Christian Henneberger

RNA editing produces glycine receptor |[alpha]|3P185L, resulting in high agonist potency

The function of supramedullary glycine receptors (GlyRs) is still unclear. Using Wistar rat collicular slices, we demonstrate GlyR-mediated inhibition of spike discharge elicited by low glycine (10 μM). Searching for the molecular basis of this phenomenon, we identified a new GlyR isoform. GlyRα3P185L, a result of cytidine 554 deamination, confers high glycine sensitivity (EC50 ∼5 μM) to neurons and thereby promotes the generation of sustained chloride conductances associated with tonic inhibition. The level of GlyRα3-C554U RNA editing is sensitive to experimentally induced brain lesion, inhibition of cytidine deamination by zebularine and inhibition of mRNA transcription by actinomycin D, but not to blockade of protein synthesis by cycloheximide. Conditional regulation of GlyRα3P185L is thus likely to be part of a post-transcriptional adaptive mechanism in neurons with enhanced excitability.

A 29-amino acid fragment of Clostridium botulinum C3 protein enhances neuronal outgrowth, connectivity, and reinnervation

Small GTPases of the Rho family play versatile roles in the formation and development of axons and dendrites, effects often studied by the Rho‐inactivating C3 transferase (C3bot) from Clostridium botulinum. Recently, we reported that transferasedeficient C3bot also exerted axonotrophic activity. Using overlapping peptides from the C3bot sequence, we identified a small peptide of 29 amino acids (covering residues 154‐182) from the C‐terminal region of C3bot that promotes both axonal and dendritic growth, as well as branching of hippocampal neurons, at sub‐micromolar concentrations. Several C3bot constructs, including the short peptide, enhanced the number of axonal segments from mid‐ to higher‐order segments. C3bot154‐182 also increased the number of synaptophysin‐expressing terminals, up‐regulated various synaptic proteins, and functionally increased the glutamate uptake. Staining against the vesicular glutamate and GABA transporters further revealed that the effect was attributable to a higher number of glutamatergic and GABAergic inputs on proximal dendrites of enhanced green fluorescent protein (EGFP)‐transfected neurons. Using organotypical slice cultures, we also detected trophic effects of C3bot154‐182 on length and density of outgrowing fibers from the entorhinal cortex that were comparable to the effects elicited by full‐length C3bot. In addition, an enhanced reinnervation was observed in a hippocampal‐entorhinal lesion model. In summary, the neurotrophic effect of C3bot is executed by a C‐terminal peptide fragment covering aa 154‐182 of C3; it triggers dendritic and axonal growth and branching as well as increased synaptic connectivity. In contrast to full‐length C3, this C3 peptide selectively acts on neurons but not on glial cells. Holtje, M., Djalali, S., Hofmann, F., Munster‐Wandowski, A., Hendrix, S., Boato, F., Dreger, S. C., Große, G., Henneberger, C., Grantyn, R., Just, I., Ahnert‐Hilger, G. A 29‐amino acid fragment of Clostridium botulinum C3 protein enhances neuronal outgrowth, connectivity, and reinnervation. FASEB J. 23, 1115–1126 (2009)

Brain-derived neurotrophic factor modulates GABAergic synaptic transmission by enhancing presynaptic glutamic acid decarboxylase 65 levels, promoting asynchronous release and reducing the number of activated postsynaptic receptors

Brain-derived neurotrophic factor is known to modulate the function of GABAergic synapses, but the site of brain-derived neurotrophic factor action is still a matter of controversy. This study was aimed at further dissecting the functional alterations produced by brain-derived neurotrophic factor treatment of GABAergic synaptic connections in cultures of the murine superior colliculus. The functional consequences of long-term brain-derived neurotrophic factor treatment were assessed by analysis of unitary evoked and delayed inhibitory postsynaptic currents in response to high frequency stimulation of single axons. It was found that brain-derived neurotrophic factor facilitated the asynchronous release, but had no effect on the probability of evoked release, the size of the readily releasable pool, and the paired-pulse behavior of evoked inhibitory postsynaptic currents. However, the amplitudes of evoked inhibitory postsynaptic currents, delayed inhibitory postsynaptic currents and miniature inhibitory postsynaptic currents were significantly reduced. Non-stationary fluctuation analysis revealed a decrease in the open channel number at the miniature/evoked inhibitory postsynaptic current peak, but no effect on the mean GABA(A) receptor single channel conductance. Quantitative immunocytochemistry uncovered a significant elevation of presynaptic levels of glutamic acid decarboxylase 65. Together, these findings indicate that brain-derived neurotrophic factor treatment induces pre- as well as postsynaptic changes. What effect predominates will depend on the presynaptic activity pattern: at low activation rates brain-derived neurotrophic factor-treated synapses display a pronounced postsynaptic depression, but at high frequencies this depression is fully compensated by an enhancement of asynchronous release.

