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(Stroke. 1998;29:499-508.)
© 1998 American Heart Association, Inc.

Original Contributions

Activation of Cerebellar Climbing Fibers Increases Cerebellar Blood Flow

Role of Glutamate Receptors, Nitric Oxide, and cGMP

Guang Yang, MD; Costantino Iadecola, MD

From the Laboratory of Cerebrovascular Biology and Stroke, Department of Neurology, University of Minnesota Medical School, Minneapolis, Minn.

Correspondence to C. Iadecola, MD, Department of Neurology, University of Minnesota Medical School, Box 295 UMHC, 420 Delaware St SE, Minneapolis, MN 55455. E-mail iadec001{at}maroon.tc.umn.edu

	Abstract

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Background—The mechanisms regulating the cerebellar microcirculation during neural activity are poorly understood. One of the major neural inputs to the cerebellar cortex is the climbing fiber (CF), a pathway that uses excitatory amino acids, including glutamate, as a transmitter. We studied whether CF activation increases cerebellar blood flow (BFcrb) and, if so, we investigated the role of glutamate receptors, nitric oxide (NO) and cGMP, in the response.

Methods—The CF were activated by harmaline administration (40 mg/kg, IP) in halothane-anesthetized rats with a cranial window placed over the cerebellar vermis. BFcrb was monitored by a laser-Doppler probe, and arterial pressure and blood gases were controlled.

Results—With Ringer superfusion, harmaline produced sustained increases in BFcrb that peaked 20 minutes after administration (+115±13%; n=6; P<.05). The increases in BFcrb were substantially reduced by superfusion with tetrodotoxin (10 µmol/L; -91±5%; n=5; P<.05 from Ringer). The response was also attenuated by the -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor inhibitor 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)-quinoxaline (100 µmol/L; -70±6%; P<.05; n=5), but not by the N-methyl-D-aspartate receptor blocker 2-amino-5-phosphonopentanoic acid (500 µmol/L; P>.05; n=5). The response was attenuated by the nonselective NO synthase (NOS) inhibitor nitro-L-arginine (1 mmol/L; -73±5%; n=6) or by 7-NI (50 mg, IP; -71±5%; n=5), a relatively selective neuronal NOS inhibitor. The soluble guanylyl cyclase inhibitor 1H-^1,2,4oxadiazolo[4,3-a]quinoxalin-1-one (100 µmol/L) attenuated the response to harmaline (-73±5; P<.05; n=6) but not to superfusion with adenosine (P>.05; n=5) or 8-bromo–cGMP (P>.05; n=5).

Conclusions—Activation of the CF system increases BFcrb. The response depends on activation of glutamate receptors and is in large part mediated by NO via stimulation of soluble guanylyl cyclase. Glutamate receptors NO and cGMP are important factors in the mechanisms of functional hyperemia in cerebellar cortex.

Key Words: cerebellum • cerebral blood flow • glutamate antagonists • hypercapnia

	Introduction

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The cerebellum, due to its relatively simple and well-characterized circuitry, is well suited to the investigation of the relationship between neural activity and blood flow. Functional brain imaging studies have demonstrated that BFcrb increases during motor and cognitive tasks, indicating that BFcrb is highly regulated and closely related to cerebellar neural activity.^{1 2 3} However, the mechanisms by which neural activity regulates BFcrb have not been studied as extensively as those of other brain regions (see references 4 and 5 for a review).

Two major excitatory synaptic inputs converge on cerebellar Purkinje cells, the only output neurons of the cerebellum: the PF and the CF. The PF are axons of cerebellar granule cells that reach to the superficial molecular layer and make synaptic contacts with Purkinje cell dendrites and molecular layer interneurons (see reference 6 for a review). The transmitter released from the PF is glutamate (see reference 7 for a review). The CF originate from the contralateral inferior olive and innervate Purkinje cell dendrites and interneurons.⁶ The transmitter released from the CF is also an excitatory amino acid, such as glutamate, aspartate, or N-acetyl-aspartyl-glutamate^{8 9} (see reference 10 for a review). The interaction between CF and PF activity modulates Purkinje cells output and is responsible for long-term depression, a phenomenon thought to subserve cerebellar plasticity and learning.¹¹

The mechanisms by which activation of the different inputs to the cerebellar cortex influences local blood flow have not been fully elucidated. Although studies in which the PF were electrically stimulated have provided an insight into the role of this system in the regulation of BFcrb,^{12 13 14 15 16 17} PF activity is unlikely to be the sole determinant of BFcrb during normal cerebellar function. The CF have a powerful synaptic association with neurons in the molecular layer and, as such, they provide an important contribution to cerebellar cortical synaptic activity.⁷ Therefore, CF activity is likely to have a significant impact on the cerebellar microcirculation.

