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1 Laboratory of Cerebrovascular Biology and Stroke, Departments of Neurology and 2 Neuroscience, University of Minnesota Medical School, Minneapolis, Minnesota 55455
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Crus II is an area of the cerebellar
cortex that receives trigeminal afferents from the perioral region. We
investigated the mechanisms of functional hyperemia in cerebellum using
activation of crus II by somatosensory stimuli as a model. In
particular, we sought to determine whether stimulation of the perioral
region increases cerebellar blood flow
(BFcrb) in crus II and, if so, whether the response depends on activation of
2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-kainate
receptors and nitric oxide (NO) production. Crus II was exposed in
anesthetized rats, and the site was superfused with Ringer. Field
potentials were recorded, and
BFcrb was measured by
laser-Doppler flowmetry. Crus II was activated by electrical stimulation of the perioral region (upper lip). Perioral stimulation evoked the characteristic field potentials in crus II and increased BFcrb (34 ± 6%; 10 Hz-25 V;
n = 6) without changing arterial
pressure. The BFcrb increases were
associated with a local increase in glucose utilization (74 ± 8%;
P < 0.05;
n = 5) and were attenuated by the
AMPA-kainate receptor antagonist
2,3-dihydroxy-6-nitro-7-sulfamoylbenzo-[f]quinoxaline (
71 ± 3%; 100 µM; P < 0.01; n = 5). The neuronal NO synthase inhibitor 7-nitroindazole (7-NI, 50 mg/kg;
n = 5) virtually abolished the
increases in BFcrb
(90 ± 2%; P < 0.01) but did not affect the amplitude of the field potentials. In
contrast, 7-NI attenuated the increase in neocortical cerebral blood
flow produced by perioral stimulation by 52 ± 6%
(P < 0.05;
n = 5). We conclude that crus II
activation by somatosensory stimuli produces localized increases in
local neural activity and BFcrb
that are mediated by activation of glutamate receptors and NO. Unlike
in neocortex, in cerebellum the vasodilation depends almost exclusively
on NO. The findings underscore the unique role of NO in the mechanisms
of synaptic function and blood flow regulation in cerebellum.
cerebral circulation; laser-Doppler flowmetry; glucose utilization; vasodilation; glutamate receptors
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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NEURAL ACTIVITY is one of the major factors regulating the cerebral circulation (see Ref. 7 for a review). Thus focal or global increases in brain activity result in increases in cerebral blood flow (CBF) that are largely proportional to the intensity of activation and highly restricted to the activated areas (e.g., Refs. 9, 11, 35). However, most studies of the "coupling" between neural activity and blood flow have been performed in the cerebral cortex (see Ref. 35 for a review), and relatively little is known about the neural regulation of flow in other brain regions, such as the cerebellum.
The cerebellar cortex includes granule neurons, Purkinje neurons, and
interneurons (basket, Golgi, and stellate cells; see Ref. 28). The
climbing fibers (CF) and the parallel fibers (PF) provide the major
input to Purkinje neurons. The CF originate from the contralateral
inferior olive, whereas the PF arise from granule cells. The granule
cells, in turn, receive inputs from brain stem nuclei and spinal cord
via the mossy fibers (34). The transmitter released from PF and CF is
glutamate or a closely related excitatory amino acid, whereas
-aminobutyric acid is the major inhibitory transmitter released from
Purkinje cells and interneurons (29). Activation of glutamate receptors
is linked to production of nitric oxide (NO), a molecular mediator that
plays an important role in synaptic function in the cerebellum (see
Ref. 5 for a review). Recent investigations have used electrical or
chemical stimulation of the PF and CF to study the relationship between
neural activity and blood flow in the cerebellum (1, 37). Although
these studies have provided an initial insight into the mechanisms of
functional hyperemia in the cerebellum, maximal stimulation of
individual input pathways is unlikely to reflect accurately the pattern
of activation that occurs during normal cerebellar function. Therefore,
it would be desirable to use a model that resembles more closely the
pattern of neural activity occurring in the working cerebellum.
In this study, we sought to gain further insights into the neural
regulation of cerebellar blood flow
(BFcrb) using somatosensory stimulation as a model of functional activation. In cerebellum, trigeminal afferents from the perioral region terminate in
noncontiguous patches in the posterior lobe, mainly in crus II (30,
34). One pathway by which these projections reach the cerebellum is via
the CF, which originate in the inferior olive and terminate in discrete
parasagittal zones (21). We found that stimulation of the perioral
region increases BFcrb in crus II.
