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An ongoing controversy in brain metabolism is whether increases in neural activity cause a local and rapid decrease in oxygen concentration (i.e., the “initial dip”) preceding functional hyperemia. This initial dip has been suggested to cause a transient increase in vascular deoxyhemoglobin with several imaging techniques and stimulation paradigms, but not consistently. Here, we investigate contributors to this initial dip in a distinct neuronal network, an olfactory bulb (OB) glomerulus most sensitive to a specific odorant (ethyl tiglate [ET]) and a site of strong activation and energy consumption upon ET stimulation. Combining two-photon fluorescence and phosphorescence lifetime microscopy, and calcium, blood flow, and pO2 measurements, we characterized this initial dip in pO2 in mice chronically implanted with a glass cranial window, during both awake and anesthetized conditions. In anesthetized mice, a transient dip in vascular pO2 was detected in this glomerulus when functional hyperemia was slightly delayed, but its amplitude was minute (0.3 SD of resting baseline). This vascular pO2 dip was not observed in other glomeruli responding nonspecifically to ET, and it was poorly influenced by resting pO2. In awake mice, the dip in pO2 was absent in capillaries as well as, surprisingly, in the neuropil. These high-resolution pO2 measurements demonstrate that in awake mice recovered from brain surgery, neurovascular coupling was too fast and efficient to reveal an initial dip in pO2.

The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a high-power femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity.

Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation.

Analysis of the spatiotemporal coupling between neuronal activity and cerebral blood flow requires the precise measurement of the dynamics of RBC flow in individual capillaries that irrigate activated neurons. Here, we use two-photon microscopy in vivo to image individual RBCs in glomerular capillaries in the rat dorsal olfactory bulb. We find that odor stimulation evokes capillary vascular responses that are odorant- and glomerulus-specific. These responses consist of increases as well as decreases in RBC flow, both resulting from independent changes in RBC velocity or linear density. Finally, measuring RBC flow with micrometer spatial resolution and millisecond temporal resolution, we demonstrate that, in olfactory bulb superficial layers, capillary vascular responses precisely outline regions of synaptic activation.

Survival and differentiation of myogenic cells grafted into infarcted myocardium have raised the hope that cell transplantation becomes a new therapy for cardiovascular diseases. The approach was further supported by transplantation of skeletal myoblasts, which was shown to improve cardiac performance in several animal species. Despite the success of myoblast transplantation and its recent trial in human, the mechanism responsible for the functional improvement remains unclear. Here, we used intracellular recordings coupled to video and fluorescence microscopy to establish whether myoblasts, genetically labeled with enhanced GFP and transplanted into rat infarcted myocardium, retain excitable and contractile properties, and participate actively to cardiac function. Our results indicate that grafted myoblasts differentiate into peculiar hyperexcitable myotubes with a contractile activity fully independent of neighboring cardiomyocytes. We conclude that mechanisms other than electromechanical coupling between grafted and host cells are involved in the improvement of cardiac function.

Mitral cell dendrites do more than passively integrate and convey synaptic potentials to the soma, they release transmitter onto local interneurones to mediate recurrent and lateral inhibition. Several mechanisms may control the level of dendritic intracellular calcium ([Ca(2+)]) and define timing for dendritic release. Here we investigated in vivo, how odor controls calcium dynamics in mitral cell dendrites by combining intracellular recording and two-photon microscopy imaging of [Ca(2+)]. During odor stimulation, two types of [Ca(2+)] changes accompany membrane potential oscillations that are phase-locked with the respiratory cycle: (i) one is graded and parallels the membrane potential, even below the threshold for action potential firing; (ii) a second is transient, triggered by sodium action potentials that invade the entire dendritic tree. These results indicate that mitral cell dendritic compartments are synchronized by action potentials and suggest that the efficacy of dendritic synapses is finely tuned by odor-evoked graded changes in [Ca(2+)].

In the hippocampus, a CA3 pyramidal cell forms excitatory synapses with thousands of other pyramidal cells and inhibitory interneurons. By using sequential paired recordings from three connected cells, we show that the presynaptic properties of CA3 pyramidal cell terminals, belonging to the same axon, differ according to the type of target cell. Activation of presynaptic group III metabotropic glutamate receptors decreases transmitter release only at terminals contacting CA1 interneurons but not CA1 pyramidal cells. Furthermore, terminals contacting distinct target cells show different frequency facilitation. On the basis of these results, we conclude that the pharmacological and physiological properties of presynaptic terminals are determined, at least in part, by the target cells.