia under ultrasound visualization. Dissection demonstrates that the medial and lateral pectoral nerves were well stained while leaving the brachial plexus cords unstained. We show that 10 ml of an injected solution is sufficient to stain all the medial and lateral pectoral nerve branches without a proximal extension to the cords of the brachial plexus. Clin. Anat. 26:49–55, 2013.

Expression of presynaptic proteins is closely correlated with the chronotopic pattern of axons in the retinotectal system of the chick

Newly synthesized presynaptic integral membrane proteins in neurons are transported in precursor vesicles from the site of protein biosynthesis in the cell body by fast axonal flow to the presynaptic terminal. We followed the path that presynaptic proteins travel on the way to their central targets of the highly ordered primary visual pathway of the chick and analyzed the developmental changes in the expression of synaptic vesicle protein 2 (SV2), synaptotagmin, and syntaxin. Immunofluorescences revealed that: (1) the onset of protein expression in the retinal ganglion cells occurs in a central to peripheral developmental pattern from embryonic day 4 (E4) onward; (2) the proteins were found first in the inner and later in the outer plexiform layer of the retina; and (3) they were redistributed from the photoreceptor inner segments and cell bodies to the terminals in the outer plexiform layer. From E4 onward, immunopositive axons for SV2, synaptotagmin, and syntaxin were found in the optic nerve, disappearing after E9 for SV2 and synaptotagmin. The optic tract was stained for SV2 and synaptotagmin between E7 and E12, for syntaxin until the posthatching period. Finally, immunoreactivities for the investigated proteins were present at the surface of the tectum from E8 onward, when first retinal axons arrived there. The present study revealed that SV2 and synaptotagmin, but not syntaxin, are expressed in a transient wave that follows the advancement of optic axons and the proteins towards the optic tectum. J. Comp. Neurol. 418:361–372, 2000.

Developmental Expression of Dynamin in the Chick Retinotectal System

Dynamin I, a GTPase involved in the endocytic cycle of synaptic vesicle membranes, is believed to support axonal outgrowth and/or synaptogenesis. To explore the temporal and spatial patterns of dynamin I distribution in neuronal morphogenesis, we compared the developmental expression of dynamin with the expression of presynaptic membrane proteins such as SV2, synaptotagmin, and syntaxin in the chick primary visual pathway. Western blots of retina and tectum revealed a steady increase of synaptotagmin and syntaxin from embryonic Day 7 (E7) to E11, whereas for the same time frame no detectable increase of dynamin was found. Later stages showed increasing amounts of all tested proteins until the first postnatal week. Immunofluorescence revealed that SV2, synaptotagmin, and syntaxin are present in retinal ganglion cell axons from E4 on. In later stages, the staining pattern in the retina and along the visual pathway paralleled the formation and maturation of axons. In contrast, dynamin is not detectable by immunofluorescence in the developing retina and optic tectum before synapse formation. Our data indicate that, in contrast to the early expression of synaptotagmin, SV2, and syntaxin during axonal growth, dynamin is upregulated after synapse formation, suggesting its function predominantly during and after synaptogenesis but not in axonogenesis.

Expression of neuroserpin in the visual cortex of the mouse during the developmental critical period

The neuronal serine protease inhibitor neuroserpin is widely expressed in the developing and adult brain. In the neocortex, neuroserpin is displayed particularly during the period of synaptic specification and refinement, indicating a role as modulator of extracellular proteolytic processes. The synaptic connections of the visual system of the mouse are shaped during early postnatal life by an activity‐dependent process. We have studied the expression of the neuronal serine protease inhibitor neuroserpin in the primary visual cortex of mice from birth until the end of the critical period by means of reverse transcription polymerase chain reaction and in situ hybridization. The localization and the level of expression were constant throughout this period. Monocular deprivation with an eyelid sutured induced a decrease in neuroserpin expression in neurons of area 17 after 1 week of deprivation, the decrease being more pronounced on the side contralateral to the closed eye. The expression of neuroserpin in the visual cortex during the critical period and its decrease in parallel to the refinement of synaptic contacts after visual deprivation suggests a regulative role of neuroserpin on these processes.

Differential expression of neuronal calcium sensor-1 in the developing chick retina

Neuronal calcium sensor‐1 (NCS‐1) is a Ca2+ binding protein that has been implicated in the regulation of neurotransmission and synaptogenesis. In this study we investigated the developmental expression and localization of NCS‐1 in the chick retina. Single‐ and double‐labeling experiments with three‐dimensional reconstruction as well as ultrastructural data of the distribution of NCS‐1 suggest that this protein is also involved in axonal process outgrowth. We found an early expression of NCS‐1 in ganglion cells and their axons, in amacrine, and in horizontal cells, whereas photoreceptors were immunonegative at embryonic stages. In the early posthatching days we found strong immunostaining for NCS‐1 in horizontal cells and their processes in the outer plexiform layer. In contrast, synaptic vesicle protein 2 (SV2) was prominent only in photoreceptor synaptic terminals. Ultrastructural analysis confirmed that NCS‐1 was localized postsynaptically in horizontal cell processes, whereas presynaptic terminals were immunonegative. However, at late posthatching days we observed that photoreceptor ribbon synapses (from rods and/or cones) also expressed NCS‐1. Thus the results support the notion that NCS‐1 is involved in neuronal process outgrowth and is localized in pre‐ and postsynaptic compartments including mature photoreceptor synapses. J. Comp. Neurol. 449:231–240, 2002.

Calretinin regulates Ca2+-dependent inactivation and facilitation of Ca(v)2.1 Ca2+ channels through a direct interaction with the α12.1 subunit.

Background: Ca2+-dependent inactivation and facilitation of Cav2.1 Ca2+ channels are major determinants of neuronal excitability and synaptic plasticity. Results: The Ca2+-binding protein calretinin interacts with Cav2.1 and inhibits Ca2+-dependent inactivation and enhances facilitation of Cav2.1. Conclusion: In addition to its role as a diffusible Ca2+ buffer, calretinin can interact with targets such as Cav2.1 and modulate their function. Significance: Calretinin-Cav2.1 interactions may shape Ca2+ signaling dynamics in neurons. Voltage-gated Cav2.1 Ca2+ channels undergo dual modulation by Ca2+, Ca2+-dependent inactivation (CDI), and Ca2+-dependent facilitation (CDF), which can influence synaptic plasticity in the nervous system. Although the molecular determinants controlling CDI and CDF have been the focus of intense research, little is known about the factors regulating these processes in neurons. Here, we show that calretinin (CR), a Ca2+-binding protein highly expressed in subpopulations of neurons in the brain, inhibits CDI and enhances CDF by binding directly to α12.1. Screening of a phage display library with CR as bait revealed a highly basic CR-binding domain (CRB) present in multiple copies in the cytoplasmic linker between domains II and III of α12.1. In pulldown assays, CR binding to fusion proteins containing these CRBs was largely Ca2+-dependent. α12.1 coimmunoprecipitated with CR antibodies from transfected cells and mouse cerebellum, which confirmed the existence of CR-Cav2.1 complexes in vitro and in vivo. In HEK293T cells, CR significantly decreased Cav2.1 CDI and increased CDF. CR binding to α12.1 was required for these effects, because they were not observed upon substitution of the II-III linker of α12.1 with that from the Cav1.2 α1 subunit (α11.2), which lacks the CRBs. In addition, coexpression of a protein containing the CRBs blocked the modulatory action of CR, most likely by competing with CR for interactions with α12.1. Our findings highlight an unexpected role for CR in directly modulating effectors such as Cav2.1, which may have major consequences for Ca2+ signaling and neuronal excitability.