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Genetically modified mouse strains that express Cre-recombinase in specific neuronal sub-populations have become widely used tools for investigating neuronal function. The Ntsr1-Cre GN220 mouse expresses this enzyme in corticothalamic neurons in layer 6 of cerebral cortex. We observed that about 7% of Cre-expressing cells in the primary visual cortex are found within the white matter bordering layer 6. By using the immunohistochemical marker for layer 6 neurons, Forkhead box protein 2 (FoxP2), and fluorescently conjugated latex beads injected into the dorsal lateral geniculate nucleus, we show that about half of these cells are similar to and could belong to the layer 6 corticothalamic neuron population. The other half seems to be a distinct white matter (WM) neuron sub-population that we estimate to constitute 2-4% of the total cortical Cre-expressing population. Staining for the neuronal marker Neuronal nuclei (NeuN) revealed that about 15-40% of WM neurons are Cre-expressing. Thus, the potential contribution from WM neurons needs to be considered when interpreting the results from experiments using the Ntsr1-Cre GN220 mouse for investigating corticothalamic neuronal function.

The Ntsr1-Cre GN220 mouse expresses Cre-recombinase in corticothalamic (CT) neurons in neocortical layer 6. It is not known if the other major types of pyramidal neurons in this layer also express this enzyme. By electrophysiological recordings in slices and histological analysis of the uptake of retrogradely transported beads we show that Cre-positive neurons are CT and not corticocortical or corticoclaustral types. Furthermore, we show that Ntsr1-Cre-positive cells are immuno-positive for the nuclear transcription factor Forkhead box protein P2 (FoxP2). We conclude that Cre-expression is limited to a specific type of pyramidal neuron: CT. However, it appears as not all CT neurons are Cre-expressing; there are indications that the penetrance of the gene is about 90%. We demonstrate the utility of assigning a specific identity to individual neurons by determining that the CT neurons are potently modulated by acetylcholine acting on both nicotinic and muscarinic acetylcholine receptors. These results corroborate the suggested function of these neurons in regulating the gain of thalamocortical transfer of sensory information depending on attentional demand and state of arousal.

The readily releasable pool of vesicles (RRP) varies in size during synaptic activity and is replenished by recruitment from the reserve pool as well as vesicle retrieval after fusion. To investigate which of these steps is rate limiting in supplying vesicles to the RRP, we measured the effects of changes in temperature in cultured hippocampal neurons, where higher average rates of release can be maintained as the temperature is increased. Using a pHluorin-based reporter of exocytosis and endocytosis (sypHy), we find that changes in temperature between 25 degrees C and 35 degrees C do not significantly alter the rate of recruitment from the reserve pool. In contrast, the time constant of endocytosis fell from approximately 17 s at 25 degrees C to approximately 10 s at 35 degrees C (Q(10) = 1.7), while the time constant of vesicle reacidification fell from approximately 5.5 s to approximately 1 s (Q(10) = 5.5). A kinetic model of the vesicle cycle constructed using measured parameters was found to describe variations in vesicle release rate observed during long trains of spikes as well as recovery from synaptic depression after bursts of activity. These results indicate that endocytosis operating with time constants of 10-15 s is the rate-limiting process determining replenishment of the RRP during long-term activity. A fast mode of vesicle retrieval could not be detected at any temperature, nor was it necessary to invoke such a mechanism to account for use-dependent changes in synaptic release probability.

The maintenance of synaptic transmission requires that vesicles are recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, but the relative importance of these has been controversial. It is established that synaptic vesicles can collapse on fusion and the machinery for retrieving this membrane by clathrin-mediated endocytosis (CME) is enriched in the presynaptic terminal. But it has also been suggested that the majority of vesicles released by physiological stimulation are recycled by a second, faster mechanism called 'kiss-and-run', which operates in 1 s or less to retrieve a vesicle before it has collapsed. The most recent evidence argues against the occurrence of 'kiss-and-run' in hippocampal synapses. First, an improved fluorescent reporter of exocytosis (sypHy), indicates that only a slow mode of endocytosis (tau = 15 s) operates when vesicle fusion is triggered by a single nerve impulse or short burst. Second, this retrieval mechanism is blocked by overexpressing the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi. Third, vesicle fusion is associated with the movement of clathrin and vesicle proteins out of the synapse into the neighbouring axon. These observations indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of retrieval in small hippocampal synapses.

