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Low-magnification high-numerical aperture objectives maximize the collection efficiency for scattered two-photon excited fluorescence (2PEF), but non-descanned detection schemes for such objectives demand optical components much bigger than standard microscope optics. Fiber coupling offers the possibility of removing bulky multi-channel detectors from the collection site, but coupling and transmission losses are generally believed to outweigh the benefits of optical fibers. We present here two new developments based on large-core fiber-optic fluorescence detection that illustrate clear advantages over conventional air-coupled 2PEF detection schemes. First, with minimal modifications of a commercial microscope, we efficiently couple the output of a 20×/NA0.95 objective to a large-core liquid light guide and we obtain a 7-fold collection gain when imaging astrocytes at 100 μm depth in acute brain slices of adult ALDH1L1-GFP mice. Second, combining 2PEF microscopy and 4-color detection on a custom microscope, mode scrambling inside a 2-mm plastic optical fiber is shown to cancel out the spatially non-uniform spectral sensitivity observed with air-coupled detectors. Spectral unmixing of images of brainbow mice taken with a fiber-coupled detector revealed a uniform color distribution of hippocampal neurons across a large field of view. Thus, fiber coupling improves both the efficiency and the homogeneity of 2PEF collection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Light scattering by tissue limits the imaging depth of two-photon microscopy and its use for functional brain imaging in vivo. We investigate the influence of scattering on both fluorescence excitation and collection, and identify tissue and instrument parameters that limit the imaging depth in the brain. (i) In brain slices, we measured that the scattering length at lambda=800 nm is a factor 2 higher in juvenile cortical tissue (P14-P18) than in adult tissue (P90). (ii) In a detection geometry typical for in vivo imaging, we show that the collected fraction of fluorescence drops at large depths, and that it is proportional to the square of the effective angular acceptance of the detection optics. Matching the angular acceptance of the microscope to that of the objective lens can result in a gain of approximately 3 in collection efficiency at large depths (>500 microm). A low-magnification (20x), high-numerical aperture objective (0.95) further increases fluorescence collection by a factor of approximately 10 compared with a standard 60x-63x objective without compromising the resolution. This improvement should allow fluorescence measurements related to neuronal or vascular brain activity at >100 microm deeper than with standard objectives.

Oxytocin and vasopressin increased the rate of firing of a class of presumed non-pyramidal neurones located in the CA1 area of rat hippocampal slices. This excitatory effect persisted in conditions of synaptic uncoupling. In contrast, pyramidal neurones were either unaffected by neurohypophysial peptides or showed one or several of the following effects: a decrease in firing rate in cells which were spontaneously active; a slight membrane hyperpolarization; and an increase in the rate of occurrence of spontaneous inhibitory postsynaptic potentials. We therefore propose that oxytocin and vasopressin excite directly a class of non-pyramidal inhibitory interneurones, whereas their observed effect on pyramidal neurones is indirect and inhibitory.

In slices of guinea pig brain, intracellular recordings were obtained from neurones of the ventromedial nucleus of the hypothalamus (VMH). [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO), an agonist selective for mu-opioid receptors, caused an inhibition of spontaneous firing activity and a membrane hyperpolarization. This effect was reversible, concentration-dependent and could be blocked by naloxone. DAGO directly inhibited VMH neurones since its effect persisted when the slice was perifused with a solution that blocks synaptic transmission. The hyperpolarization induced by DAGO was associated with a marked decrease in membrane input resistance and it was reversed in polarity at membrane potentials 30-40 mV more negative than the resting potential. A chloride current did not contribute to the hyperpolarization brought about by DAGO. We conclude that DAGO inhibits VMH neurones, probably by opening membrane potassium channels.

Extracellular recordings were obtained from spontaneously active neurones located in the dorsal motor nucleus of vagus nerve ( DMX ) in slices of the rat brainstem. Oxytocin applied to the bath at concentrations of 10(-7) M or 10(-6) M excited 79% of these cells in a concentration-dependent, reversible manner. The remaining cells were unaffected. The stimulatory effect of oxytocin was reversibly antagonized by a synthetic structural analogue known to block the peripheral, endocrine effects of neurohypophysial peptides. A selective oxytocic agonist was as potent as oxytocin, whereas vasopressin exerted a much weaker effect. We therefore suggest that neurones located in DMX are endowed with receptors for oxytocin.

The effect of arginine vasopressin (AVP) on neurones in the dorsal cochlear nucleus (DCN) of young guinea-pigs of either sex was investigated in brainstem slices. Most impaled neurones fired in a regular manner, either spontaneously or following a depolarizing current injection. AVP, at concentrations of 10-1000 nM, excited 19/19 neurones from male and 16/19 neurones from female animals. This effect of AVP was concentration-dependent and could be mimicked by the V1 agonist [Phe2,Orn8]VT. Oxytocin was less potent than AVP and a selective V2 agonist, deamino-DAVP, was without effect. Thus, a class of DCN neurones is probably endowed with functional V1 vasopressin receptors. By making use of an antibody raised against the vasopressin-related glycopeptide, dense AVP-like immunoreactivity was found in the DCN of young animals of either sex.