A Novel Technique for Visualizing and Analyzing the Cerebral Vasculature in Rodents.

We introduce a novel protocol to stain, visualize, and analyze blood vessels from the rat and mouse cerebrum. This technique utilizes the fluorescent dye, DiI, to label the lumen of the vasculature followed by perfusion fixation. Following brain extraction, the labeled vasculature is then imaged using wide-field fluorescence microscopy for axial and coronal images and can be followed by regional confocal microscopy. Axial and coronal images can be analyzed using classical angiographic methods for vessel density, length, and other features. We also have developed a novel fractal analysis to assess vascular complexity. Our protocol has been optimized for adult rat, adult mouse, and neonatal mouse studies. The protocol is efficient, can be rapidly completed, stains cerebral vessels with a bright fluorescence, and provides valuable quantitative data. This method has a broad range of applications, and we demonstrate its use to study the vasculature in assorted models of acquired brain injury.

Gonadotropin-Releasing Hormone (GnRH) Neuron Excitability Is Regulated by Estradiol Feedback and Kisspeptin.

Gonadotropin-releasing hormone (GnRH) neurons produce the central output controlling fertility and are regulated by steroid feedback. A switch from estradiol negative to positive feedback initiates the GnRH surge, ultimately triggering ovulation. This occurs on a daily basis in ovariectomized, estradiol-treated (OVX+E) mice; GnRH neurons are suppressed in the morning and activated in the afternoon. To test the hypotheses that estradiol and time of day signals alter GnRH neuron responsiveness to stimuli, GFP-identified GnRH neurons in brain slices from OVX+E or OVX female mice were recorded during the morning or afternoon. No differences were observed in baseline membrane potential. Current-clamp revealed GnRH neurons fired more action potentials in response to current injection during positive feedback relative to all other groups, which were not different from each other despite reports of differing ionic conductances. Kisspeptin increased GnRH neuron response in cells from OVX and OVX+E mice in the morning but not afternoon. Paradoxically, excitability in kisspeptin knock-out mice was similar to the maximum observed in control mice but was unchanged by time of day or estradiol. A mathematical model applying a Markov Chain Monte Carlo method to estimate probability distributions for estradiol- and time of day-dependent parameters was used to predict intrinsic properties underlying excitability changes. A single identifiable distribution of solutions accounted for similar GnRH neuron excitability in all groups other than positive feedback despite different underlying conductance properties; this was attributable to interdependence of voltage-gated potassium channel properties. In contrast, redundant solutions may explain positive feedback, perhaps indicative of the importance of this state for species survival.SIGNIFICANCE STATEMENT Infertility affects 15%-20% of couples; failure to ovulate is a common cause. Understanding how the brain controls ovulation is critical for new developments in both infertility treatment and contraception. Gonadotropin-releasing hormone (GnRH) neurons are the final common pathway for central neural control of ovulation. We studied how estradiol feedback regulates GnRH excitability, a key determinant of neural firing rate using laboratory and computational approaches. GnRH excitability is upregulated during positive feedback, perhaps driving increased neural firing rate at this time. Kisspeptin increased GnRH excitability and was essential for estradiol regulation of excitability. Modeling predicts that multiple combinations of changes to GnRH intrinsic conductances can produce the firing response in positive feedback, suggesting the brain has many ways to induce ovulation.

Traumatic brain injury results in acute rarefication of the vascular network.

The role of the cerebrovascular network and its acute response to TBI is poorly defined and emerging evidence suggests that cerebrovascular reactivity is altered. We explored how cortical vessels are physically altered following TBI using a newly developed technique, vessel painting. We tested our hypothesis that a focal moderate TBI results in global decrements to structural aspects of the vasculature. Rats (naïve, sham-operated, TBI) underwent a moderate controlled cortical impact. Animals underwent vessel painting perfusion to label the entire cortex at 1 day post TBI followed by whole brain axial and coronal images using a wide-field fluorescence microscope. Cortical vessel network characteristics were analyzed for classical angiographic features (junctions, lengths) wherein we observed significant global (both hemispheres) reductions in vessel junctions and vessel lengths of 33% and 22%, respectively. Biological complexity can be quantified using fractal geometric features where we observed that fractal measures were also reduced significantly by 33%, 16% and 13% for kurtosis, peak value frequency and skewness, respectively. Acutely after TBI there is a reduction in vascular network and vascular complexity that are exacerbated at the lesion site and provide structural evidence for the bilateral hemodynamic alterations that have been reported in patients after TBI.

Excitability and Burst Generation of AVPV Kisspeptin Neurons Are Regulated by the Estrous Cycle Via Multiple Conductances Modulated by Estradiol Action.

