Two modes of vesicle recycling in the rat calyx of Held.

Vesicle recycling was studied in the rat calyx of Held, a giant brainstem terminal involved in sound localization. Stimulation of brain slices containing the calyx-type synapse with a high extracellular potassium ion concentration in the presence of horseradish peroxidase resulted within several minutes in a reduction of the number of neurotransmitter vesicles and in the appearance of labeled endosome-like structures. After returning to normal solution, the endosome-like structures disappeared over a period of several minutes, whereas simultaneously the number of labeled vesicles increased. A comparison with afferent stimulation suggested that the endosome-like structures normally do not participate in the vesicle cycle. Afferent stimulation at 5 Hz resulted in sustained synaptic transmission, without vesicle depletion but with an estimated endocytotic activity of <0.2 synaptic vesicles per active zone per second. At 20 Hz, the presynaptic action potentials generally failed during prolonged stimulation. In identified synapses, the number of vesicles labeled by photoconversion after stimulation at 5 Hz in the presence of the styryl dye RH414 was much lower than the number of vesicles that were released, as determined by measuring EPSCs. No more than approximately 5% of the vesicles were labeled after 20 min stimulation at 5 Hz, whereas this stimulation protocol was sufficient to largely destain a terminal after previous loading. The results support a scheme for recycling in which two different modes coexist. At physiological demands, a pool of approximately 5% of all vesicles provides sufficient vesicles for release. During intense stimulation, such as occurs in the presence of high extracellular K+, the synapse resorts to bulk endocytosis, a very slow mode of recycling.

Altered brain stem responsivity to duodenal pain after a single stressful experience.

A single session of foot shock stress produces stable and long lasting sensitization of behavioral, hormonal and intestinal motility responses to novel stressful stimuli in laboratory rats. This is reflected in increased expression of the activity marker protein Fos in brain areas involved, following an external stressor. We present data from awake, freely moving rats in which a silicone balloon was surgically implanted in the duodenum. Firstly, cardiovascular reflexes to distentions were studied using telemetry with surgically implanted transmitters, 2 weeks after a single, 15-min session of foot shocks. The distentions caused characteristic, bi-phasic responses in both mean arterial blood pressure and heart rate that were not different between preshocked and control animals. Secondly, the numbers of Fos immunopositive cells were quantified in selected brain areas, 1 h after repeated distention of the duodenum. We found an increase in distention-induced Fos in preshocked rats in the nucleus tractus solitarius and a weaker effect in the central nucleus of the amygdala. This could be a first indication that altered visceral afferent processing in previously stressed rats, found earlier for the colon, may be a general and not an organ-specific phenomenon.

Single-cell analysis of peptide expression and electrophysiology of right parietal neurons involved in male copulation behavior of a simultaneous hermaphrodite.

Male copulation is a complex behavior that requires coordinated communication between the nervous system and the peripheral reproductive organs involved in mating. In hermaphroditic animals, such as the freshwater snail Lymnaea stagnalis, this complexity increases since the animal can behave both as male and female. The performance of the sexual role as a male is coordinated via a neuronal communication regulated by many peptidergic neurons, clustered in the cerebral and pedal ganglia and dispersed in the pleural and parietal ganglia. By combining single-cell matrix-assisted laser mass spectrometry with retrograde staining and electrophysiology, we analyzed neuropeptide expression of single neurons of the right parietal ganglion and their axonal projections into the penial nerve. Based on the neuropeptide profile of these neurons, we were able to reconstruct a chemical map of the right parietal ganglion revealing a striking correlation with the earlier electrophysiological and neuroanatomical studies. Neurons can be divided into two main groups: (i) neurons that express heptapeptides and (ii) neurons that do not. The neuronal projection of the different neurons into the penial nerve reveals a pattern where (spontaneous) activity is related to branching pattern. This heterogeneity in both neurochemical anatomy and branching pattern of the parietal neurons reflects the complexity of the peptidergic neurotransmission involved in the regulation of male mating behavior in this simultaneous hermaphrodite.

Sequential changes in synaptic vesicle pools and endosome-like organelles during depolarization near the active zone of central nerve terminals.

During periods of high-frequency stimulation the maintenance of synaptic transmission depends on a continued supply of synaptic vesicles. Local recycling in the terminals ensures synaptic vesicle replenishment, but the intermediate steps are still a matter of debate. We analyzed changes in synaptic vesicle pools and endosome-like organelles near the active zone in central nerve terminals during depolarization at the ultrastructural level by electron microscopy. A short, 100 ms, depolarization-induced recruitment of synaptic vesicles was observed from a reserve pool to a recruited pool, within 150 nm of the active zone, and the docked pool at the active zone was increased as well. Prolonged, 15 s or 3 min, depolarization decreased the total amount of synaptic vesicles, which was accompanied by a parallel increase in size and amount of endosome-like organelles. After a period of rest, the number of endosome-like organelles decreased and the amount of synaptic vesicles was restored to control level. The endocytotic nature of part of the endosome-like organelles after 15 s and 3 min depolarization was indicated by their labeling with extracellularly added horseradish peroxidase (HRP). In addition, a small number of synaptic vesicles entrapped HRP under these conditions. After repolarization, the number of HRP-loaded endosome-like structures decreased. Simultaneously, a strong increase in amount of HRP-loaded small vesicles did occur. These results indicate that during sub-second depolarization, synaptic vesicles were rapidly recruited from the reserve pool to replenish the releasable pool, whereas prolonged depolarization (s-min) induced local endocytosis in at least two ways, i.e. either directly as vesicles or via endosome-like organelles from which synaptic vesicles were reformed.

Functional analysis of a recurrent missense mutation in Notch3 in CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular dementia characterised by recurrent ischemic strokes in the deep white matter. Mutations in the gene encoding the cell surface receptor, Notch3, have been identified in CADASIL patients, and accumulation of the extracellular domain of Notch3 has been demonstrated in affected vessels. Almost all CADASIL mutations alter the number of cysteine residues in the epidermal growth factor (EGF)-like repeats in the extracellular domain of the protein. OBJECTIVES: To understand the functional consequences of a recurrent CADASIL mutation on furin processing, cell surface expression, ligand binding, and activation of a downstream effector CBF1 by the Notch3 receptor. METHODS: We expressed wild type and mutant Notch3 receptors in cultured cells and examined cell surface expression of the proteins. We also applied a new flow cytometry based approach to semi-quantitatively measure binding to three Notch ligands. Additionally, we used a well characterised co-culture system to examine ligand dependent activation of transcription from a CBF1-luciferase reporter construct. RESULTS: These studies revealed subtle abnormalities in furin processing of the mutant receptor, although both heterodimeric and full length receptors are present on the cell surface, are capable of interacting with soluble forms of three ligands, Delta1, Delta4, and Jagged1, and retain the ability to activate CBF1 in a ligand dependent manner. CONCLUSIONS: By comparison with other mutant forms of Notch3, these data indicate that individual CADASIL mutations can have disparate effects on Notch3 expression and function.