Alzheimer's disease (AD) is the major cause of dementia. During the development of AD, neurofibrillary tangles progress in a fixed pattern, starting in the transentorhinal cortex followed by the hippocampus and cortical areas. In contrast, the deposition of β-amyloid (Aβ) plaques, which are the other histological hallmark of AD, does not follow the same strict spatiotemporal pattern, and it correlates poorly with cognitive decline. Instead, soluble Aβ oligomers have received increasing attention as probable inducers of pathogenesis. In this study, we use microinjections into electrophysiologically defined primary hippocampal rat neurons to demonstrate the direct neuron-to-neuron transfer of soluble oligomeric Aβ. Additional studies conducted in a human donor–acceptor cell model show that this Aβ transfer depends on direct cellular connections. As the transferred oligomers accumulate, acceptor cells gradually show beading of tubulin, a sign of neurite damage, and gradual endosomal leakage, a sign of cytotoxicity. These observations support that intracellular Aβ oligomers play a role in neurodegeneration, and they explain the manner in which Aβ can drive disease progression, even if the extracellular plaque load is poorly correlated with the degree of cognitive decline. Understanding this phenomenon sheds light on the pathophysiological mechanism of AD progression. Additional elucidation will help uncover the detailed mechanisms responsible for the manner in which AD progresses via anatomical connections and will facilitate the development of new strategies for stopping the progression of this incapacitating disease.

Accurate measurement of synaptic vesicle exocytosis and endocytosis is crucial to understanding the molecular basis of synaptic transmission. The fusion of a pH-sensitive green fluorescent protein (pHluorin) to various synaptic vesicle proteins has allowed the study of synaptic vesicle recycling in real time. Two such probes, synaptopHluorin and sypHy, have been imaged at synapses of hippocampal neurons in culture. The combination of these reporters with techniques for molecular interference, such as RNAi allows for the study of molecules involved in synaptic vesicle recycling. Here the authors describe methods for the culture and transfection of hippocampal neurons, imaging of pHluorin-based probes at synapses and analysis of pHluorin signals down to the resolution of individual synaptic vesicles. The original publication is available at www.springerlink.com: Stephen J Royle, Björn Granseth, Benjamin Odermatt, Aude Derevier and Leon Lagnado, Imaging phluorin-based probes at hippocampal synapses, 2008, Methods in Molecular Biology; Membrane Trafficking, (457), 293-303. http://dx.doi.org/10.1007/978-1-59745-261-8 Copyright: Humana Press http://www.springer.com/

In young rats the cartilaginous epiphyses forming the knee joint are supplied with blood vessels and peptidergic sensory nerve fibres through the perichondrium and cartilage canals. In the present study we show that cartilage-related dorsal root ganglion neurones co-cultured with perichondrial cells develop extensive neurite trees and express calcitonin gene-related peptide (CGRP) and substance P (SP) in in vivo-like proportions using retrograde tracing and immunohistochemistry. Moreover, whole cell patch clamp recordings from these cells showed that the majority is depolarised by application of H + -ions. These results are compatible with the hypothesis that a local imbalance of blood flow and metabolism during normal skeletal maturation may cause tissue acidosis eliciting release of CGRP/SP from sensory nerve endings.

SummaryThe maintenance of synaptic transmission requires that vesicles be recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, including a fast “kiss-and-run” mechanism that releases neurotransmitter through a fusion pore. Using an improved fluorescent reporter comprising pHluorin fused to synaptophysin, we find that only a slow mode of endocytosis (τ = 15 s) operates at hippocampal synapses when vesicle fusion is triggered by a single nerve impulse or short burst. This retrieval mechanism is blocked by overexpression of the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi, and it is associated with the movement of clathrin and vesicle proteins out of the synapse. These results indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of vesicle retrieval after physiological stimuli.

Hedgehog (Hh) signaling is a key regulatory pathway during development and also has a functional role in mature neurons. Here, we show that Hh signaling regulates the odor response in adult Drosophila olfactory sensory neurons (OSNs). We demonstrate that this is achieved by regulating odorant receptor (OR) transport to and within the primary cilium in OSN neurons. Regulation relies on ciliary localization of the Hh signal transducer Smoothened (Smo). We further demonstrate that the Hh- and Smo-dependent regulation of the kinesin-like protein Cos2 acts in parallel to the intraflagellar transport system (IFT) to localize ORs within the cilium compartment. These findings expand our knowledge of Hh signaling to encompass chemosensory modulation and receptor trafficking. Funding Agencies|Swedish Foundation for Strategic Research [F06-0013]