Therefore, in the present study we sought to define the contribution of CF activity to local blood flow. In particular, we studied whether CF activation increases BFcrb and, if so, we sought to determine the mechanisms and the mediators of the vasodilation. We found that activation of the CF by harmaline elicits profound increases in BFcrb that are mediated by glutamate receptors and are substantially attenuated by inhibitors of NOS or soluble guanylyl cyclase. The data indicate that activity in the CF system is an important determinant of BFcrb and provide additional evidence that glutamatergic neurotransmission, NO, and cGMP are involved in regulation of the cerebellar microcirculation during neural activity.

	Materials and Methods

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General Surgical Procedures
Experimental protocols were approved by the Institutional Animal Care Committee. Studies were performed on 81 male Sprague-Dawley rats (Sasco, Omaha, Neb) weighing 290 to 380 g. Rats were anesthetized with 5% halothane in 100% oxygen. After induction of anesthesia, the concentration of halothane was reduced to 1% to 2%. Catheters were inserted into both femoral arteries, into the left femoral vein, and into the trachea. Animals were then placed on a stereotaxic frame (model 1404, D. Kopf Instruments) mounted on a vibration-free table (TMC), and artificially ventilated with an oxygen-nitrogen mixture by a mechanical ventilator (model 638, Harvard Apparatus). The oxygen concentration in the mixture was adjusted to obtain an arterial PO₂ of 120 to 140 mm Hg (Table ). Body temperature was maintained at 37±0.5°C using a heating lamp thermostatically controlled by a rectal probe (model 73A-TA, YSI). One of the arterial catheters was used for continuous recording of arterial pressure and heart rate on a strip-chart recorder (model 716P, Grass Instruments), while the other arterial catheter was used for blood sampling. Arterial PCO₂, PO₂, and pH were measured at several different times on 100 µL of blood using a blood gas analyzer (model 178, Ciba-Corning). At the end of the surgical procedures the halothane concentration was reduced to 1%. Because animals were not paralyzed, the level of anesthesia was assessed by testing corneal reflexes and motor responses to tail pinch.

View this table: [in this window] [in a new window]   Table 1. Arterial Pressure and Blood Gas Values of the Rats Studied

Monitoring of BFcrb by Laser-Doppler Flowmetry
A small craniotomy (3x3 mm) was performed in the interparietal bone using a dental drill. The dura was carefully removed and the cerebellar vermis exposed (lobule VI). The cranial window was continuously superfused with Ringer at a rate of 0.33 mL/min using miniperistaltic pumps.¹³ As in previous studies, solutions were equilibrated with 100% O₂ and 5% CO₂ (pH 7.3 to 7.4) and warmed to 37°C.¹³ Temperature, pH, and PCO₂ of the superfusion solution were checked by sampling from the brain surface. BFcrb was monitored using a laser-Doppler flowmeter (model BPM 403A, Vasamedic).¹³ The flow probe (tip diameter 0.8 mm) was mounted on a micromanipulator (Kopf) and positioned 0.5 mm above the pial surface. The analog output of the flowmeter (time constant 5 seconds) was fed into a DC amplifier (model 7P1, Grass Instruments) and displayed on the polygraph. After a 10- to 20-minute stabilization period, probe position and reactivity of the preparation were tested at each site by determining the cerebrovascular reactivity to inhalation of 5% CO₂. Once a suitable placement was obtained, the probe was left at that site for the duration of the experiment. Changes in BFcrb were calculated as a percentage of the baseline value determined at the end of the experiment.

Experimental Protocol
After completion of the surgery, the flow probe was placed in the cranial window, and the window was superfused with Ringer. Arterial blood gase values were then adjusted. Studies commenced when arterial pressure, arterial blood gase values, and flow signal were in a steady state. In these experiments the CF were activated by systemic administration of the alkaloid harmaline.^{18 19} Harmaline is thought to inhibit the serotoninergic input to the inferior olive, leading to rhythmic activity in olivary neurons and in the CF.²⁰ Harmaline has been used extensively to activate CF in several different animal models (see reference 21 for a review).