The preovulatory secretory surge of gonadotropin-releasing hormone (GnRH) is crucial for fertility and is regulated by a switch of estradiol feedback action from negative to positive. GnRH neurons likely receive estradiol feedback signals via ERα-expressing afferents. Kisspeptin neurons in anteroventral periventricular nucleus (AVPV) are thought to be critical for estradiol-positive feedback induction of the GnRH surge. We examined the electrophysiological properties of GFP-identified AVPV kisspeptin neurons in brain slices from mice on the afternoon of diestrus (negative feedback) and proestrus (positive feedback, time of surge). Extracellular recordings revealed increased firing frequency and action potential bursts on proestrus versus diestrus. Whole-cell recordings were used to study the intrinsic mechanisms of bursting. Upon depolarization, AVPV kisspeptin neurons exhibited tonic firing or depolarization-induced bursts (DIB). Both tonic and DIB cells exhibited bursts induced by rebound from hyperpolarization. DIB occurred similarly on both cycle stages, but rebound bursts were observed more often on proestrus. DIB and rebound bursts were both sensitive to Ni(2+), suggesting that T-type Ca(2+) currents (I Ts) are involved. I T current density was greater on proestrus versus diestrus. In addition to I T, persistent sodium current (I NaP) facilitated rebound bursting. On diestrus, 4-aminopyridine-sensitive potassium currents contributed to reduced rebound bursts in both tonic and DIB cells. Manipulation of specific sex steroids suggests that estradiol induces the changes that enhance AVPV kisspeptin neuron excitability on proestrus. These observations indicate cycle-driven changes in circulating estradiol increased overall action potential generation and burst firing in AVPV kisspeptin neurons on proestrus versus diestrus by regulating multiple intrinsic currents.

GABAergic transmission to kisspeptin neurons is differentially regulated by time of day and estradiol in female mice.

Gonadotropin-releasing hormone (GnRH) secretion is regulated by estradiol feedback. This feedback switches from negative to positive in females; this switch depends on time of day in many species. Estradiol feedback is likely conveyed via afferents. Kisspeptin neurons of the arcuate nucleus and anteroventral-periventricular region (AVPV) may differentially regulate GnRH neurons during negative and positive feedback, respectively. We tested estradiol and time of day regulation of GABAergic transmission and postsynaptic response to GABA in these two populations using transgenic mice with GFP-identified kisspeptin neurons. Ovariectomized (OVX) mice treated or not with estradiol (E) were studied in the AM (negative feedback) or PM (positive feedback). GABAA receptor reversal potential was unaffected by time of day or estradiol. GABA depolarized the membrane potential of arcuate neurons from OVX+E mice; this response was blunted in cells from OVX mice. GABA hyperpolarized AVPV kisspeptin neurons, except in the OVX PM group in which GABA did not alter membrane potential attributable to a PM hyperpolarization of baseline membrane potential. In both kisspeptin neuron populations from OVX mice, the frequency of GABAergic spontaneous postsynaptic currents was increased in the PM; this increase was blunted by estradiol. In arcuate, but not AVPV, kisspeptin neurons, estradiol reduced miniature postsynaptic current amplitude independent of time of day. Using nonstationary fluctuation analysis and diazepam to manipulate GABAA receptor apparent affinity, the decrease in arcuate miniature postsynaptic current amplitude was attributed to decreased number of receptors bound by GABA. Time of day and estradiol feedback thus both target presynaptic and postsynaptic mechanisms to differentially regulate kisspeptin neurons via GABAergic transmission.

Albumin therapy enhances collateral perfusion after laser-induced middle cerebral artery branch occlusion: a laser speckle contrast flow study.

Laser speckle contrast (LSC) was used to compare the extent of cortical ischemia in two inbred mouse strains that differed in their degree of collateral circulation, after laser occlusion of the distal middle cerebral artery, and after treatment with 25% albumin (ALB) or saline (control). Sequential LSC images acquired over ∼90 minutes were coaligned, converted to relative flow, and normalized to baseline. After 3-day survival, infarction was quantified by triphenyl tetrazolium chloride or magnetic resonance imaging. In the sparsely collateralized BALB/c strain, mean flow fell to 13% to 14% and 33% to 34% of baseline in central (core) and peripheral (penumbral) regions of interest, and ALB treatment at 30 minutes enhanced perfusion in both regions by ∼2-fold relative to saline, restoring flow to the benign-oligemic range centrally, and to the hyperemic range peripherally. The ALB-induced increment in parenchymal perfusion was disproportionate to the subtle flow increase in the occluded artery itself, suggesting that ALB improved collateral circulation. Cortical infarction in BALB/c mice was reduced 45% by ALB treatment. In contrast to BALB/c mice, the better-collateralized CD-1 strain developed milder ischemia, had smaller infarcts, and showed no differential benefit of ALB. We conclude that where native collateralization is insufficient (BALB/c strain), ALB treatment exerts a significant therapeutic effect after ischemia by augmenting collateral perfusion.

Fluorescence patterning in films of a photoswitchable BODIPY-spiropyran dyad.