The increase in BFcrb is
restricted to the activated region, is associated with increases in
local glucose utilization (GU), and is dependent on activation of
2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-kainate
receptors. In crus II, the BFcrb
response elicited by perioral stimulation is virtually abolished by
inhibition of NO synthase (NOS), whereas in somatosensory cortex NOS
inhibition attenuates the CBF increase by 
50%. The data suggest
that NO plays a more prominent role in the mechanisms coupling
functional activity to blood flow in cerebellum than in cerebral cortex.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Methods for monitoring blood flow using laser-Doppler flowmetry (LDF) for recording of field potentials and for determination of GU have been described previously (16, 17) and are summarized below.
General Surgical Procedures
Studies were performed on 58 male Sprague-Dawley rats (Sasco, Omaha, NE) weighing ~300 g. Rats were anesthetized with 5% halothane in an oxygen-nitrogen mixture. After induction of anesthesia, the concentration of halothane was reduced to 1-2%. Catheters were inserted in both femoral arteries, in the left femoral vein, and in the trachea. Animals were then placed on a stereotaxic frame (model 1404; D. Kopf Instruments, Tujunga, CA) mounted on a vibration-free table (TMC, Peabody, MA) and artificially ventilated with a oxygen-nitrogen mixture by a mechanical ventilator (model 638; Harvard Apparatus, South Natick, MA). The oxygen concentration in the mixture was adjusted to obtain an arterial PO2 of 140-160 mmHg (Table 1). Body temperature was maintained at 37 ± 0.5°C using a heating lamp thermostatically controlled by a rectal probe (model 73A-TA; YSI, Yellow Springs, OH). One of the arterial catheters was used for continuous recording of arterial pressure and heart rate on a chart recorder (model 716P; Grass, Quincy, MA), whereas the other arterial catheter was used for blood sampling. Arterial PCO2, PO2, and pH were measured at multiple times on 100 µl of blood using a blood gas analyzer (model 248; Chiron Diagnostics, Norwood, MA). At the end of the surgical procedures, the halothane concentration was reduced to 1%. Because animals were not paralyzed, the adequacy of the level of anesthesia was assessed by testing corneal reflexes and motor responses to tail pinch.
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Perioral Stimulation and Monitoring of Blood Flow and Field Potentials
A small craniotomy (3 × 3 mm) was performed in the interparietal bone using a dental drill. The dura was carefully removed, and the left cerebellar hemisphere was exposed (crus I and II). In studies in which CBF was monitored in the somatosensory cortex, a small craniotomy (3 × 3 mm) was performed 2-3 mm posterior to bregma and 6 mm lateral to the midline. The cranial window was continuously superfused with Ringer or drugs at a rate of 0.33 ml/min (see Ref. 15 for Ringer composition and Ref. 17). The turnover rate of the window volume was 20-30 s. As in previous studies, solutions were equilibrated with 100% O2 and 5% CO2 (pH 7.3-7.4) and warmed to 37°C (17). Two needle electrodes (distance between electrodes: 0.5 cm) were inserted in the perioral region (upper lip) for delivering low-intensity electrical stimuli (5-30 V; 2-16 Hz; see Ref. 4). For BFcrb studies, stimuli were negative square waves (pulse duration 0.3 ms) delivered from a Grass S88 stimulator through a stimulus isolation unit (PSIU6; Grass).Field potentials were recorded in crus II by glass micropipettes (tip
diameter 30-50 µm) filled with 2 M NaCl (resistance 2-5
M
) and were inserted at a depth of 400 µm. Another micropipette served as reference electrode. The signal from the micropipettes was
fed through an extracellular microelectrode amplifier (7P5; Grass),
displayed on an oscilloscope, and digitized using a computerized data
acquisition system (MacAdios IIjr; GW Instruments). In studies of field
potentials, PF were stimulated at the rate of 1/s (100 µA; pulse
duration 0.3 ms). In each trial, 20 traces were acquired, averaged, and
stored for off-line analysis (Superscope software; GW Instruments).
