A novel method to quantify cutaneous vascular innervation.

INTRODUCTION: To develop a new method to quantify the density of nerves, vessels, and the neurovascular contacts, we studied skin biopsies in diabetes and control subjects. METHODS: Skin biopsies with dual immunofluorescent staining were used to visualize nerves and blood vessels. The density of nerves, vessels, and their neurovascular contacts were quantified with unbiased stereology. Results were compared with examination findings, validated questionnaires, and autonomic function. RESULTS: In tissue from 19 controls and 20 patients with diabetes, inter-rater and intra-rater intraclass correlation coefficients were high (>0.85; P < .001) for all quantitative methods. In diabetes, the nerve densities (P < .05), vessel densities (P < .01), and the neurovascular densities (P < .01) were lower compared with 20 controls. Results correlated with autonomic function, examination and symptom scores. DISCUSSION: We report an unbiased, stereological method to quantify the cutaneous nerve, vessel and neurovascular density and offer new avenues of investigation into cutaneous neurovascular innervation in health and disease.

Granular Layer Neurons Control Cerebellar Neurovascular Coupling Through an NMDA Receptor/NO-Dependent System.

Neurovascular coupling (NVC) is the process whereby neuronal activity controls blood vessel diameter. In the cerebellum, the molecular layer is regarded as the main NVC determinant. However, the granular layer is a region with variable metabolic demand caused by large activity fluctuations that shows a prominent expression of NMDA receptors (NMDARs) and nitric oxide synthase (NOS) and is therefore much more suitable for effective NVC. Here, we show, in the granular layer of acute rat cerebellar slices, that capillary diameter changes rapidly after mossy fiber stimulation. Vasodilation required neuronal NMDARs and NOS stimulation and subsequent guanylyl cyclase activation that probably occurred in pericytes. Vasoconstriction required metabotropic glutamate receptors and CYP ω-hydroxylase, the enzyme regulating 20-hydroxyeicosatetraenoic acid production. Therefore, granular layer capillaries are controlled by the balance between vasodilating and vasoconstricting systems that could finely tune local blood flow depending on neuronal activity changes at the cerebellar input stage. SIGNIFICANCE STATEMENT: The neuronal circuitry and the biochemical pathways that control local blood flow supply in the cerebellum are unclear. This is surprising given the emerging role played by this brain structure, not only in motor behavior, but also in cognitive functions. Although previous studies focused on the molecular layer, here, we shift attention onto the mossy fiber granule cell (GrC) relay. We demonstrate that GrC activity causes a robust vasodilation in nearby capillaries via the NMDA receptors-neuronal nitric oxide synthase signaling pathway. At the same time, metabotropic glutamate receptors mediate 20-hydroxyeicosatetraenoic acid-dependent vasoconstriction. These results reveal a complex signaling network that hints for the first time at the granular layer as a major determinant of cerebellar blood-oxygen-level-dependent signals.

Spatiotemporal distribution of neurovascular alignment in remodeling adult rat mesentery microvascular networks.

An emerging area of microvascular research focuses on the links between neural and vascular patterning. However, the functional dependence between vascular and neural growth in adult tissues remains underinvestigated. The objective of this study was to determine the spatial and temporal coordination between vascular and neural networks over a time course of adult microvascular growth. Mesentery tissues from adult male Wistar rats were harvested prior to stimulation, and 2, 10 and 30 days after angiogenesis stimulated by mast cell degranulation. Tissues were immunolabeled for PECAM (endothelial cell marker) and class III ß-tubulin (peripheral nerve marker). Neurovascular alignment was quantified per vessel category: arterioles (>20 µm), pre-capillary arterioles (10-20 µm), post-capillary venules (10-20 µm), venules (>20 µm), capillaries (<10 µm) and capillary sprouts. Neurovascular alignment along pre-capillary arterioles, capillaries, post-capillary venules and venules was decreased compared to unstimulated levels on days 2 and 10. These decreases inversely correlated with increases in vessel density per vessel category. By day 30, alignment either returned to unstimulated levels or was increased compared to day 10. These results suggest that neurovascular alignment arises after microvascular network growth and is present along arterioles, venules and even capillaries.

Oscillating neuro-capillary coupling during cortical spreading depression as observed by tracking of FITC-labeled RBCs in single capillaries.