A BODIPY-spiropyran dyad was embedded within poly(methyl methacrylate) films spin-coated on glass slides. Visible illumination of the resulting materials excites selectively the BODIPY fragment, which then deactivates radiatively by emitting light in the form of fluorescence. Ultraviolet irradiation promotes the isomerization of the spiropyran component to the corresponding merocyanine. This photoinduced transformation activates electron and energy transfer pathways from the fluorescent to the photochromic fragment. Consistently, the BODIPY fluorescence is effectively suppressed within the photogenerated isomer. As a result, ultraviolet illumination with a laser, producing a doughnut-shaped spot on the sample, confines the fluorescent species within the doughnut hole. This behavior is an essential requisite for the implementation of super-resolution imaging schemes based on fluorescence photodeactivation. Thus, the operating principles governing the photochemical and photophysical response of this molecular switch can ultimately lead to the development of innovative probes for fluorescence nanoscopy.

Albumin therapy augments the effect of thrombolysis on local vascular dynamics in a rat model of arteriolar thrombosis: a two-photon laser-scanning microscopy study.

BACKGROUND AND PURPOSE: Results of our recent pilot clinical trial suggest that the efficacy of thrombolytic therapy in acute ischemic stroke may be enhanced by the coadministration of high-dose albumin. Here, we explored the microvascular hemodynamic effects of this combined therapy in a laboratory model of cortical arteriolar thrombosis. METHODS: We studied the cortical microcirculation of physiologically monitored rats in vivo by two-photon laser-scanning microscopy after plasma-labeling with fluorescein-dextran. We induced focal thrombosis in 30- to 50-microm cortical arterioles by laser irradiation and measured arteriolar flow velocity by repeated line-scanning. At 30 minutes post-thrombosis, we treated animals with the thrombolytic agent, reteplase, which was coadministered with either human albumin, 2 g/kg, or with saline control. RESULTS: Baseline arteriolar flow velocity averaged 3.8+/-0.7 mm/s, was immediately reduced by thrombosis to 22% to 25% of control values, and remained unchanged before treatment. Subthrombolytic doses of reteplase combined with saline led to a median increase in flow velocity to 37% of control distal to the thrombus (P=nonsignificant versus pretreatment). By contrast, reteplase combined with albumin therapy resulted in a prompt, highly significant increase of median flow velocity to 58% of control levels (P=0.013 versus reteplase+saline), which remained significantly higher than the reteplase+saline group at multiple time-points over the subsequent hour. CONCLUSIONS: The beneficial effect of subthrombolytic doses of reteplase on microvascular hemodynamics distal to a cortical arteriolar thrombosis is markedly enhanced by the coadministration of high-dose albumin therapy; these results have important clinical implications for the management of patients with acute ischemic stroke.

Separate populations of receptor cells and presynaptic cells in mouse taste buds.

Taste buds are aggregates of 50-100 cells, only a fraction of which express genes for taste receptors and intracellular signaling proteins. We combined functional calcium imaging with single-cell molecular profiling to demonstrate the existence of two distinct cell types in mouse taste buds. Calcium imaging revealed that isolated taste cells responded with a transient elevation of cytoplasmic Ca2+ to either tastants or depolarization with KCl, but never both. Using single-cell reverse transcription (RT)-PCR, we show that individual taste cells express either phospholipase C beta2 (PLCbeta2) (an essential taste transduction effector) or synaptosomal-associated protein 25 (SNAP25) (a key component of calcium-triggered transmitter exocytosis). The two functional classes revealed by calcium imaging mapped onto the two gene expression classes determined by single-cell RT-PCR. Specifically, cells responding to tastants expressed PLCbeta2, whereas cells responding to KCl depolarization expressed SNAP25. We demonstrate this by two methods: first, through sequential calcium imaging and single-cell RT-PCR; second, by performing calcium imaging on taste buds in slices from transgenic mice in which PLCbeta2-expressing taste cells are labeled with green fluorescent protein. To evaluate the significance of the SNAP25-expressing cells, we used RNA amplification from single cells, followed by RT-PCR. We show that SNAP25-positive cells also express typical presynaptic proteins, including a voltage-gated calcium channel (alpha1A), neural cell adhesion molecule, synapsin-II, and the neurotransmitter-synthesizing enzymes glutamic acid decarboxylase and aromatic amino acid decarboxylase. No synaptic markers were detected in PLCbeta2 cells by either amplified RNA profiling or by immunocytochemistry. These data demonstrate the existence of at least two molecularly distinct functional classes of taste cells: receptor cells and synapse-forming cells.

Horizontal spread of activity in neocortical inhibitory networks.

In the presence of 4-aminopyridine (4-AP) and excitatory amino acid receptor blockers, GABAergic networks in the neocortex give rise to large spontaneous GABA-mediated depolarizations. We used voltage-sensitive dye techniques to explore the network properties of depolarizing GABA responses. Voltage-sensitive dye signals demonstrated that the superficial layers support the propagation of depolarizing GABA responses, with only minimal signals detected in deeper cortical layers. GABA responses propagated at a speed of 2.7 +/- 0.2 mm/s, a rate intermediate to fast synaptic transmission and spreading depression. Changes in the extracellular potassium concentration altered the propagation speed of the depolarizing GABA response. Taken together, these data support a role for both direct synaptic action of GABA at GABA(A) receptors and nonsynaptic mechanisms in the generation and propagation of depolarizing GABA responses.