CBF in somatosensory cortex and BFcrb in crus II were monitored using a laser-Doppler flowmeter (model BPM 403A; Vasamedic Flowmeter, St. Paul, MN; see Ref. 17). The LDF probe (tip diameter 0.8 mm) was mounted on a micromanipulator (Kopf) and was positioned 0.5 mm above the pial surface. The analog output of the flowmeter was fed into a direct current amplifier (model 7P1; Grass) and displayed on the polygraph. To avoid pulsatile variations in the flow signal, a long time constant was used (5 s). After a 10- to 20-min stabilization period, probe position and reactivity of the preparation were tested at each site by determining the cerebrovascular reactivity to inhalation of 5% CO2. Changes in flow were calculated as percentage of the baseline value determined at the end of the experiment.
Cerebral GU
GU was determined by the 2-[14C]deoxyglucose (2-DG) method with quantitative autoradiography (32). As described in detail elsewhere (17), 2-DG (12.5 µCi/100 g in 1 ml 0.9% NaCl; New England Nuclear) was infused intravenously for 40 s using an infusion pump. Simultaneously, ~70 µl of arterial blood were collected every 5-10 s during the first minute, at 90 s, and at 2, 3, 5, 10, 15, 25, 35, and 45 min. Blood samples were centrifuged and stored on ice. Forty-five minutes later, rats were killed by an intravenous bolus of saturated KCl. The brain was then rapidly removed and frozen in isopentane cooled to30°C with dry ice. Serial sections (20 µm) were cut through the cerebellum using a cryostat (Hacker-Bright).
Sections were picked up from the blade with coverslips, mounted on
glass slides, and apposed to X-ray film (DuPont) together with
calibrated 14C standards (17). Ten
days later, the film was developed using an automatic developer
(Kodak), and the optical density (OD) of regions of interest was
determined bilaterally on four adjacent sections using a computerized
image analyzer (MCID system; Imaging Research). At the site of
stimulation, OD was measured in all of the sections in which a focal
increase in density was observed. OD was transformed in
14C concentration (nCi/g) using
the standards on the film (17). Radioactivity (nCi/g) of plasma samples
was determined by liquid scintillation counting as previously described
(17). Plasma glucose was measured using a glucose analyzer (Beckman).
GU (µmol · 100 g
1 · min1)
was calculated from the OD of the regions of interest and the arterial
time course of 2-DG using the equation developed by Sokoloff et al.
(32).
Experimental Protocol
Relationship between stimulus parameters and blood flow in cerebellar and cerebral cortex. In these experiments (n = 6), the cranial window was superfused with Ringer. Stimulating electrodes were inserted, and the LDF probe was placed on crus II. The preparation was then allowed to stabilize. Arterial blood gases were adjusted and maintained in a steady state (Table 1). The perioral region was stimulated for 40 s, and the corresponding BFcrb increases were recorded in ipsilateral crus II. Multiple stimulations were delivered, and at each stimulation the intensity of the stimulus was varied (5-30 V) while the stimulus frequency was maintained at 10 Hz. Next, the frequency of the stimulus was increased (2-16 Hz) while the intensity was maintained at 25 V. In separate rats (n = 5), the relationship between stimulation parameters and CBF in the somatosensory cortex was studied. In these studies, the stimulus intensity was varied between 3 and 12 V, and stimulus frequency was varied between 3 and 12 Hz.Effect of tetrodotoxin, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo-[f]quinoxaline, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, and 7-nitroindazole on the field potentials evoked by crus II activation. In these experiments, the effect of the Na+ channel blocker tetrodotoxin (TTX), of the AMPA-kainate receptor inhibitor 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo-[f]quinoxaline (NBQX), and of the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) on the field potentials produced by crus II activation was studied. The relatively selective neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI) was also studied. TTX (10 µM) and NBQX (100 µM) were dissolved in normal Ringer. ODQ was dissolved in DMSO and diluted in Ringer (37). The final concentration of DMSO was <0.1%. 7-NI was suspended in oil and administered intraperitoneally at a dose of 50 mg/kg (18, 20). All solutions were prepared daily and used fresh. Agents were tested in separate groups of rats at concentrations previously found to be effective in this preparation (16-18, 20, 37). Field potentials were first tested during superfusion with Ringer. After obtaining stable baseline responses, the superfusion solution was changed to TTX (n = 3), NBQX (n = 3), or ODQ (n = 3), and field potentials were tested again at 15-min intervals until a stable effect was observed. In experiments in which 7-NI was used (n = 3), field potentials were tested before and 30-45 min after intraperitoneal administration of the drug.
