Autaptic cultures of single hippocampal granule cells of mice and rats.

When a single neuron is grown on a small island of glial cells, the neuron forms synapses onto itself. The so-called autaptic culture systems have proven extremely valuable in elucidating basic mechanisms of synaptic transmission, as they allow application of technical approaches that cannot be used in slice preparations. However, this method has been almost exclusively used for pyramidal cells and interneurons. In this study, we generated autaptic cultures from granule cells isolated from the dentate gyrus of rodent hippocampi. Our subsequent morphological and functional characterisation of these cells confirms that this culture model is suitable for investigating basic mechanisms of granule cell synaptic transmission. Importantly, the autosynaptic connectivity allows recordings of pure mossy fibre miniature EPSCs, which are not possible in slice preparations. Further, by fast application of hypertonic sucrose solutions it is possible to directly measure the readily releasable pool and to calculate the probability of vesicular release.

C3 peptide enhances recovery from spinal cord injury by improved regenerative growth of descending fiber tracts.

Functional recovery and regeneration of corticospinal tract (CST) fibers following spinal cord injury by compression or dorsal hemisection in mice was monitored after application of the enzyme-deficient Clostridium botulinum C3-protein-derived 29-amino-acid fragment C3bot(154-182). This peptide significantly improved locomotor restoration in both injury models as assessed by the open-field Basso Mouse Scale for locomotion test and Rotarod treadmill experiments. These data were supported by tracing studies showing an enhanced regenerative growth of CST fibers in treated animals as visualized by anterograde tracing. Additionally, C3bot(154-182) stimulated regenerative growth of raphespinal fibers and improved serotonergic input to lumbar alpha-motoneurons. These in vivo data were confirmed by in vitro data, showing an enhanced axon outgrowth of alpha-motoneurons and hippocampal neurons cultivated on normal or growth-inhibitory substrates after application of C3bot(154-182). The observed effects were probably caused by a non-enzymatic downregulation of active RhoA by the C3 peptide as indicated by pull-down experiments. By contrast, C3bot(154-182) did not induce neurite outgrowth in primary cultures of dorsal root ganglion cells. In conclusion, C3bot(154-182) represents a novel, promising tool to foster axonal protection and/or repair, as well as functional recovery after traumatic CNS injury.

Age-dependent axonal expression of potassium channel proteins during development in mouse hippocampus.

The development of the hippocampal network requires neuronal activity, which is shaped by the differential expression and sorting of a variety of potassium channels. Parallel to their maturation, hippocampal neurons undergo a distinct development of their ion channel profile. The age-dependent dimension of ion channel occurrence is of utmost importance as it is interdependently linked to network formation. However, data regarding the exact temporal expression of potassium channels during postnatal hippocampal development are scarce. We therefore studied the expression of several voltage-gated potassium channel proteins during hippocampal development in vivo and in primary cultures, focusing on channels that were sorted to the axonal compartment. The Kv1.1, Kv1.2, Kv1.4, and Kv3.4 proteins showed a considerable temporal variation of axonal localization among neuronal subpopulations. It is possible, therefore, that hippocampal neurons possess cell type-specific mechanisms for channel compartmentalization. Thus, age-dependent axonal sorting of the potassium channel proteins offers a new approach to functionally distinguish classes of hippocampal neurons and may extend our understanding of hippocampal circuitry and memory processing.

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 transferase-deficient 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 submicromolar concentrations. Several C3bot constructs, including the short peptide, enhanced the number of axonal segments from mid- to higher-order segments. C3bot(154-182) 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 C3bot(154-182) 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.

Role of Rho GTPase in astrocyte morphology and migratory response during in vitro wound healing.

