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Division of Neurobiology, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York 10021
Submitted 16 January 2003 ; accepted in final form 4 March 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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cerebral circulation; cerebellum; laser-Doppler flowmetry; vasodilation; glutamate
Nitric oxide (NO), a potent vasodilator released during synaptic activity, has emerged as an important factor in the mechanisms of functional hyperemia (9, 20). Evidence for the involvement of NO seems particularly strong in the cerebellar cortex, wherein glutamate-evoked NO release is thought to play a critical role in synaptic signaling (for a review, see Ref. 18). Thus NO has been implicated in the increase in cerebellar blood flow (BFcrb) produced by activation of the major inputs to Purkinje cells, parallel fibers (PF) and climbing fibers (CF) (1, 2, 14, 38). Furthermore, NO has been proposed to mediate the increases in BFcrb produced by activation of trigeminal afferents terminating in a region of the posterior lobe of the cerebellum termed Crus II (33–35).
However, the evidence supporting a role of NO in functional hyperemia in the cerebellum is based exclusively on studies using pharmacological inhibitors (1, 2, 10, 14, 35), agents that have been reported to have unrelated effects that may confound the interpretation of the results (e.g., Refs. 19 and 26). Although NO synthase (NOS) inhibitors have provided important insights into the role of NO in cerebrovascular regulation, these agents do not completely inhibit neuronal NOS (nNOS) activity in vivo, and their isoform selectivity is not absolute (3). Therefore, it would be important to provide independent evidence of the role of NO in cerebellar hyperemia by using an approach not based on pharmacological agents. Mice lacking selected isoforms of NOS have been useful in exploring the role of NO in cerebrovascular regulation (5, 16). In the present study, we used nNOS-null mice (12) to more specifically investigate the role of nNOS in the mechanisms of functional hyperemia in the cerebellum. We found that the increases in BFcrb produced in Crus II by topical superfusion of glutamate, by stimulation of the CF, or by somatosensory activation are markedly attenuated in nNOS-null mice. In contrast, BFcrb responses not mediated by nNOS, such as those evoked by ACh or adenosine, are preserved. These findings in nNOS-deficient mice, in concert with studies using pharmacological inhibitors (2, 35), provide strong evidence that nNOS-derived NO is a critical mediator in the mechanisms of glutamate-dependent hyperemia in the cerebellum.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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All experimental methods were approved by the Institutional Animal Care and Use Committee. Studies were performed in 2- to 3-mo-old nNOS-null mice that were obtained from a colony developed from breeding pairs provided by Dr. Paul Huang (12). All mice were genotyped. Homozygous nNOS-deficient (nNOS-/-) and wild-type (nNOS+/+) littermates were studied. Mice were anesthetized with 5% halothane in 100% oxygen. After induction of anesthesia, the concentration of halothane was reduced to 1–2%. Catheters were inserted in the femoral artery [polyethylene (PE)-10] and in the trachea (PE-90, length 6 mm). Animals were then placed in a stereotaxic frame (Kopf Instruments; Tujunga, CA) mounted on a vibration-free table (TMC; Peabody, MA). Mice were artificially ventilated with an oxygen-nitrogen mixture by a mechanical ventilator (SAR-830, CWI; Ardmore, PA). The oxygen concentration in the mixture was adjusted to maintain the arterial PO2 between 120 and 150 mmHg (Table 1). End-tidal CO2 was continuously monitored by using a CO2 analyzer (Capstar-100, CWI) (36). Body temperature was maintained at 37 ± 0.5°C by using a heating lamp thermostatically controlled by a rectal probe (model 73A-TA, Yellow Springs Instruments; Yellow Springs, OH). The arterial catheter was used for continuous recording of arterial pressure and heart rate on a chart recorder (model 716P, Grass; Quincy, MA) and for blood sampling. At the end of the surgical procedures, the halothane concentration was reduced to 1%. Because mice were not paralyzed, the adequacy of the level of anesthesia was assessed by testing corneal reflexes and motor responses to tail pinch. Throughout the experiment, two to three samples (50 µl) of arterial blood were collected for blood gas analysis. Such blood removal did not affect arterial pressure.
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Techniques used for monitoring BFcrb in anesthetized mice have been described previously (36). A small hole (3 x 3 mm) was drilled in the occipital region to expose the Crus II, and the dura was carefully removed. The cranial window was continuously superfused with Ringer solution (pH 7.3–7.4, 37°C) at a rate of 0.33 ml/min (36). BFcrb was monitored by using a laser-Doppler flowmeter (model BPM 403A, Vasamedic; St. Paul, MN). The flow probe (tip diameter 0.8 mm) was mounted on a micromanipulator (Kopf Instruments) and positioned 0.5 mm above the pial surface. The analog output of the flowmeter was amplified (model 7P1, Grass DC amplifier) and displayed on the polygraph. Changes in BFcrb were calculated as a percentage of the baseline flow. The value for zero flow was determined at the end of the experiment after the heart was stopped with an overdose of halothane.