Effect of NBQX and ODQ on the increase in BFcrb evoked by crus II activation. In these experiments, the effect on BFcrb of NBQX or ODQ was studied. After obtaining a stable flow response to crus II activation, NBQX (100 µM) or ODQ (100 µM) was superfused on the cerebellar cortex. Each agent was tested in a separate group of rats. The perioral region was stimulated (5-25 V; 10 Hz; 40 s) immediately before and at 15-min intervals until an effect on the BFcrb response was observed. Data reported in Figs. 1-9 represent effects on BFcrb occurring after 45-60 min of superfusion. The effect of NBQX on the increase in BFcrb produced by systemic hypercapnia (PCO2 50-60 mmHg) was also studied. Hypercapnia was produced by adding 5% CO2 to the circuit of the ventilator (16). The effect of ODQ or NBQX on the increase in BFcrb produced by the cGMP-independent vasodilator adenosine (100 and 1,000 µM; see Ref. 37) was also tested. In experiments using ODQ, Ringer containing 0.1% DMSO was used as vehicle.
Effect of crus II activation on GU. Rats were surgically prepared, and blood gases were adjusted until they reached a steady state. Crus II was activated with intermittent trains of stimuli applied to the perioral region (1 s on and 1 s off) at 25 V and 10 Hz. This stimulation paradigm was found in preliminary studies to provide a stable increase in BFcrb sustained for at least 30 min (n = 4; data not shown). 2-DG was infused intravenously 30 s after the start of the stimulation. Perioral stimulation was continued until, 45 min later, rats were killed. Brains were removed and processed for quantitative autoradiography. Effect of 7-NI on the increase in cerebellar or neocortical blood flow produced by perioral stimulation. In these experiments, we used 7-NI to study the role of nNOS-derived NO in the cerebellar and neocortical hyperemia evoked by perioral stimulation. 7-NI was administered intraperitoneally at a dose of 50 mg/kg (n = 6). We have previously demonstrated that 7-NI at this dose attenuates NOS activity in cerebral cortex and cerebellum without affecting endothelial NOS function (18, 20). The BFcrb response to perioral stimulation was tested before and 30-45 min after administration of 7-NI or vehicle (oil, n = 5). Stimulus parameters were 5-30 V at 10 Hz for BFcrb in crus II and 3-12 V at 7 Hz for neocortical blood flow in the somatosensory cortex. The effect of 7-NI on the increase in BFcrb produced by adenosine (100 and 1,000 µM; see Ref. 37) was also tested (n = 5).Effect of nitro-L-arginine on the increase in BFcrb evoked by crus II activation. In these experiments, we studied the effect of the nonselective NOS inhibitor nitro-L-arginine (L-NA) on the increase in BFcrb produced by crus II activation. Responses to crus II activation were tested before and 60 min after superfusion with Ringer containing L-NA (1 mM, n = 6). This concentration inhibits cerebellar NOS activity at the site of superfusion by >90% (16).
Data Analysis
Data in text, Tables 1 and 2, and Figs. 1-9 are presented as means ± SE. Comparisons between two groups were evaluated by the Student's t-test. Multiple comparisons were evaluated by ANOVA and Tukey's test (Systat, Evanston, IL).| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of Crus II Activation on BFcrb
Perioral stimulation (15-25 V, 1/s; n = 5) produced polyphasic field potentials in crus II reflecting mossy fibers (P1, N1) and granule cell activation (Fig. 1A, N2; see Ref. 4). These potentials were abolished by superfusion with TTX (Fig. 1A) and could be recorded from crus II and not lobule 6. Perioral stimulation increased BFcrb in crus II (Fig. 1B; n = 6). The magnitude of the elevation depended on the intensity of stimulation and was greatest at a frequency of 10 Hz (34 ± 5%; Fig. 2). The increases in BFcrb were independent of changes in arterial pressure (Fig. 2) and were restricted to a selected portion of crus II (Fig. 1C). The increases in BFcrb produced by crus II activation, but not those elicited by hypercapnia, were abolished by superfusion with TTX (Fig. 1B). TTX did not affect the increase in BFcrb produced by adenosine topically superfused at 100 µM (Ringer: 41 ± 2%; TTX: 44 ± 4%; n = 5; P > 0.05) or 1,000 µM (Ringer: 67 ± 7%; TTX: 63 ± 4%; n = 5; P > 0.05). Somatosensory stimulation produced by gently stroking the perioral region also increased BFcrb in crus II, albeit less than electrical stimulation (18 ± 1%; P < 0.01; n = 5).