Small Rho GTPases are key regulators of the cytoskeleton in a great variety of cells. Rho function mediates morphological changes as well as locomotor activity. Using astrocyte cultures established from neonatal mice we investigated the role of Rho in process formation during astrocyte stellation. Using a scratch-wound model, we examined the impact of Rho on a variety of morphological and functional variables such as stellation and migratory activity during wound healing. C3 proteins are widely used to study cellular Rho functions. In addition, C3 derived from Clostridium botulinum (C3bot) is considered selectively to promote neuronal regeneration. Because the latter requires a balanced activity of neurones and glial cells, the effects of C3 protein on glial cells such as astrocytes have to be considered carefully. Low nanomolar concentrations of C3 proteins significantly promoted process outgrowth and increased process branching. Besides enzymatic inactivation of Rho by ADP-ribosylation, changes in protein levels of the various Rho GTPases may also contribute to the observed effects. Furthermore, incubation of scratch-wounded astrocyte cultures with C3bot accelerated wound healing. By inhibiting the Rho downstream effector ROCK with the selective inhibitor Y27632 we were able to demonstrate that the accelerated wound closure resulted from both enhanced polarized process formation and increased migratory activity of astrocytes into the lesion site. These results suggest that Rho negatively regulates astrocytic process growth and migratory responses after injury and that its inactivation by C3bot in nanomolar concentrations promotes astrocyte migration.

Area-specific effects of brain-derived neurotrophic factor (BDNF) genetic ablation on various neuronal subtypes of the mouse brain.

The effects of brain-derived neurotrophic factor (BDNF) on the development of presynaptic terminals and of neuronal subtypes in various brain areas were studied in BDNF-knockout (BDNF-/-) mice at postnatal days 15-17. Western analysis revealed no changes in the overall amount of a variety of synaptic proteins in BDNF-/- mice as compared to wild type mice. In addition, the complex between the vesicular proteins, synaptophysin and synaptobrevin, as well as their respective homodimers were unaltered. Moreover, no changes in the density of neurons were found in, e.g., the CA3 region of the hippocampus and the nucleus nervi facialis of BDNF-/- mice. However, cholinergic cells were reduced by 20% in the medial septum of BDNF-/- mice associated with a decrease in the activity of choline acetyltransferase and protein levels of nerve growth factor in the hippocampus by 16% and 44%, respectively. In the striatum, however, the total number of cholinergic cells were comparable in both groups, although the activity of choline acetyltransferase was decreased by 46%. In GABAergic interneurons, the expression of neuropeptides in various brain areas was differentially affected by BDNF deletion as revealed by immunohistochemistry. In the hippocampus and cortex of BDNF-/- mice, the density of neuropeptide Y-, somatostatin-, and parvalbumin-immunoreactive cells was drastically reduced, whereas the density of calretinin-positive cells was increased. The extent of these changes in neuropeptide-containing cells varied among hippocampal subregions. In the striatum, only the density of parvalbumin-immunoreactive cells was decreased by approximately 45%. In conclusion, BDNF deficiency is accompanied by a differential dysregulation in the expression of neuropeptides and calcium-binding proteins in otherwise intact GABAergic and glutamatergic neurons in a region-specific manner.

Axonal sorting of Kir3.3 defines a GABA-containing neuron in the CA3 region of rodent hippocampus.

Hippocampal interneurons comprise a heterogeneous group of locally acting GABAergic neurons. In addition to their variability in cotransmitter content and receptor profile, they express a variety of potassium channels that specify their individual properties. Here we describe a new type of large GABA-containing neuron in rodent hippocampus that is characterized by an axonal sorting of the potassium channel Kir3.3. The parent cell bodies of the Kir3.3-positive axons are located in CA3, as assessed by primary cultures derived from hippocampal subareas. At postnatal day 14 these neurons appear at the border between stratum oriens and stratum pyramidale of CA3, from where their axons pass through stratum pyramidale to join the mossy fiber tract. In adult hippocampus, high levels of Kir3.3 channel protein exist in axons that run with the mossy fiber tract. Kir3.3 and the vesicular GABA transporter could be identified in subpopulations of large synaptic terminals that probably derive from Kir3.3 neurons. Axonal sorting of Kir3.3 appears to be typical of a group of large inhibitory neurons, including Purkinje cells and a novel type of interneuron in CA3. Kir3.3 neurons might modulate the activity of CA3 circuitries and consequently memory processing in the hippocampus.