Effect of TTX, NBQX, and AP-5 on the Increases in BFcrb Produced by Harmaline
Harmaline (40 mg/kg, IP) was administered, and the changes in BFcrb were monitored continuously for up to 90 minutes after administration. In one group of rats (n=6), the window was superfused with Ringer and the effects of harmaline on BFcrb were determined. The effects of the sodium channel blocker TTX, of the -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor inhibitor NBQX, or the NMDA receptor antagonist AP-5 were tested in separate groups of rats. First, the reactivity of the preparation to hypercapnia (PCO₂=50 to 60 mm Hg) was tested while the window was superfused with Ringer. CO₂ was introduced into the circuit of the respirator for 2 to 3 minutes and the changes in BFcrb were monitored.¹³ After PCO₂ had returned to baseline, TTX (10 µmol/L; n=5), NBQX (100 µmol/L; n=5), or AP-5 (500 µmol/L; n=5) was superfused for 30 minutes, and the reactivity of BFcrb to hypercapnia was tested again. Drugs were dissolved in Ringer and were applied at concentrations found to be effective in previous studies.^{12 15} In another group of rats (n=6), the effect of AP-5 on the increase in BFcrb produced by glutamate was studied. Glutamate (200 nmol/200 nL) was microinjected into the cerebellar cortex using a micropipette connected to a pressurized microinjection system, and the resulting changes in BFcrb were monitored (see reference 16 for a detailed description). AP-5 (500 µmol/L) was then superfused for 30 minutes, and the effect of glutamate microinjection was tested again. Multiple injections of glutamate using this technique have been shown to elicit reproducible increases in BFcrb.¹⁶

Effect of Nitro-Arginine on the Increases in BFcrb Produced by Harmaline
The cranial window was first superfused with Ringer and then the reactivity of BFcrb to hypercapnia was tested. The nonselective NOS inhibitor L-NA (1 mmol/L; n=5) was then superfused for 45 to 60 minutes and the reactivity to hypercapnia assessed again. This L-NA concentration inhibits cerebellar NOS activity in the field of superfusion by more than 90%.¹³ Harmaline was then administered and BFcrb monitored. In other rats (n=5), the effect of the inactive isomer of nitro-arginine D-NA on the BFcrb response to harmaline was studied. In additional rats (n=5), the effect of L-NA on the increase in BFcrb produced by topical application of the NO donor SNAP (100 µmol/L) or of adenosine (1 mmol/L) was tested. Drugs were topically applied until the increase in BFcrb reached a steady state (usually 3 to 5 minutes).

Effect of 7-NI on the Increases in BFcrb Produced by Harmaline
The relatively selective neuronal NOS (nNOS) inhibitor 7-NI (50 mg/kg, IP; in oil) was administered 30 minutes before harmaline. This concentration of 7-NI reduces cerebellar NOS activity by 70%.¹⁴ The effect of vehicle (oil; n=5) on the elevations in BFcrb produced by harmaline and the effect of 7-NI on the increase in BFcrb produced by hypercapnia (n=6) or topical application of SNAP (100 µmol/L; n=5) were studied in separate groups of rats.

Effect of ODQ on the Increase in BFcrb Produced by Harmaline
The soluble guanylyl cyclase inhibitor ODQ (100 µmol/L)²² was superfused on the cranial window for 45 minutes and then harmaline was administered (n=5). ODQ was dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in the solution was adjusted to be less than 0.05%.²³ In five rats the effect of ODQ on the vasodilation produced by SNAP (100 µmol/L), adenosine (1 mmol/L), or the cGMP analogue 8-Br–cGMP(100 µmol/L) was studied. In five additional rats the effect of ODQ on the vasodilation produced by hypercapnia was tested.

Data Analysis
Data in text, the table, and figures are presented as mean±SE. Comparisons between two groups were evaluated by paired or unpaired Student's t tests as appropriate. Multiple comparisons were evaluated by ANOVA and Tukey's test (Systat Inc).

	Results

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Effect of Harmaline on BFcrb
Administration of harmaline (40 mg/kg, IP) produced fine tremors restricted to the whiskers and the facial muscles. Harmaline elicited a profound increase in BFcrb that began 5 minutes after administration, peaked between 15 and 20 minutes later (+115±13%; n=6), and was still present (+25±7%) at 90 minutes (Fig 1A; P<.05 from time zero; ANOVA and Tukey's test). The increases in BFcrb were not associated with changes in arterial pressure or blood gas values (Fig 1B; Table).

View larger version (15K): [in this window] [in a new window]   Figure 1. A, Effect of harmaline (40 mg/kg; IP) on BFcrb measured by laser-Doppler flowmetry in halothane-anesthetized rats equipped with a cranial window. During superfusion with normal Ringer, harmaline produced profound increases in BFcrb that reached a peak 20 minutes after administration and were still present at 90 minutes (mean±SE). Superfusion with the sodium channel blocker TTX nearly blocked the response (P<.05 from Ringer at each time point; t test), suggesting that the flow increase is mediated by synaptic activity. B, Effect of harmaline on systemic arterial pressure (AP) in rats in which the cranial window was superfused with Ringer. Note that harmaline had no effect on AP.