Rapid genotyping of newborn gene mutant mice

One important aspect of utilizing transgenic mice is the need to genotype them in order to distinguish mice that carry a disrupted gene or a transgene from mice that do not. Current methods for genotyping include isolation of genomic DNA from tail biopsies followed by PCR amplification. Particularly, both digestion of tail tissue using proteinase K as well as resuspension of purified DNA are time-consuming and were usually carried out overnight. Here, we describe a rapid and robust method for the genotyping of bdnf targeted mice which allows us to determine the genotype of newborn mice at the day of birth within 6 h. After a freezing-thawing step tail tissue is digested in less than 2 h, and the DNA is precipitated, resuspended and ready for PCR in about 60 min. The method could be easily adapted to a variety of different mutant mice and especially should benefit neuroscientists interested in using animals with known genotype very early in postnatal development.

GluR- and TrkB-mediated maturation of GABAA receptor function during the period of eye opening

Synapse maturation includes the shortening of postsynaptic currents, due to changes in the subunit composition of respective transmitter receptors. Patch clamp experiments revealed that GABAergic inhibitory postsynaptic currents (ISPCs) of superior colliculus neurons significantly shorten from postnatal day (P)1 to P21. The change started after P6 and was steepest between P12 and P15, i.e. around eye opening. It was accompanied by enhanced sensitivity to zolpidem and increased expression of GABAAR α1 mRNA, whereas the level of α3 mRNA decreased. This result is consistent with the hypothesis that the IPSC kinetics of developing collicular neurons is determined by the level of α1/α3. As α1/α3 peaked when N‐methyl‐D‐aspartate receptor (NMDAR)‐mediated synaptic currents reached their maximum (P12) it was asked whether NMDAR activity can shape the kinetics of GABAergic IPSCs. Cultured collicular neurons were treated with NMDA or NMDAR block, and it was found that the former resulted in faster and the latter in slower IPSC decay. Group I mGluR blockade had no effect. Experiments with bdnf–/– mice revealed that, with some delay, the increase of α1/α3 mRNA also occurred in the chronic absence of brain‐derived neurotrophic factor (BDNF) and, again, this was accompanied by the shortening of IPSCs. In addition, there was an age‐dependent depression of IPSC amplitudes by endogenous BDNF, which might reflect the developmental increase in the expression of GABAAR γ2L, as opposed to γ2S. Together, these experiments suggest that the GABAAR α subunit switch and the associated change in the IPSC kinetics were specifically controlled by NMDAR activity and independent on the signalling through group I mGluRs or TrkB.

Cajal–Retzius cells in the mouse neocortex receive two types of pre- and postsynaptically distinct GABAergic inputs

Cajal–Retzius (CR) cells are principal cells of layer I in the developing neocortex. They are able to generate action potentials, make synaptic contacts in layer I and receive excitatory GABAergic inputs before birth. Although CR cells participate in neuronal network activity in layer I, the properties of their synaptic inputs are not yet characterized. We recorded miniature (mIPSCs) and evoked (eIPSCs) postsynaptic currents using the whole‐cell patch‐clamp technique. Most of CR cells displayed two types of mIPSCs, namely those with fast (mIPSCF) and slow (mIPSCS) rise kinetics. The mIPSCF mean amplitude was significantly larger than that of mIPSCS, while their decay rates were not different. Peak‐scaled non‐stationary noise analysis revealed that mIPSCS and mIPSCF differed in their weighted single‐channel conductance. In addition, zolpidem (100 nm), a modulator of α1 subunit‐containing GABAA receptors, selectively affected mIPSCS suggesting that different postsynaptic GABAA receptors mediate mIPSCF and mIPSCS. eIPSCs also split into two populations with different rise kinetics. Fast eIPSCs (eIPSCF) displayed higher paired‐pulse ratio (PPR) and lower GABA release probability than slowly rising eIPSCs (eIPSCS). As CGP55845, a GABAB receptor antagonist, eliminated the observed difference in PPR, the lower release probability at IPSCF connections probably reflects a stronger tonic GABAB receptor‐mediated inhibition of IPSCF synapses. At low (0.1 Hz) stimulation frequency both inputs can effectively convert presynaptic action potentials into postsynaptic ones; however, only IPSCF connections reliably transfer the presynaptic activity patterns at higher stimulation rates. Thus, CR cells receive two GABAergic inputs, which differ in the quantal amplitude, the probability of GABA release and the frequency dependence of signal transfer.