Coupling between capillary red blood cell (RBC) movements and neuronal dysfunction during cortical spreading depression (CSD) was examined in rats by employing a high-speed camera laser-scanning confocal fluorescence microscope system in conjunction with our Matlab domain software (KEIO-IS2). Following microinjection of K(+) onto the surface of the brain, changes in electroencephalogram (EEG), DC potential and tissue optical density were all compatible with the occurrence of a transient spreading neuronal depression. RBC flow in single capillaries was not stationary. Unpredictable redistribution of RBCs at branches of capillaries was commonly observed, even though no change in diameter was apparent at the reported site of the capillary sphincter and no change of arteriolar-venule pressure difference was detected. There appeared to be a slow morphological change of astroglial endfeet. When local neurons were stunned transiently by K(+) injection, the velocity and oscillation frequency of RBCs flowing in nearby capillaries started to decrease. The flow in such capillaries was rectified, losing oscillatory components. Sluggish floating movements of RBCs in pertinent capillaries were visualized, with occasional full stops. When CSD subsided, RBC movements recovered to the original state. We postulate that neuronal depolarization blocks oscillatory signaling to local capillaries via low-shear plasma viscosity increases in the capillary channels, and a complex interaction between the RBC surface and the buffy coat on the capillary wall surface increases the capillary flow resistance. Then, when CSD subsides and oscillatory neuronal function is recovered, the normal physiological conditions are restored.

Excitatory effects of the puberty-initiating peptide kisspeptin and group I metabotropic glutamate receptor agonists differentiate two distinct subpopulations of gonadotropin-releasing hormone neurons.

Activation of the G-protein-coupled receptor GPR54 by kisspeptins during normal puberty promotes the central release of gonadotropin-releasing hormone (GnRH) that, in turn, leads to reproductive maturation. In humans and mice, a loss of function mutations of GPR54 prevents the onset of puberty and leads to hypogonadotropic hypogonadism and infertility. Using electrophysiological, morphological, molecular, and retrograde-labeling techniques in brain slices prepared from vGluT2-GFP and GnRH-GFP mice, we demonstrate the existence of two physiologically distinct subpopulations of GnRH neurons. The first subpopulation is comprised of septal GnRH neurons that colocalize vesicular glutamate transporter 2 and green fluorescent protein and is insensitive to metabotropic glutamate receptor agonists, but is exquisitely sensitive to kisspeptin which closes potassium channels to dramatically initiate a long-lasting activation in neurons from prepubertal and postpubertal mice of both sexes. A second subpopulation is insensitive to kisspeptin but is uniquely activated by group I metabotropic glutamate receptor agonists. These two physiologically distinct classes of GnRH cells may subserve different functions in the central control of reproduction and fertility.

beta-Adrenergic regulation of adenosine 3',5'-monophosphate concentration in brain microvessels.

Norepinephrine increases the concentration of adenosine 3',5'-monophosphate (cyclic AMP) in an incubated suspension of brain microvessels. This response can be matched by other drugs that stimulate the beta receptors, but the alpha-adrenergic agonist phenylephrine is without effect; beta-adrenergic blockade abolishes the response while alpha-adrenergic blockade produces no change. The data support the contention that cerebral capillary function is subject to adrenergic neural control.

The Pericyte of the Pancreatic Islet Regulates Capillary Diameter and Local Blood Flow.

Efficient insulin secretion requires a well-functioning pancreatic islet microvasculature. The dense network of islet capillaries includes the islet pericyte, a cell that has barely been studied. Here we show that islet pericytes help control local blood flow by adjusting islet capillary diameter. Islet pericytes cover 40% of the microvasculature, are contractile, and are innervated by sympathetic axons. Sympathetic adrenergic input increases pericyte activity and reduces capillary diameter and local blood flow. By contrast, activating beta cells by increasing glucose concentration inhibits pericytes, dilates islet capillaries, and increases local blood flow. These effects on pericytes are mediated by endogenous adenosine, which is likely derived from ATP co-released with insulin. Pericyte coverage of islet capillaries drops drastically in type 2 diabetes, suggesting that, under diabetic conditions, islets lose this mechanism to control their own blood supply. This may lead to inadequate insulin release into the circulation, further deteriorating glycemic control.

Origins of the neurovascular bundle: interactions between developing nerves and blood vessels in embryonic chick skin.