Effect of TTX and Glutamate Receptor Inhibition on the Increase in BFcrb Produced by Harmaline
To determine whether the increases in flow were related to synaptic activity, the effect of TTX was studied. TTX (10 µmol/L; n=5) produced a small but significant reduction in resting BFcrb (before: 11.0±0.7, after: 9.0±0.3 perfusion units; P<.05; t test; n=5) and attenuated substantially the increase in BFcrb elicited by harmaline (P<.05 from Ringer at each time point) (Fig 1A). Twenty minutes after administration of harmaline, the BFcrb increase was attenuated by 95±5% (P<.05) (Fig 1A). However, TTX did not affect the increase in flow produced by hypercapnia (Ringer's: +69±8%; TTX: +71±8%; P>.05; n=5).

Excitatory amino acids mediate synaptic transmission in the CF system.¹⁰ Therefore, we investigated whether the increase in BFcrb produced by harmaline was related to activation of glutamate receptors. Superfusion with the AMPA receptor antagonist NBQX (100 µmol/L) did not affect resting BFcrb (before: 8.2±0.3; after: 7.6±0.3 perfusion units; P>.05; t test; n=5), but attenuated substantially the BFcrb response to harmaline (-70±6%; P<.05; n=5) (Fig 2A). NBQX did not affect the increase in BFcrb produced by hypercapnia (before: +58±5%; after: +68±5%; P>.05; n=5). In contrast, the NMDA receptor antagonist AP-5 (500 µmol/L) did not attenuate the increase in BFcrb produced by harmaline or hypercapnia (Fig 2; P>.05 from Ringer, ANOVA; n=5). However, AP-5 inhibited the increase in BFcrb-produced microinjection of glutamate (200 nmol/200 nL) into the cerebellar molecular layer (Fig 2B; P<.05; n=6).

View larger version (19K): [in this window] [in a new window]   Figure 2. A, Effect of glutamate receptor antagonists on the increase in BFcrb produced by harmaline. The NMDA receptor inhibitor AP-5 does not affect the increase in BFcrb (P>.05 from Ringer superfusion; ANOVA) (see Fig 1 for BFcrb response during Ringer superfusion). In contrast, the AMPA receptor inhibitor NBQX attenuates the increase in flow substantially (P<.05 from AP-5 at each time point; t test). B, The NMDA receptor antagonist AP-5 does not affect the vasodilation produced by hypercapnia (PCO2=50 to 60 mm Hg; see Table for values) but attenuates the increase in BFcrb produced by microinjection of glutamate into the cerebellar molecular layer. The data provide evidence that AP-5 is effective in inhibiting glutamate receptors.

Effect of NOS Inhibition on the Increases in BFcrb Produced by Harmaline
The data presented above indicate that the increase in BFcrb produced by harmaline is mediated by activation of a glutamate receptor. Because glutamate receptor activation increases blood flow through NO production,^{14 24 25 26} we studied the effect of NOS inhibitors on the vascular response to harmaline administration. Superfusion with the nonselective NOS inhibitor L-NA (1 mmol/L) reduced resting BFcrb (before: 10.5±0.4; after: 8.9±0.4 perfusion units; P<.05; t test; n=5) and attenuated the increase in BFcrb produced by harmaline by 73±5% (P<.05; n=5) (Fig 3A). L-NA also attenuated the BFcrb response to hypercapnia (-48±10%; P<.05), but not to topical application of the NO donor SNAP or adenosine (P>.05; n=5) (Fig 3B). In contrast, the inactive isomer D-NA did not affect the increase in BFcrb produced by harmaline (Fig 3A; P>.05 from Ringer; ANOVA; n=5). Others have reported that L-NA attenuates the increase in BFcrb produced by adenosine.²⁷ However, in our experimental preparation adenosine behaved as a NO-independent vasodilator. The observation that ODQ does not affect the response to adenosine also supports this conclusion (see below).

View larger version (22K): [in this window] [in a new window]   Figure 3. A, Effect D-NA, L-NA and ODQ on the increases in BFcrb produced by hypercapnia. The inactive stereoisomer of nitroarginine, D-NA, does not affect the response (P>.05 from Ringer; ANOVA)(see Fig 1 for BFcrb response during Ringer superfusion). The nonselective NOS inhibitor L-NA and the guanylyl cyclase inhibitor ODQ attenuate the response substantially (P<.05 from D-NA; ANOVA and Tukey's test). The data suggest that the increase in BFcrb produced by harmaline is, in great part, mediated by NO and cGMP. B, L-NA attenuates the increase in BFcrb produced by hypercapnia (P<.05; t test) but does not reduce the response to topical application of the NO donor SNAP or of the NO-independent vasodilator adenosine.

