Stephan W. Schwarzacher

Neuroligin 2 Drives Postsynaptic Assembly at Perisomatic Inhibitory Synapses through Gephyrin and Collybistin

In the mammalian CNS, each neuron typically receives thousands of synaptic inputs from diverse classes of neurons. Synaptic transmission to the postsynaptic neuron relies on localized and transmitter-specific differentiation of the plasma membrane with postsynaptic receptor, scaffolding, and adhesion proteins accumulating in precise apposition to presynaptic sites of transmitter release. We identified protein interactions of the synaptic adhesion molecule neuroligin 2 that drive postsynaptic differentiation at inhibitory synapses. Neuroligin 2 binds the scaffolding protein gephyrin through a conserved cytoplasmic motif and functions as a specific activator of collybistin, thus guiding membrane tethering of the inhibitory postsynaptic scaffold. Complexes of neuroligin 2, gephyrin and collybistin are sufficient for cell-autonomous clustering of inhibitory neurotransmitter receptors. Deletion of neuroligin 2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.

Neuroanatomical characteristics of the human pre-Bötzinger complex and its involvement in neurodegenerative brainstem diseases

The pre-Bötzinger complex has been identified as an essential part of the medullary respiratory network in mammals. Although well described in experimental animals, its localization in the human brain has remained elusive. Using serially sectioned brainstems from 19 normal individuals and patients suffering from neurodegenerative diseases (multiple system atrophy, n = 10; spinocerebellar ataxia type 3, n = 8), we have identified a circumscribed area of the ventrolateral medulla that represents the human homologue of the pre-Bötzinger complex and have mapped its longitudinal and horizontal extents. The presumed human pre-Bötzinger complex is characterized by an aggregation of loosely scattered, small and lipofuscin-rich neurons, which contain neurokinin 1 receptor as well as somatostatin, but are negative for markers of monoaminergic neurons and of motoneurons. In brains of patients suffering from multiple systems atrophy (with central respiratory deficits but without swallowing problems), pre-Bötzinger complex neurons were reduced, whereas pharyngeal motoneurons of the ambigual nucleus were not affected. In contrast, in brains of patients with spinocerebellar ataxia 3 (no reported central respiratory deficits but with dysphagia), pre-Bötzinger complex neurons were preserved, whereas ambigual motoneurons, which control swallowing, were diminished. These pathoanatomical findings support the view, that affection of the central respiratory network, including the pre-Bötzinger complex, contributes to breathing disorders in multiple system atrophy, whereas damage to ambigual motoneurons is important for pathogenesis of breathing disturbances and dysphagia in patients with spinocerebellar ataxia type 3. On the basis of these findings, the putative human homologue of the pre-Bötzinger complex can now be reliably delineated on pigment-Nissl-stained sections, making neuropathological investigations of central respiratory disturbances feasible.

Increased Dentate Gyrus Excitability in Neuroligin-2-Deficient Mice in Vivo

The postsynaptic adhesion protein neuroligin-2 (NL2) is selectively localized at inhibitory synapses. Here, we studied network activity in the dentate gyrus of NL2-deficient mice following perforant path (PP) stimulation in vivo. We found a strong increase in granule cell (GC) excitability. Furthermore, paired-pulse inhibition (PPI) of the population spike, a measure for γ-aminobutyric acid (GABA)ergic network inhibition, was severely impaired and associated with reduced GABA(A) receptor (GABA(A)R)-mediated miniature inhibitory postsynaptic currents recorded from NL2-deficient GCs. In agreement with these functional data, the number of gephyrin and GABA(A)R clusters was significantly reduced in the absence of NL2, indicating a loss of synaptic GABA(A)Rs from the somata of GCs. Computer simulations of the dentate network showed that impairment of perisomatic inhibition is able to explain the electrophysiological changes observed in the dentate circuitry of NL2 knockout animals. Collectively, our data demonstrate for the first time that deletion of NL2 increases excitability of cortical neurons in the hippocampus of intact animals, most likely through impaired GABA(A)R clustering.

A role for the spine apparatus in LTP and spatial learning

Long-term potentiation (LTP) of synaptic strength is a long-lasting form of synaptic plasticity that has been linked to information storage. Although the molecular and cellular events underlying LTP are not yet fully understood, it is generally accepted that changes in dendritic spine calcium levels as well as local protein synthesis play a central role. These two processes may be influenced by the presence of a spine apparatus, a distinct neuronal organelle found in a subpopulation of telencephalic spines. Mice lacking spine apparatuses (synaptopodin-deficient mice) show deficits in LTP and impaired spatial learning supporting the involvement of the spine apparatus in synaptic plasticity. In our review, we consider the possible roles of the spine apparatus in LTP1 (protein synthesis-independent), LTP2 (translation-dependent and transcription-independent) and LTP3 (translation- and transcription-dependent) and discuss the effects of the spine apparatus on learning and memory.

Impairment of in vivo theta‐burst long‐term potentiation and network excitability in the dentate gyrus of synaptopodin‐deficient mice lacking the spine apparatus and the cisternal organelle

The function of the spine apparatus in dendritic spines and the cisternal organelles in axon initial segments is little understood. The actin‐associated protein, synaptopodin, is essential for the formation of these organelles which are absent in synaptopodin −/− mice. Here, we used synaptopodin −/− mice to explore the role of the spine apparatus and the cisternal organelle in synaptic plasticity and local circuit excitability in response to activation of the perforant path input to the dentate gyrus in vivo. We found impaired long‐term potentiation following theta‐burst stimulation, whereas tetanus‐evoked LTP was unaffected. Furthermore, paired‐pulse inhibition of the population spike was reduced and granule cell excitability was enhanced in mutants, hence revealing an impairment of local network inhibition. In summary, our data represent the first electrophysiological evidence that the lack of the spine apparatus and the cisternal organelle leads to a defect in long‐term synaptic plasticity and alterations in local circuit control of granule cell excitability under adult in vivo conditions.