Functional hallmarks of GABAergic synapse maturation and the diverse roles of neurotrophins.

Functional impairment of the adult brain can result from deficits in the ontogeny of GABAergic synaptic transmission. Gene defects underlying autism spectrum disorders, Rett’s syndrome or some forms of epilepsy, but also a diverse set of syndromes accompanying perinatal trauma, hormonal imbalances, intake of sleep-inducing or mood-improving drugs or, quite common, alcohol intake during pregnancy can alter GABA signaling early in life. The search for therapeutically relevant endogenous molecules or exogenous compounds able to alleviate the consequences of dysfunction of GABAergic transmission in the embryonic or postnatal brain requires a clear understanding of its site- and state-dependent development. At the level of single synapses, it is necessary to discriminate between presynaptic and postsynaptic alterations, and to define parameters that can be regarded as both suitable and accessible for the quantification of developmental changes. Here we focus on the performance of GABAergic synapses in two brain structures, the hippocampus and the superior colliculus, describe some novel aspects of neurotrophin effects during the development of GABAergic synaptic transmission and examine the applicability of the following rules: (1) synaptic transmission starts with GABA, (2) nascent/immature GABAergic synapses operate in a ballistic mode (multivesicular release), (3) immature synaptic terminals release vesicles with higher probability than mature synapses, (4) immature GABAergic synapses are prone to paired pulse and tetanic depression, (5) synapse maturation is characterized by an increasing dominance of synchronous over asynchronous release, (6) in immature neurons GABA acts as a depolarizing transmitter, (7) synapse maturation implies inhibitory postsynaptic current shortening due to an increase in alpha1 subunit expression, (8) extrasynaptic (tonic) conductances can inhibit the development of synaptic (phasic) GABA actions.

Early onset of glutamatergic and GABAergic synaptic activity in the visual layers of the rodent superior colliculus.

During postnatal development, the retinocollicular pathway undergoes activity‐dependent refinement, resulting in the precise retinotopic map seen in adults. Previous studies established that retinal efferents reach the mouse superior colliculus (SC) by embryonic day 16. Morphologically, synapses were found in the rat SC before birth. As part of an extended project aimed at understanding the development of synaptic transmission in the visual layers of the SC, we report here the presence of functionally active synapses immediately after birth. Circuit activity in mouse SC neurons was detected in horizontal slices of the visual layers using cell‐attached voltage clamp. The spontaneous discharge of action potentials was abolished by glutamatergic blockers and facilitated by bicuculline, showing that circuit activity is based on synaptic transmission and that the action of γ‐aminobutyric acid is inhibitory. Using whole‐cell voltage clamp, spontaneous glutamatergic postsynaptic currents as well as miniature GABAergic postsynaptic currents were recorded on postnatal day 1. Excitatory and inhibitory postsynaptic currents could also be evoked by electrical stimulation. Glutamatergic postsynaptic currents comprised both (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid and N‐methyl‐d‐aspartate receptor‐mediated components. The early function of glutamatergic and GABAergic synaptic transmission in the visual layers of SC suggests that SC neurons are able to process information originating from retinal axons immediately after birth.

Synaptic Potentiation at Basal and Apical Dendrites of Hippocampal Pyramidal Neurons Involves Activation of a Distinct Set of Extracellular and Intracellular Molecular Cues

In the central nervous system, several forms of experience-dependent plasticity, learning and memory require the activity-dependent control of synaptic efficacy. Despite substantial progress in describing synaptic plasticity, mechanisms related to heterogeneity of synaptic functions at local circuits remain elusive. Here we studied the functional and molecular aspects of hippocampal circuit plasticity by analyzing excitatory synapses at basal and apical dendrites of mouse hippocampal pyramidal cells (CA1 region) in acute brain slices. In the past decade, activity of metalloproteinases (MMPs) has been implicated as a widespread and critical factor in plasticity mechanisms at various projections in the CNS. However, in the present study we discovered that in striking contrast to apical dendrites, synapses located within basal dendrites undergo MMP-independent synaptic potentiation. We demonstrate that synapse-specific molecular pathway allowing MMPs to rapidly upregulate function of NMDARs in stratum radiatum involved protease activated receptor 1 and intracellular kinases and GTPases activity. In contrast, MMP-independent scaling of synaptic strength in stratum oriens involved dopamine D1/D5 receptors and Src kinases. Results of this study reveal that 2 neighboring synaptic systems differ significantly in extracellular and intracellular cascades that control synaptic gain and provide long-searched transduction pathways relevant for MMP-dependent synaptic plasticity.