To study the role of nNOS in the response, the relatively selective nNOS inhibitor 7-NI was tested. We have previously demonstrated that in this preparation 7-NI inhibits nNOS without affecting eNOS-dependent vascular responses.^{14 16} 7-NI reduced resting BFcrb (before: 8.7±0.6; after: 6.3±0.3 perfusion units; P<.05; t test; n=6) and attenuated the increase in BFcrb produced by harmaline (-71±5%; P<.05; n=6) or hypercapnia (-63±6%; P<.05; n=6) but not to SNAP superfusion (P>.05; n=5; Fig 4). Thus, the BFcrb response to harmaline is attenuated by both L-NA and 7-NI.

View larger version (18K): [in this window] [in a new window]   Figure 4. A, Effect of the relatively selective nNOS inhibitor 7-NI on the increase in BFcrb produced by harmaline. Administration of vehicle (oil) does not affect the increase in BFcrb (P>.05; ANOVA) (see Fig 1 for BFcrb response during Ringer superfusion). 7-NI attenuates the response to harmaline markedly (P<.05 from vehicle at each time point; t test). The data suggest that the NOS isoform involved in the response is nNOS. B, 7-NI reduces the response to hypercapnia (P<.05; t test) but does not affect the vasodilation produced by the NO donor SNAP.

Effect of the Guanylyl Cyclase Inhibitor ODQ on the Increase in BFcrb Produced by Harmaline
One of the mechanisms by which NO produces vascular relaxation is by activating soluble guanylyl cyclase and increasing cGMP in vascular smooth muscles (see reference 28 for a review). Therefore, in these experiments we studied the effect of the soluble guanylyl cyclase inhibitor ODQ²² on the increase in BFcrb produced by harmaline. ODQ (100 µmol/L) increased resting BFcrb (before: 10.0±0.7; after: 12.0±0.9 perfusion units; P<.05; t test; n=6) and inhibited the response to harmaline (-73±5%; P<.05; n=6) (Fig 3A) or hypercapnia (-49±7%; P<.05; n=6) (Fig 5). ODQ attenuated the increase in BFcrb produced by SNAP (P<.05; n=6), but did not affect the response to the cAMP-dependent vasodilator adenosine (P>.05; n=5) or to the cGMP analogue 8-Br–cGMP (P>.05; n=6) (Fig 5). The finding that ODQ did not affect the vasodilation produced by adenosine supports the hypothesis that the vasodilation produced by this nucleoside is independent of NO and cGMP.

View larger version (20K): [in this window] [in a new window]   Figure 5. Effect of ODQ on the increase in BFcrb produced by the NO donor SNAP, hypercapnia, cGMP, and the NO-independent vasodilator adenosine. ODQ attenuated the increase in BFcrb produced by SNAP and hypercapnia but did not affect the response to cGMP or adenosine. The data suggest that in this preparation ODQ acts as a selective inhibitor of soluble guanylyl cyclase.

	Discussion

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We have investigated the mechanisms of the increases in BFcrb produced by activation of the CF. The CF provide a strong excitatory synaptic input to the cerebellar Purkinje cells. The CF originate from the contralateral inferior olive, project directly to the cerebellar molecular layer, and make multiple synaptic contacts with Purkinje cell dendrites and molecular layer interneurons.⁶ Despite the fact that a Purkinje cell receives inputs only from a single CF,⁶ CF activation produces powerful synaptic responses in Purkinje cell dendrites²⁹ (see reference 30 for a review). CF-induced Purkinje cell discharges are associated with increases in cerebellar glucose utilization.³¹ We have found that activation of the CF using harmaline elicits profound increases in BFcrb that are independent of changes in arterial pressure and blood gases. The increases in BFcrb are protracted in time and are larger in magnitude than those produced by stimulation of the PF, hypercapnia, or topical application of vasodilators.^{13 14 15 16 32} The flow increase is virtually abolished by TTX, indicating that it is mediated by enhanced synaptic activity. The latter finding also suggests that direct vascular effects of harmaline are unlikely to contribute to the vasodilation. These observations indicate that the synaptic activity evoked from the CF has profound effects on the microvascular flow of the cerebellar cortex and that CF activity is a major determinant of BFcrb. Harmaline could also activate other neural pathways in addition to the CF. However, mapping of neural activity either by microelectrode recordings or 2-deoxyglucose autoradiography suggests that the activation produced by harmaline involves predominantly the inferior olive–CF system.^{18 19 31} It is, therefore, likely that the increases in BFcrb produced by harmaline reflect largely CF activity. The data provide evidence that CF activity is an important factor in the elevations in BFcrb that occur during normal cerebellar function.^{1 2 3}