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Effect of Crus II Activation on Local GU
In these experiments, we investigated whether the increases in BFcrb produced by crus II activation were associated with increases in GU. Perioral stimulation increased GU in the ipsilateral trigeminal nucleus (+127%; P < 0.03; n = 5) and crus II (+74%; Fig. 3 and Table 2). No changes in GU (P > 0.05) were observed in the vestibular complex, reticular formation, dentate nucleus, or cerebellar vermis (Table 2).
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Effect of NBQX, 7-NI, and ODQ on the Increase in BFcrb Evoked by Crus II Activation
We then sought to define the mechanisms of the increase in BFcrb produced by crus II activation. In agreement with previous studies (4), superfusion with the AMPA-kainate receptor antagonist NBQX attenuated the field potentials produced by perioral stimulation in crus II (Fig. 4). NBQX did not influence resting BFcrb (Ringer: 12.9 ± 1.1 perfusion units; NBQX: 14.8 ± 0.9; P > 0.05 paired t-test; n = 5), but it attenuated the increase in BFcrb produced by crus II activation (71 ± 5% at 25 V;
P < 0.01). However, as reported
previously (37), NBQX did not attenuate significantly the increase in
BFcrb produced by hypercapnia
(Ringer: +70 ± 7%; NBQX: 61 ± 11%;
P > 0.05) or adenosine at 100 µM
(Ringer: 39 ± 7%; NBQX: 38 ± 3%;
n = 5;
P > 0.05) or 1,000 µM (Ringer: 60 ± 9%; NBQX: 63 ± 6%; n = 5;
P > 0.05).
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To study the involvement of NO in the
BFcrb response to crus II
activation, we used the nNOS inhibitor 7-NI. 7-NI reduced resting
BFcrb from 14 ± 1 to 11 ± 1 perfusion units (P < 0.05; n = 6). 7-NI did not influence the
field potentials evoked by crus II activation, but it did reduce the
increase in BFcrb by 90% (25 V;
10 Hz; P < 0.01; Fig.
5). In the same rats, 7-NI had no effect on
the increase in BFcrb produced by
topical application of the NO donor
S-nitroso-N-acetylpenicillamine
(SNAP; before 7-NI: 68 ± 8%; after 7-NI: 58 ± 5%;
P > 0.05). 7-NI did not affect the
increase in BFcrb produced by
adenosine at 100 µM (Ringer: 41 ± 2%; 7-NI: 44 ± 4%;
n = 5;
P > 0.05) or 1,000 µM (Ringer: 64 ± 8%; 7-NI: 60 ± 9%;
n = 5;
P > 0.05).
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The increase in BFcrb produced by
crus II activation did not differ before and after administration of
the vehicle in which 7-NI was suspended (oil; Fig.
6A;
n = 5). The nonselective NOS inhibitor
L-NA reduced resting
BFcrb from 12 ± 1 to 10 ± 1 perfusion units (P < 0.05;
n = 5) and attenuated the increase in
CBF produced by crus II activation by 86% (25 V;
P < 0.05; Fig.
6B). L-NA did not affect
the vasodilation produced by topical application of SNAP (Ringer: 52 ± 5%; L-NA: 54 ± 6%;
P > 0.05).
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ODQ, a guanylyl cyclase inhibitor (10), was then used to establish
whether activation of guanylyl cyclase contributes to the increase in
flow. ODQ did not affect resting
BFcrb (before ODQ: 13 ± 1 perfusion units; after ODQ: 12 ± 2;
P > 0.05;
n = 5). However, ODQ attenuated the
increase in BFcrb produced by crus
II activation by 63% (25 V; 10 Hz; P < 0.01) without influencing the field potentials (Fig.
7). ODQ did not affect the vasodilation produced by the cGMP-independent vasodilator adenosine topically applied at a concentration of 100 µM (vehicle: 37 ± 6%; ODQ: 37 ± 4%; n = 5;
P > 0.05) or 1,000 µM (vehicle: 69 ± 6%; ODQ: 65 ± 5%; n = 5;
P > 0.05).