Growth cones of nerves and endothelial cells of blood vessels are closely analogous in their migratory behavior, and they are both set a similar task during the early development of a limb. Both must invade the mesenchyme to form ramifying networks of large nerves and vessels. Both systems must densely pervade certain regions of the developing limb, such as muscle rudiments, and both form dense cutaneous plexuses at precisely the same depth beneath the epidermis. Moreover, adult tissues show many examples of neurovascular bundles in which nerves and blood vessels run closely parallel and branch in a correlated fashion, suggesting some interdependence during development. We have examined the interrelationship between developing nerves and blood vessels in chick wing skin because it allows a particularly convenient two-dimensional analysis of the two systems which can be revealed simultaneously in the same preparation by injection of Indian ink combined with silver-staining. We show that nerves do not use blood vessels as pathways along which to crawl, but that there are two other ways in which neurovascular associations arise: in some situations nerves and blood vessels follow the same route because they are responding independently to the same mesenchymal cues; and in some situations nerves induce blood vessels to remodel around them.

Suprachiasmatic efferents avoid phenestrated capillaries but innervate neuroendocrine cells, including those producing dopamine.

The key role of the suprachiasmatic nucleus in the diurnal regulation of anterior pituitary hormone secretions, including PRL, is well established. However, the pathway via suprachiasmatic signals reach the pituitary is ill defined. To determine whether suprachiasmatic efferents innervate neuroendocrine cells, the anterograde tracer, Phaseolus vulgaris leukoagglutinin, was injected iontophoretically into the suprachiasmatic nucleus in parallel with ip administration of fluorogold (20 mg/BW in saline). After visualization of anterogradely labeled processes with a dark blue chromogen. Vibratome sections were immunostained for fluorogold. As fluorogold labeling resulted in dense immunopositive granules without diffuse cytoplasmic labeling, selected sections were further immunostained for cytoplasmic tyrosine hydroxylase (dopamine). Anterogradely labeled suprachiasmatic efferents were observed in the medial preoptic area, periventricular regions, and the lateral aspects of the arcuate and ventromedial nuclei of the hypothalamus, whereas the median eminence and organum vasculosum laminae terminalis lacked labeled suprachiasmatic projections. All of the aforementioned regions contained a high number of cells immunoreactive for fluorogold. However, immunolabeling for fluorogold revealed no retrogradely labeled (ergo neuroendocrine) cells in the suprachiasmatic nucleus. Retrogradely labeled cells in all of these hypothalamic sites, with the exception of the median eminence and organum vasculosum laminae terminalis, were targets of suprachiasmatic nucleus axon terminals. In the preoptic area, anterior hypothalamus, periventricular area, and arcuate nucleus, subpopulations of dopamine cells were retrogradely labeled. In all of these areas, both retrogradely labeled and nonlabeled dopamine cells were frequently found to be in contact with dark blue, anterogradely labeled suprachiasmatic efferents. Electron microscopic examination confirmed the putative connections to be synaptic. This experiment provided evidence that the circadian pacemaker suprachiasmatic nucleus sends efferents onto neuroendocrine cells, but has no contacts with fenestrated capillaries. It was found that a population of median eminence-projective cells targeted by suprachiasmatic axons in the hypothalamus contains dopamine. These observations indicate no direct effect of the circadian pacemaker on the anterior hypophysis, but offer an indirect pathway via circadian signals, mediated by hypothalamic neural systems, that may regulate pituitary hormone secretion, in particular PRL.

Innervation of capillaries by local neurons in the cat hypothalamus: a light microscopic study with horseradish peroxidase.

The protein tracer horseradish peroxidase (HRP) has been used in an attempt to define the cell bodies of origin of "nonadrenergic" varicose axons which terminate on the walls of hypothalamic capillaries. Capillaries in this region are also known to receive direct axonal contacts from adrenergic neurons in the pontine locus ceruleus. Solutions of HRP were infused into the lateral ventricles of adult cats of either sex and permitted to circulate in the cerebrospinal fluid spaces for 10 min, 20 min, or 2 h. During these periods HRP entered the perivascular spaces around penetrating arterioles and spread into the surrounding extracellular spaces of the hypothalamus. Certain neurons in the periarteriolar neuropil were consistently labeled by the tracer after all three circulation periods. These cells, including all of their processes, could be visualized in detail. Most neurons, by contrast, did not accumulate HRP. The axons of some tracer-filled neurons terminated on the walls of capillaries in the immediate vicinity of the penetrating arteriole. The arrangement and distribution of these cells suggest that they may provide a substrate for local neural influences on the hypothalamic microcirculation.