We then began to study the mechanisms of the increase in flow evoked by CF activation. Synaptic transmission in the CF system is mediated by excitatory amino acids, which act on glutamate receptors on Purkinje cell dendrites and interneurons.^{33 34 35} We, therefore, tested the hypothesis that activation of glutamate receptors initiates the increase in flow produced by CF activation. It was found that the vascular response to harmaline is attenuated by NBQX, an AMPA receptor blocker, but not by the NMDA receptor blocker AP-5. The lack of effectiveness of AP-5 could not be attributed to insufficient dose or poor penetration of the drug because AP-5 attenuates the increase in BFcrb produced by glutamate microinjection. These observations suggest that the increases in flow evoked from CF activation are mediated largely by activation of AMPA receptors. The observation that NBQX does not block the flow increase completely raises the possibility that metabotropic glutamate receptors or other receptors are also involved in the response. Metabotropic glutamate receptors are present on Purkinje cell dendrites, and they are linked to NO production.^{36 37} However, future studies are required to define the role of these receptors in the flow response to harmaline.

Evidence exists that activation of glutamate receptors in the cerebellum is coupled to NO production^{38 39 40} (see reference 41 for a review). Therefore, we studied whether NO, a potent vasodilator, participates in the increase in BFcrb produced by harmaline. The BFcrb response to harmaline was attenuated by the NOS inhibitor L-NA and by the relatively selective inhibitor of nNOS 7-NI. The effect of L-NA or 7-NI could not be attributed to a nonspecific loss of vascular reactivity because these agents did not affect the increase in BFcrb produced by the NO donor SNAP or by the NO-independent vasodilator adenosine. L-NA has been reported to also inhibit ATP-sensitive K⁺ channels.⁴² The possibility that activation of K⁺ channels contributes to the vasodilation cannot be ruled out on the basis of the present study. However, 7-NI, a NOS inhibitor that is structurally unrelated to L-NA⁴³ and that inhibits NOS by a mechanism different from that of L-NA,⁴⁴ attenuates the BFcrb response to harmaline in a fashion nearly identical to that of L-NA. This observation supports the notion that the effect of L-NA is related to NOS inhibition and not to other factors. In addition, the inactive isomer of nitro-arginine, D-NA, does not attenuate the vasodilation produced by harmaline. This finding suggests that the effect of L-NA is stereoselective and that it provides additional support to the contention that the attenuation of the response by L-NA is related to NOS inhibition.

One of the mechanisms by which NO produces vasodilation is activation of soluble guanylyl cyclase and cGMP production (see reference 28 for a review). To determine whether cGMP is involved in the vasodilation produced by harmaline, we used the recently introduced soluble guanylyl cyclase inhibitor ODQ.^{22 23} It was found that ODQ attenuates the BFcrb response to harmaline without affecting the vasodilation produced by the cGMP analogue 8-Br–cGMP or the guanylyl cyclase-independent vasodilator adenosine. These observations indicate that the effect of ODQ is related to guanylyl cyclase inhibition. The finding that the attenuation by ODQ of the BFcrb to harmaline is virtually identical in magnitude to that produced by NOS inhibition suggests that the NO-dependent component of the response is mediated by activation of guanylyl cyclase and not by other effects of NO, resulting in smooth muscle relaxation. ⁴⁵ The cells responsible for the production of NO during CF activation remain to be defined. The observation that L-NA and 7-NI attenuate the response to harmaline administration by a similar degree is consistent with a neuronal source of NO. In the cerebellar molecular layer, NOS is present in interneurons, mainly basket and stellate cells, but not in Purkinje cells.^{46 47 48} It is, therefore, likely that NO is produced by molecular layer interneurons.

The evidence presented above suggests the following mechanism for the increase in BFcrb produced by CF activation. CF activation produces depolarization of Purkinje cells and interneurons. The associated increase in intracellular calcium activates NOS in interneurons, resulting in production of NO. NO, or a closely related chemical specie, diffuses to local blood vessels and activates soluble guanylyl cyclase, resulting in vasodilation. However, NO is unlikely to be the sole factor responsible for the vasodilation. The observations that NOS or guanylyl cyclase inhibition attenuates but does not abolish the increase in BFcrb produced by harmaline suggests that a component of the vasodilation is independent of NO/cGMP. However, the component of the response independent of NO is relatively small. The mechanisms of such NO-independent component remain to be defined. In the PF system, adenosine is responsible for the portion of the vasodilation not mediated by NO.¹⁷ Adenosine, a potent cerebrovasodilator,⁴⁹ is present in Purkinje cells⁵⁰ and could conceivably be released also during CF-induced synaptic activity. However, the role of adenosine in the response will have to be addressed in future studies.