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Effect of 7-NI on the Increase in Neocortical CBF Produced by Perioral Stimulation
In these experiments, we studied the effect of 7-NI on the increase in somatosensory cortex CBF produced by perioral stimulation. We first studied the relationship between stimulation parameters and CBF response. The greatest increases in CBF were obtained at 10 V and 7 Hz (Fig. 8; n = 5). We then investigated the effect of 7-NI on the CBF response. 7-NI reduced resting CBF from 12 ± 1 to 10 ± 1 perfusion units ( n = 5; P < 0.05) and attenuated the increase in CBF produced by perioral stimulation by 52% (10 V; 7 Hz; P < 0.01; Fig. 9).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Novel Findings of the Study and Relationship to Previous Investigations
We sought to investigate further the mechanisms of the local regulation of the cerebellar microcirculation during neural activity. Although it is well known that neural activity is a major determinant of CBF, most investigations have focused on the cerebral cortex, and less attention has been paid to the neural mechanisms regulating the cerebellar microcirculation (see Ref. 17 for a review). Previous investigations have used activation of the PF or CF inputs to Purkinje neurons as a model to study the relationship between synaptic activity and BFcrb (1, 16, 17, 19, 36, 37). Although these studies have provided an insight into the mechanisms regulating BFcrb during neural activation, stimulation of individual inputs to Purkinje cells is unlikely to reflect accurately the physiological pattern of neural activity that occurs during "natural" cerebellar function. In this study, therefore, we developed a new model of functional hyperemia in cerebellar cortex based on activation of somatosensory inputs to the cerebellum. Specifically, we examined the changes in BFcrb produced by activation of crus II, an area of the cerebellar cortex that receives sensory afferents from the region of the face (30). We found that electrical or mechanical stimulation of the perioral region increases BFcrb in crus II. The increases depend on the intensity and frequency of the stimulus, are blocked by TTX, and are associated with focal elevations in GU. These observations suggest that the increases in BFcrb are related to increases in local synaptic activity and energy metabolism.We then investigated the transmitters and vascular mediators involved
in the increase in BFcrb. We found
that inhibition of AMPA-kainate receptors by NBQX attenuates the field
potentials and reduces the increase in
BFcrb produced by crus II
activation. To determine whether NO, a potent vasodilator released by
activation of glutamate receptors, is involved in the vascular
response, the relatively selective nNOS inhibitor 7-NI was used. It was found that 7-NI nearly blocks the increase in
BFcrb without affecting the field
potentials evoked by crus II activation. In contrast, in somatosensory
cortex, 7-NI attenuated the CBF response to perioral stimulation only
by 50%. NO produces vasodilation, mainly by activating soluble
guanylyl cyclase (see Ref. 7 for a review). To determine whether the
vasodilation is dependent on soluble guanylyl cyclase activation, we
used the inhibitor ODQ (10). We found that ODQ attenuates the increase
in BFcrb without affecting the
field potentials evoked by crus II activation. These data, collectively, indicate that the increases in
BFcrb initiated by activation of
crus II are mediated by activation of glutamate receptors via
production of NO and cGMP.
Exclusion of Potential Sources of Artifacts
The increases in BFcrb produced by crus II activation cannot be a consequence of changes in arterial pressure or blood gases because these parameters were closely monitored and did not differ among groups. Similarly, the elevation in BFcrb elicited by perioral stimulation is not secondary to a global arousal reaction because the changes in flow and GU were not diffuse but were restricted to a subregion of crus II. Furthermore, the attenuation of the response to crus II activation by NBQX, ODQ, and 7-NI cannot be attributed to nonspecific effects of these agents, resulting in vasoparalysis. NBQX, while attenuating the flow response to crus II activation, did not affect the vasodilation produced by hypercapnia or adenosine. ODQ attenuated the response to crus II activation but not the increase in BFcrb produced by the cGMP-independent vasodilator adenosine, whereas 7-NI and L-NA blocked the response to crus II activation but did not affect the vasodilation produced by the NO donor SNAP or adenosine. The effect of 7-NI is unlikely to result from inhibition of other isoforms of NOS. Whereas the inducible or "immunological" isoform of NOS is not expressed in the intact cerebellum (27), we have previously demonstrated that the endothelial isoform of NOS is not inhibited by 7-NI in this preparation (18). The relatively high concentration of L-NA used in this study (1 mM) was needed to assure penetration of the drug in sufficient concentrations to inhibit NOS in the cerebellar parenchyma. Such concentration