Localized dynamic changes in cortical blood flow with whisker stimulation corresponds to matched vascular and neuronal architecture of rat barrels.

The hypothesis that functional groups of neurons in whisker barrels are linked to a modular organization of cortical vessels was tested. Endovascular casts demonstrated cortical capillary networks resembling the whisker barrel pattern that were fed from the middle cerebral artery. In histological sections, dense capillaries apparently were confined to single barrels and were supplied by one or a few penetrating arterioles. The barrel field in cortical layer IV was localized in relation to surface arteriovenous patterns. Living vessels were imaged through a closed cranial window under anesthesia with a fluorescence microscope and SIT or ICCD cameras. After intracarotid injections of fluorescein isothiocyanatedextrans, saline, or 3 microns latex beads, changes in arteriolar diameter, arteriovenous transit times (AVTTs), and bead velocities were measured. When row C whiskers were stroked at 4-5 Hz for 1 min, blood flow increased in arterioles that supplied contralateral row C barrels as demonstrated by postmortem histology. AVTTs slowed significantly in vessels supplying adjacent cortex. We hypothesize that cerebral vascular units supply individual whisker barrels and are functionally linked to them for precise focal regulation of cerebral blood flow.

On the possible relationship of cortical microvascular pathology to blood brain barrier changes in Alzheimer's disease.

A group of alterations in the structure of cortical microvessels is described in the brain tissue of neuropathologically confirmed patients with Alzheimer's disease. These changes include irregular thickening of the vessel walls, infiltration with amyloid and the serum P component of amyloid, loss of the perivascular neural plexus and the frequent occurrence of pits and lacunae in vessel walls. The possibility that these alterations may be related to blood brain barrier defects is suggested.

A histochemical study of the innervation of cerebral blood vessels in the turtle.

Specific histochemical techniques for the demonstration of noradrenaline and of acetylcholinesterase have been used to study the distribution of adrenergic and cholinergic nerves to the cerebral blood vessels of turtle, Geoclemys reevesii. The major and medium-sized cerebral arteries were supplied with dense adrenergic nerve plexuses, the plexuses were particularly dense in the medium-sized pial arteries of very thick vascular wall, indicating the functional significance of these arteries in the cerebral circulation. The parenchymal arterioles and capillaries were also supplied with adrenergic nerves. On the other hand, the cholinergic innervation was less dense than the adrenergic one and the acetylcholinesterase activity of the nerve fibres was remarkably weak. By contrast, the parenchymal small arterioles and capillaries exhibited heavy acetylcholinesterase activity on the vascular wall and, in addition, the capillaries were supplied with the well-stained acetylcholinesterase-positive nerve fibres. These fibres and also the adrenergic fibres associating with the capillaries appear to be of central origin. It is suggested that the cholinergic mechanisms in the parenchymal small vessels also play an important role in the cerebral circulation. The basophil leucocytes observed abundantly in the blood of turtle emitted an intensive greenish yellow fluorescence after formaldehyde gas-treatment.

An ultrastructural analysis of neurites in the basal lamina of capillaries in the chinchilla cochlear nucleus.

In an ultrastructural study of the chinchilla anteroventral cochlear nucleus (AVCN), we found innervated capillaries in the layer of granule cells that comprises the dorsolateral portion of the AVCN and forms a cap over the principal portion of the nucleus. In 66% of 215 capillaries we examined in the granule cell layer of various levels of the AVCN, we found structures having ultrastructural features of axons that (1) were within the brain parenchyma and were in direct contact with the pericapillary basal lamina, or (2) were separate fro, the adjacent neuropil and, often in the company of astrocytic processes, were completely enveloped by the pericapillary basal lamina. An analysis of serial sections confirmed that neurites within the pericapillary basal lamina were in continuity with neurites in the brain parenchyma. Most neurites within the basal lamina of capillaries were next to pericytes, but some neurites were next to endothelial cells. None of the neurites adjoining capillaries had the abundance of synaptic vesicles typical of autonomic vasomotor nerve endings and synaptic terminals. Consequently, they may be sensory, responding perhaps to changes in hydrostatic pressure or chemical composition of the blood or cerebrospinal fluid. However, we cannot exclude the possibility that neurites which accompany capillaries in the AVCN terminate elsewhere. In the AVCN the innervation of capillaries is restricted to the superficial layer of granule cells. In none of 177 capillaries of the principal portion of the AVCN did we find an example of a neurite in contact with the pericapillary basal lamina, a pericyte, or an endothelial cell, although it was sometimes necessary to examine specimens at various angles of tilt to confirm that the pericapillary glial sleeve was continuous. Furthermore, we found no innervated blood vessels among 266 capillaries examined in the granular and molecular layers of the cerebellar cortex.