Increased network excitability and impaired induction of long-term potentiation in the dentate gyrus of collybistin-deficient mice in vivo

Collybistin (Cb), a brain-specific guanine nucleotide exchange factor, has been shown to be essential for the gephyrin-dependent clustering of a specific set of GABA(A) receptors at inhibitory postsynaptic sites. Here, we examined whether the lack of Cb affects synaptic properties and neuronal activity in the intact hippocampus by monitoring network activity in the dentate gyrus of Cb-deficient mice after perforant-path stimulation in vivo. We found a decreased threshold for evoked population spikes of granule cells, indicating their increased excitability. Paired-pulse inhibition of the population spike, a measure for somatic GABAergic network inhibition, was enhanced. Mutant mice exhibited steeper slopes of field excitatory postsynaptic potentials, consistent with a reduced dendritic inhibition. In addition, the induction of long-term potentiation (LTP) was reduced. In line with these functional changes, the number of postsynaptic gephyrin and GABA(A) receptor clusters in the Cb-deficient dentate gyrus was significantly decreased. In conclusion, our data provide the first evidence that Cb-deficiency leads to significant changes of GABAergic inhibition, network excitability and synaptic plasticity in vivo.

3D-reconstruction and functional properties of GFP-positive and GFP-negative granule cells in the fascia dentata of the Thy1-GFP mouse.

Granule cells of the mouse fascia dentata are widely used in studies on neuronal development and plasticity. In contrast to the rat, however, high‐resolution morphometric data on these cells are scarce. Thus, we have analyzed granule cells in the fascia dentata of the adult Thy1‐GFP mouse (C57BL/6 background). In this mouse line, single neurons in the granule cell layer are GFP‐labeled, making them amenable to high‐resolution 3D‐reconstruction. First, calbindin or parvalbumin‐immunofluorescence was used to identify GFP‐positive cells as granule cells. Second, the dorsal‐ventral distribution of GFP‐positive granule cells was studied: In the dorsal part of the fascia dentata 11% and in the ventral part 15% of all granule cells were GFP‐positive. Third, GFP‐positive and GFP‐negative granule cells were compared using intracellular dye‐filling (fixed slice technique) and patch‐clamp recordings; no differences were observed between the cells. Finally, GFP‐positive granule cells (dorsal and ventral fascia dentata) were imaged at high resolution with a confocal microscope, 3D‐reconstructed in their entirety and analyzed for soma size, total dendritic length, number of segments, total number of spines and spine density. Sholl analysis revealed that dendritic complexity of granule cells is maximal 150–200 μm from the soma. Granule cells located in the ventral part of the hippocampus revealed a greater degree of dendritic complexity compared to cells in the dorsal part. Taken together, this study provides morphometric data on granule cells of mice bred on a C57BL/6 background and establishes the Thy1‐GFP mouse as a tool to study granule cell neurobiology.

Neuroligin-1 regulates excitatory synaptic transmission, LTP and EPSP-spike coupling in the dentate gyrus in vivo

Neuroligins are transmembrane cell adhesion proteins with a key role in the regulation of excitatory and inhibitory synapses. Based on previous in vitro and ex vivo studies, neuroligin-1 (NL1) has been suggested to play a selective role in the function of glutamatergic synapses. However, the role of NL1 has not yet been investigated in the brain of live animals. We studied the effects of NL1-deficiency on synaptic transmission in the hippocampal dentate gyrus using field potential recordings evoked by perforant path stimulation in urethane-anesthetized NL1 knockout (KO) mice. We report that in NL1 KOs the activation of glutamatergic perforant path granule cell inputs resulted in reduced synaptic responses. In addition, NL1 KOs displayed impairment in long-term potentiation. Furthermore, field EPSP-population spike (E-S) coupling was greater in NL1 KO than WT mice and paired-pulse inhibition was reduced, indicating a compensatory rise of excitability in NL1 KO granule cells. Consistent with changes in excitatory transmission, NL1 KOs showed a significant reduction in hippocampal synaptosomal expression levels of the AMPA receptor subunit GluA2 and NMDA receptor subunits GluN1, GluN2A and GluN2B. Taken together, we provide first evidence that NL1 is essential for normal excitatory transmission and long-term synaptic plasticity in the hippocampus of intact animals. Our data provide insights into synaptic and circuit mechanisms of neuropsychiatric abnormalities such as learning deficits and autism.

CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice.

Despite extensive investigations of Cbl‐interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85Δex2) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85Δex2 animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post‐synaptic compartment of striatal neurons in which it co‐clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85Δex2 mice.

Functional genomics suggest neurogenesis in the adult human olfactory bulb.

Abstract The human olfactory bulb displays high morphologic dynamics changing its volume with olfactory function, which has been explained by active neurogenetic processes. Discussion continues whether the human olfactory bulb hosts a continuous turnover of neurons. We analyzed the transcriptome via RNA quantification of adult human olfactory bulbs and intersected the set of expressed transcriptomic genes with independently available proteomic expression data. To obtain a functional genomic perspective, this intersection was analyzed for higher-level organization of gene products into biological pathways established in the gene ontology database. We report that a fifth of genes expressed in adult human olfactory bulbs serve functions of nervous system or neuron development, half of them functionally converging to axonogenesis but no other non-neurogenetic biological processes. Other genes were expectedly involved in signal transmission and response to chemical stimuli. This provides a novel, functional genomics perspective supporting the existence of neurogenesis in the adult human olfactory bulb.