of L-NA virtually abolishes cerebellar NOS activity in the field of superfusion (16). However, 1 mM L-NA does not have nonspecific vascular effects in this preparation (16). The possibility has been raised that L-NA also inhibits ATP-dependent K+ channels (23). However, in our preparation, the effect of L-NA is unlikely to be due to inhibition of K+ channels rather than NOS, because 7-NI, an NOS inhibitor structurally unrelated to L-NA, and ODQ, a guanylyl cyclase inhibitor, also attenuate the response. The reproducibility in time of the BFcrb response to crus II activation is demonstrated by the fact that flow increases were not different when tested before and 30-45 min after administration of the vehicle for 7-NI. Therefore, the findings of the present study cannot be attributed to cerebrovascular actions of systemic variables, instability of the preparation, or nonspecific vascular effects of the pharmacological agents used.Neural Mechanisms of the Increases in BFcrb
Perioral stimulation activates trigeminal afferents that reach crus II via the mossy fibers-granule cell-PF pathway and the inferior olive-CF pathway (28, 34). The observation that the field potentials and BFcrb increases are attenuated by the AMPA-kainate receptor inhibitor NBQX is consistent with the hypothesis that the response depends on glutamate release and AMPA-kainate receptor activation in crus II. The present study did not address the question of whether N-methyl-D-aspartate (NMDA) receptors are involved in the electrophysiological and hemodynamic response evoked by crus II activation. However, previous investigations suggest that NMDA receptors do not participate in the hemodynamic responses evoked by activation of the PF or the CF (1, 37). Because these pathways are both activated during perioral stimulation, it is conceivable that NMDA receptors do not contribute to the response. However, this critical issue needs to be addressed in future experiments in which the specific glutamate receptor subtypes mediating the hemodynamic changes are identified.Because glutamate is not vasoactive (6), it is unlikely that this transmitter is directly responsible for relaxation of vascular smooth muscles and vasodilation. The observations that the increase in BFcrb is inhibited markedly by the nNOS inhibitor 7-NI and by the guanylyl cyclase inhibitor ODQ suggest that AMPA-kainate receptor activation elicits NO production and that, in turn, NO produces vasodilation by activating soluble guanylyl cyclase. However, it is surprising that ODQ attenuates the flow response less than 7-NI. A possible explanation for this finding is that superfusion with ODQ (100 µM) did not completely block guanylyl cyclase activity in our preparation. In support of this hypothesis is the observation that ODQ, at the doses used in the present study, does not completely block responses to guanylyl activation by SNAP in the rat cerebellum (37). Similarly, ODQ substantially attenuates, but does not block, the vasodilation produced by the NO-generating agents nitroprusside or ACh (8). On the other hand, NO could produce vasodilation by mechanisms independent of guanylyl cyclase activation and cGMP (see, for example, Ref. 2). Therefore, the discrepancy between the effect of ODQ and 7-NI or L-NA remains, in part, unexplained.
It is of interest to compare the effects of NOS inhibition on the flow increase elicited by crus II activation with that evoked by activation of the PF and CF, the major input pathways to Purkinje cells. Although electrical stimulation of the PF produces increases in BFcrb that depend on activation of AMPA-kainate receptors and NO, NO is responsible for ~50% of the flow increase (1, 16). Activation of the CF, either by systemic administration of the alkaloid harmaline or by electrical stimulation of the inferior olive, also increases BFcrb via AMPA-kainate receptor activation and NO production (37). However, the component of the vascular response that is dependent on NO, ~70%, is greater than in PF stimulation (37). In the present study, we found that NO is responsible for ~90% of the increase in BFcrb produced by crus II activation. It would therefore seem that CF activity plays a prominent role in the increases in BFcrb produced in crus II by somatosensory stimuli. This possibility is supported by preliminary observations that lesions of the inferior olive attenuate the increase in neural activity produced by perioral stimulation, estimated by optical imaging of crus II (12). Also, the stimulation frequency at which maximal BFcrb increases were obtained in crus II corresponds to the spontaneous rhythmic activity of inferior olivary neurons (25). The energy demands of restoring the ionic equilibrium after complex spikes, such as those generated by CF activation, have been estimated at 100-1,000 times that of simple spikes (13, 14). This high energy demand is consistent with the CF system being a major factor in the BFcrb increase. Therefore, CF inputs to Purkinje cells might be the major determinant of synaptic activity and blood flow in the cerebellar cortex during natural cerebellar function.