Neurohemal contact in the internal zone of the rabbit median eminence.

The concept of neurosecretion as the mechanism by which neural control of adenohypophyseal function is accomplished was based on the observation that long capillary loops penetrate deeply into the supraopticohypophyseal tract as it passes through the median eminence internal zone. However, neural contact upon these capillary loops has not been demonstrated in the mammalian median eminence. The present transmission electron microscopic investigation of the rabbit median eminence demonstrates neurohemal contact in the median eminence internal zone. Axons containing small lucent vesicles 53.3 +/- 3.28 nm in diameter (mean +/- SEM) or small lucent and large granular vesicles with a mean diameter of 122.4 (+/- 3.28 nm) in their terminals make neurohemal contact with capillary loops in the internal zone and form a cuff about them. These terminals resemble terminals found in the external zone. Intravenous injection of the false neurotransmitter 5-hydroxydopamine (5-OH-DA) renders small lucent vesicles granular in both the external and internal zone. The effect of 5-OH-DA injection is abolished by concurrent reserpine administration. Whereas large granular vesicles in many terminals become lucent after reserpine administration, in others they remained electron dense. Viewed in the light of previous studies our findings suggest that the internal plexus arises from the external plexus and invaginates the neuropil carrying connective tissue and parvicellular axon terminals of aminergic and peptidergic systems from the external zone into the internal zone, that some elements making neurohemal contact with long capillary loops are terminals of the noradrenergic reticular infundibular tract arising outside the hypothalamus in the brainstem, and that long capillary loops form a system of repeating microvascular modules which markedly increase the surface available for neurohemal contact.

Prevention of arteriolar rarefaction in the spontaneously hypertensive rat by exposure to simulated high altitude.

Microvascular alterations associated with reversal of hypertension by exposure to simulated high altitude were investigated in the gracilis muscle of the spontaneously hypertensive rat (SHR). Male, 4-week-old SHR were either placed in a hypobaric chamber (SHR-HA) at 430 mmHg or maintained at ambient barometric pressure (SHR-SL) for 8-10 or 12-14 weeks. Measurements of microvascular diameter and density were made by closed-circuit television microscopy. Mean arterial blood pressure was significantly lower in the SHR-HA groups. Functional arteriolar rarefaction occurred in SHR-SL at 8-10 weeks while structural arteriolar rarefaction occurred after 12-14 weeks. In contrast, neither functional nor structural rarefaction of arterioles was observed in the SHR-HA groups. Although functional rarefaction of capillaries did occur in SHR-HA, it was less than that in SHR-SL. Total capillary density, however, was similar in SHR-SL and SHR-HA after 12-14 weeks. Vasoconstriction was decreased in SHR-HA and vasodilated arteriolar diameters were greater than those of SHR-SL. These results show that simulated high altitude lowered blood pressure in SHR and resulted in a microcirculatory bed resembling that seen in a normotensive animal.

Light- and electron-microscopic characterization of electrophysiologically-identified, horseradish peroxidase-injected magnocellular neuroendocrine cells in goldfish preoptic nucleus.