Stimulation of the PF increases BFcrb, an effect that is also thought to be mediated by glutamate receptors and, in part, NO.^{12 13 17} However, there are important differences between the BFcrb response evoked from PF or CF stimulation. First, the magnitude of the flow increase produced by CF activation (100%) is greater than that produced by PF stimulation (50%). The larger flow response is likely to reflect the strong synaptic interaction between CF and Purkinje cells and interneurons.²⁹ Second, the part of the NO-dependent component of the vasodilation elicited by CF activation (70%) is larger than that of the vasodilation produced by PF stimulation (50%). This observation suggests that the contribution to the flow response of molecular layer interneurons, the presumed cellular source of NO, is greater during activation of the CF than the PF. However, the possibility that these differences are related to the method used to activate these pathways, electrical stimulation for the PF versus harmaline for the CF, cannot be ruled out.

Another new finding of the present study is that the guanylyl cyclase inhibitor ODQ attenuates the response to hypercapnia. NO has been implicated in the mechanisms of the vasodilation produced by hypercapnia in several species.^{51 52 53 54 55} However, the lack of selective and specific guanylyl cyclase inhibitors precluded the need to test more directly the role of cGMP in the response. Commonly used guanylyl cyclase inhibitors, such as methylene blue and LY83583, are not suitable because they also inactivate NO by producing reactive oxygen species.⁵⁶ Furthermore, methylene blue inhibits NOS directly.⁵⁷ ODQ is a guanylyl cyclase inhibitor that does not have the drawbacks of methylene blue or LY83583.^{22 23} The observation that ODQ attenuates the hypercapnic vasodilation provides evidence that cGMP production is required for a sizable component of the flow response and provides additional support to the hypothesis that NO is involved in the mechanisms of the hypercapnic vasodilation.

In summary, we have demonstrated that activation of the cerebellar CF by harmaline increases local blood flow substantially. The effect is markedly reduced by TTX and by NBQX, suggesting that the response is mediated by glutamatergic synaptic transmission. In addition, the flow increase is attenuated by the NOS inhibitors L-NA and 7-NI or by the guanylyl cyclase inhibitor ODQ. Collectively, the data indicate that the increase in BFcrb produced by harmaline is initiated by excitatory amino acids released from the CF through the production of NO and cGMP. We conclude that CF activity is an important factor in the local control of blood flow in the cerebellar cortex and that glutamate, NO, and cGMP are critical mediators in the regulation of flow during neural activity in the cerebellar cortex.

	Selected Abbreviations and Acronyms

 

AP-5 = 2-amino-5-phosphonopentanoic acid BFcrb = cerebellar blood flow 8-Br–cGMP = 8-bromo-cGMP CF = climbing fibers NBQX = 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)-quinoxaline NMDA = N-methyl-D-aspartate NO = nitric oxide nNOS = neuronal NOS NOS = NO synthase ODQ = 1H-1,2,4oxadiazolo[4,3-a]quinoxalin-1-one PF = parallel fibers SNAP = S-nitroso-N-acetylpenicillamine TTX = tetrodotoxin

	Acknowledgments

 
This study was supported by NIH grant NS 31318. Dr Iadecola is an Established Investigator of the American Heart Association. Karen MacEwan provided excellent editorial assistance.

Received September 2, 1997; revision received October 14, 1997; accepted October 17, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
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Editorial Comment

Role of Glutamate Receptors, Nitric Oxide, and cGMP

Frank M. Faraci, PhD, Guest Editor

Department of Internal Medicine, Cardiovasular Division, University of Iowa College of Medicine, Iowa City, Iowa

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Although it is well know that increases in neuronal activity are associated with local increases in perfusion, the mechanism(s) that mediates this increase in cerebral blood flow has been difficult to fully define.¹ A series of studies over the last 9 years support the concept that neuronal release of the potent vasodilator nitric oxide (NO) functions to increase local blood flow.

Relative to other organs, the brain produces large quantities of NO. Under normal conditions, the majority of this NO is produced by the neuronal isoform of NO-synthase (nNOS).² A major stimulus for production of NO by nNOS in neurons is activation of glutamate receptors. For example, recent molecular analysis revealed physical coupling (via post-synaptic density proteins) of nNOS to one subtype of glutamate receptor, the NMDA receptor.³ Activation of NMDA receptors increases activity of nNOS, while inhibitors of NMDA receptors or NO-synthase attenuate basal and stimulated NO production in a variety of models, including brain of awake animals (see references 4 and 5 for examples).