Cellular Sources of NO in the Response to Crus II Activation
The cellular sources of NO during crus II activation have not been established and cannot be defined on the basis of the results of the present study. In the cerebellar molecular layer of rodents, NOS is present mainly in interneurons and stellate and basket cells but not in Purkinje cells (3, 22, 33). It is therefore likely that, during crus II activation, NO is produced by stellate and basket cells activated by inputs from CF and PF. There is evidence that molecular layer interneurons may be an important source of NO during PF stimulation. Stimulation of the PF in transgenic mice with targeted disruption of Purkinje cells (PO3 mice) produces increases in BFcrb smaller than those observed in nontransgenic littermates (36). However, at variance with intact mice, the residual component of the BFcrb increase is entirely dependent on NO (36). Considering that molecular layer interneurons are morphologically intact in PO3 mice (36), it is most likely that stellate and/or basket cells are the source of NO. On the other hand, NO could also be released directly from PF terminals (24, 31). In one study in which NO-sensitive electrodes were used, NO production from PF could not be attenuated by AMPA-kainate receptor inhibitors (31). Because in the present study the BFcrb flow increase could be attenuated substantially by AMPA-kainate receptor inhibition, it is unlikely that the PF are the major source of NO during somatosensory activation. However, this possibility needs to be tested experimentally.Role of NO in the response to somatosensory activation
in neocortex. Most studies of functional hyperemia have
focused on the cerebral cortex, and very little is known about the
mechanisms regulating blood flow during synaptic activity in other
brain regions. Using a model of somatosensory activation that increases flow both in cerebral cortex and cerebellar cortex, we were able to
study the role of NO in the vascular response to synaptic activity in
these two regions. We found that, under identical experimental conditions, NOS inhibition by 7-NI attenuates the increase in flow
produced by somatosensory stimulation more in cerebellar cortex than in
cerebral cortex. We have previously demonstrated that 7-NI (50 mg/kg
ip) produces comparable degrees of NOS inhibition in cerebellum
(60%; see Ref. 18) and cerebral cortex (70%; see Ref. 20). The
finding that NOS inhibition affects functional hyperemia more in
cerebellum than in neocortex has two major implications. First, it
provides evidence that the mechanisms responsible for coupling neural
activity to blood flow are regionally specific. Thus the role that
vasoactive neurotransmitters and neuromodulators play in functional
hyperemia depends on the brain region. Second, it suggests that, during
somatosensory activation, NO plays a more prominent role in the
regulation of flow in cerebellum than in cerebral cortex. This is not
surprising considering that NO is most abundant in cerebellum and plays
a critical role in cerebellar synaptic function (5). As for the
involvement of glutamate receptors in the mechanisms of functional
hyperemia in neocortex, preliminary data suggest that both NMDA and
non-NMDA receptors are involved in the vasodilation produced by sciatic
nerve stimulation in the rat somatosensory cortex (26). Therefore, in
neocortex as in cerebellum, glutamate receptors play a prominent role
in the mechanisms of functional hyperemia.
Conclusions
We have investigated the mechanisms of functional hyperemia in cerebellar cortex using activation of crus II by somatosensory stimulation as a model. We found that stimulation of trigeminal afferents from the region of the face increases BFcrb in crus II. The increases in flow are dependent on local synaptic activity, are associated with increased GU, and are attenuated by inhibition of AMPA-kainate receptors. Furthermore, the flow response is virtually abolished by nNOS inhibition and markedly attenuated by inhibition of soluble guanylyl cyclase. In contrast, the increase in CBF produced by the same somatosensory stimulus in cerebral cortex is reduced only by ~50% by NOS inhibition. Although the evidence suggests that NO plays a prominent role in the mechanisms of coupling synaptic activity to blood flow in the cerebellar cortex, it also indicates that the mechanisms regulating cerebral perfusion during synaptic activity are regionally distinct.| |
ACKNOWLEDGEMENTS |
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We thank Dr. Jon Gottesman for helpful discussion.
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FOOTNOTES |
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This work was supported by National Science Foundation Grants NS-31318 and NS-38252 and by the Strategic Initiative in Neuroscience, Academic Health Center, University of Minnesota.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. Iadecola, Dept. of Neurology, Univ. of Minnesota Medical School, Box 295 UMHC, 420 Delaware St. SE, Minneapolis, MN 55455 (E-mail: iadec001{at}tc.umn.edu).
Received 8 March 1999; accepted in final form 30 June 1999.
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