We recorded intracellularly from neurons in the goldfish preoptic nucleus which were antidromically identified by electrical stimulation of the pituitary gland and marked by intracellular injection of horseradish peroxidase for subsequent localization. At the light-microscopic level, labeled neurons resembled profiles of Golgi-impregnated neurons and lay in the magnocellular portion of the preoptic nucleus. Densely labeled axons and dendrites projected to the lateral forebrain bundle, the medial forebrain bundle, fiber tracts in the preoptico-hypophysial tract, small blood vessels and capillaries, the ependymal lining of the third ventricle and toward the preoptic neurons. Occasionally, a lightly-labeled, large perikaryon lay adjacent to a large, heavily-labeled magnocellular neuron. Ultrastructural examination of these identified cells revealed dense reaction product in neuronal perikarya and processes. Heavily labeled perikarya had elaborate networks of endoplasmic reticulum, extensive Golgi apparatus, occasional somatic spines and infrequent axo-somatic contacts from unlabeled neurons. These labeled perikarya which were frequently in close somatic apposition with unlabeled profiles were sometimes adjacent to a large, lightly-labeled perikaryon. A thin glial sheath separated most labeled neurons and processes from brain capillary endothelium. Labeled dendrites had heavily labeled spines and axo-dendritic contacts from unlabeled neurons. Labeled axons abutted unlabeled-axons and -dendrites. Synaptic boutons innervating labeled structures always contained small clear synaptic vesicles and some boutons also contained large dense-core vesicles. These results demonstrate the complex connections of goldfish preoptic magnocellular neuroendocrine cells with other neurons, fiber systems, brain capillaries, ventricular ependyma and the pituitary and provide further support for non-endocrine as well as endocrine functions of magnocellular neurons.

Functional immunohistochemistry of neuropeptides and nitric oxide synthase in the nerve fibers of the supratentorial dura mater in an experimental migraine model.

The supratentorial cerebral dura of the albino rat is equipped with a rich sensory innervation both in the connective tissue and around blood vessels, which includes nociceptive axons and their terminals; these display intense calcitonin gene-related peptide (CGRP) immunoreactivity. Stereotactic electrical stimulation of the trigeminal (Gasserian) ganglion, regarded as an experimental migraine model, caused marked increase and disintegration of club-like perivascular CGRP-immunopositive nerve endings in the dura mater and induced an apparent increase in the lengths of CGRP-immunoreactive axons. Intravenous administration of sumatriptan or eletriptan, prior to electrical stimulation, prevented disintegration of perivascular terminals and induced accumulation of CGRP in terminal and preterminal portions of peripheral sensory axons. Consequently, immunopositive terminals and varicosities increased in size; accumulation of axoplasmic organelles resulted in the "hollow" appearence of numerous varicosities. Since triptans exert their anti-migraine effect by virtue of agonist action on 5-HT(1D/B) receptors, we suggest that these drugs prevent the release of CGRP from perivascular nerve terminals in the dura mater by an action at 5-HT(1D/B) receptors. Nitroglycerine (NitroPOHL), given subcutaneously to rats, induces increased beading of nitric oxide synthase (NOS)-immunoreactive nerve fibers in the supratentorial cerebral dura mater, and an apparent increase in the number of NOS-immunoreactive nerve fibers in the dural areas supplied by the anterior and middle meningeal arteries, and the sinus sagittalis superior. Structural alterations of nitroxidergic axons innervating blood vessels of the dura mater support the idea that nitric oxide (NO) is involved in the induction of headache, a well-known side effect of coronary dilator agents.

Simulation of motor unit recruitment and microvascular unit perfusion: spatial considerations.

Muscle fiber activity is the principal stimulus for increasing capillary perfusion during exercise. The control elements of perfusion, i.e., microvascular units (MVUs), supply clusters of muscle fibers, whereas the control elements of contraction, i.e., motor units, are composed of fibers widely scattered throughout muscle. The purpose of this study was to examine how the discordant spatial domains of MVUs and motor units could influence the proportion of open capillaries (designated as perfusion) throughout a muscle cross section. A computer model simulated the locations of perfused MVUs in response to the activation of up to 100 motor units in a muscle with 40,000 fibers and a cross-sectional area of 100 mm2. The simulation increased contraction intensity by progressive recruitment of motor units. For each step of motor unit recruitment, the percentage of active fibers and the number of perfused MVUs were determined for several conditions: 1) motor unit fibers widely dispersed and motor unit territories randomly located (which approximates healthy human muscle), 2) regionalized motor unit territories, 3) reversed recruitment order of motor units, 4) densely clustered motor unit fibers, and 5) increased size but decreased number of motor units. The simulations indicated that the widespread dispersion of motor unit fibers facilitates complete capillary (MVU) perfusion of muscle at low levels of activity. The efficacy by which muscle fiber activity induced perfusion was reduced 7- to 14-fold under conditions that decreased the dispersion of active fibers, increased the size of motor units, or reversed the sequence of motor unit recruitment. Such conditions are similar to those that arise in neuromuscular disorders, with aging, or during electrical stimulation of muscle, respectively.