Because NO is lipid- and water-soluble, it can easily diffuse extracellularly and influence local vascular tone. Using a bioassay system, Garthwaite et al provided the first direct evidence that neurons release NO in response to activation of NMDA receptors in quantities sufficient to relax vascular muscle in vitro.⁶ We subsequently reported that local dilatation of cerebral arterioles in response to activation of NMDA receptors in vivo is mediated by NO.⁷ Since our initial observation, several other laboratories have confirmed that glutamate or glutamate analogues produce NO-mediated vasodilatation in brain (see references 8-12 for examples).

The study presented here makes an important contribution in this area. Previous in vivo studies that focused on cerebral vascular responses have been performed almost exclusively in the cerebral cortex. In this study, the experimental approach took advantage of the fact that the neuronal circuitry and neurochemisty of the cerebellar cortex are simpler than that in many other brain regions and have been relatively well characterized. The results indicate that increases in cerebellar blood flow in response to activation of climbing fibers (using harmaline) was inhibited by an antagonist of the AMPA subtype of glutamate receptor and inhibitors of NO-synthase (including one selective for nNOS). The approach using harmaline is attractive because it allows one to examine mechanisms that mediate vascular responses to endogenous release of an excitatory amino acid which activates glutamate receptors. Thus endogenous neurotransmitter release and subsequent activation of glutamate receptors produced NO-mediated vasodilatation.

An additional goal was to examine the mechanism by which NO increased blood flow. Soluble guanylyl cyclase in vascular muscle is known to be a key molecular target for NO. Previous studies have provided evidence that relaxation of cerebral vessels to NO, produced endogenously by endothelium or perivascular neurons, is mediated in large part by activation of soluble guanylyl cyclase.^{13 14} In the present study, increases in cerebellar blood flow in response to activation of climbing fibers was also reduced markedly by a selective inhibitor of soluble guanylyl cyclase. Thus these findings provide additional support for the concept that vasodilatation in brain in response to activation of glutamate receptors is mediated by neuronally derived NO acting on soluble guanylyl cyclase in cerebral vascular muscle.

	Selected Abbreviations and Acronyms

 

AP-5 = 2-amino-5-phosphonopentanoic acid BFcrb = cerebellar blood flow 8-Br–cGMP = 8-bromo-cGMP CF = climbing fibers NBQX = 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)-quinoxaline NMDA = N-methyl-D-aspartate NO = nitric oxide nNOS = neuronal NOS NOS = NO synthase ODQ = 1H-1,2,4oxadiazolo[4,3-a]quinoxalin-1-one PF = parallel fibers SNAP = S-nitroso-N-acetylpenicillamine TTX = tetrodotoxin

Received September 2, 1997; revision received October 14, 1997; accepted October 17, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Iadecola C. Regulation of the cerebral microcirculation during neural activity: is nitric oxide the missing link? Trends Neurosci.. 1993;16:206–214.[Medline] [Order article via Infotrieve]

2. Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC. Targeted disruption of the neuronal nitric oxide synthase gene. Cell.. 1993;75:1273–1286.[Medline] [Order article via Infotrieve]

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6. Garthwaite J, Charles SL, Chess-Williams R. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature.. 1988;336:385–388.

7. Faraci FM, Breese KR. Nitric oxide mediates vasodilatation in response to activation of N-methyl-D-aspartate receptors in brain. Circ Res.. 1993;72:476–480.

8. Meng W, Tobin JR, Busija DW. Glutamate-induced cerebral vasodilation is mediated by nitric oxide through N-methyl-D-aspartate receptors. Stroke.. 1995;26:857–863.

9. Yang G, Iadecola C. Glutamate microinjections in cerebellar cortex reproduce cerebrovascular effects of parallel fiber stimulation. Am J Physiol.. 1996;271:R1568–R1575.[Abstract/Free Full Text]

10. Northington FJ, Tobin JR, Koehler RC, Traystman RJ. In vivo production of nitric oxide correlates with NMDA-induced cerebral hyperemia in newborn sheep. Am J Physiol.. 1995;269:H215–H221.[Abstract/Free Full Text]

11. Mayhan WG, Didion SP. Glutamate-induced disruption of the blood-brain barrier in rats. Stroke. 1996;27:965–970.

12. Wilderman MJ, Armstead WM. Role of neuronal NO synthase in relationship between NO and opioids in hypoxia-induced pial artery dilation. Am J Physiol.. 1997;273:H1807–H1815.[Abstract/Free Full Text]

13. Sobey CG, Faraci FM. Effects of a novel inhibitor of guanylyl cyclase on dilator responses of mouse cerebral arterioles. Stroke. 1997;28:837–843.

14. Gonzalez C, Barroso C, Martin C, Gulbenkian S, Estrada C. Neuronal nitric oxide synthase activation by vasoactive intestinal peptide in bovine cerebral arteries. J Cerebral Blood Flow Metab.1997;17:977–984.

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