Cerebellar Activation and Monitoring of Field Potentials
The Crus II was activated by electrical stimulation of the upper lip with
needle electrodes (distance between electrodes 0.5 cm). Stimuli were negative
square waves (pulse duration 0.3 ms) delivered from a stimulator (model S88,
Grass) through a stimulus isolation unit (model PSIU6, Grass). Field
potentials evoked in the Crus II by upper lip stimulation (1/s, 20 V) were
recorded by glass micropipettes (tip diameter 5–10 µm) filled with 2
M NaCl (resistance 2–5 M
) and inserted at a depth of
300–400 µm. The signal from the micropipettes was amplified (model
7P5, Grass microelectrode amplifier), displayed on an oscilloscope, and
digitized by using a computerized data-acquisition system (MacAdios II Jr, GW
Instruments; Sommerville, MA). In each trial, 10 traces were acquired,
averaged, and stored for off-line analysis (Superscope software, GW
Instruments) (36). Field
potentials and BFcrb were recorded in separate groups of mice.
Experimental Protocol
After the surgical procedures were completed, the superfusion with Ringer solution was started, and blood gases were adjusted (Table 1). The electrodes for stimulation of the upper lip were inserted, and the animal was allowed to stabilize for 30 min. Stimulation was started when hemodynamic and respiratory parameters reached a steady state.
Effect of perioral stimulation on BFcrb in
Crus II of nNOS-null mice. To activate Crus II, the upper lip was
stimulated for 30- to 40-s epochs with increasing current intensities
(5–25 V, 10 Hz) or frequencies (4–16 Hz, 25 V), and the evoked
increase in BFcrb was monitored in the ipsilateral Crus II. Crus II
activation produces increases in BFcrb that reach a plateau after
30 s of stimulation (35).
The BFcrb increase was measured at the level of the plateau.
Effect of hypercapnia or adenosine on BFcrb in nNOS-null mice. Two levels of hypercapnia (PCO2 = 40–45 or 55–60 mmHg) were produced by introducing CO2 into the circuit of the ventilator. A stable level of hypercapnia was maintained until the BFcrb increase reached a steady state. In these experiments, the rate of BFcrb rise per millimeter of mercury of PCO2 was consistent with that reported in the literature (for a review, see Ref. 32). In experiments in which adenosine was studied, this nucleoside (100 and 1,000 µM) was superfused on the Crus II until the BFcrb increase reached a steady state, which usually took 3–5 min. The superfusion solution was then switched back to normal Ringer solution.
Effect of glutamate or acetylcholine on BFcrb in nNOS-null mice. Glutamate (5 and 10 µM) or acetylcholine (5 and 10 µM) was superfused on the Crus II until a stable increase in BFcrb was obtained, usually 3–5 min. After the BFcrb increase was recorded, the solution was switched to normal Ringer solution.
Effect of harmaline on BFcrb in nNOS-null mice. Harmaline is an alkaloid that activates the CF by antagonizing serotoninergic inputs to the inferior olive, the site wherein CF originate (17). Methods for assessment of the effect of harmaline on BFcrb have been described in detail previously (38). Briefly, harmaline (20 mg/kg) was injected intraperitoneally, and the BFcrb increase was recorded for the following 90 min. In some mice, harmaline produced fine tremors restricted to the facial whiskers (38). Because of the prolonged BFcrb monitoring period, particular care was exercised to monitor arterial pressure and control blood gases.
Data Analysis
Data are presented as means ± SE. Multiple comparisons were evaluated by ANOVA and Tukey's test (Systat; Evanston, IL). Two-group comparisons were evaluated by two-tailed Student's t-test. Differences were considered significant for probability values of <0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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First, we sought to verify that NO-dependent BFcrb responses were attenuated in nNOS-deficient mice. In the cerebellum, glutamatergic synaptic activity releases NO (30) and increases BFcrb (37). Therefore, we examined whether the increases in BFcrb produced by glutamate are attenuated in nNOS-deficient mice. As a control, we also tested the effect of ACh, a transmitter that in the mouse cerebral microcirculation increases BFcrb through activation of endothelial NOS (eNOS) (29, 31). In nNOS+/+ mice (n = 6), glutamate increased BFcrb (Fig. 1). The increase in BFcrb was markedly attenuated in nNOS-/- mice (P < 0.05, n = 6; Fig. 1). In contrast, the increase in BFcrb produced by ACh did not differ between nNOS+/+ and nNOS-/- mice (n = 7 mice/group, P > 0.05; Fig. 1).
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Effect of Perioral Stimulation on BFcrb in nNOS-Null Mice
In nNOS+/+ mice (n = 6), activation of the Crus II by perioral stimulation increased BFcrb (Fig. 2). The increases in flow were related to the intensity and frequency of stimulation and were greatest (+28 ± 3%) at 25 V and 10 Hz. In nNOS-/- mice (n = 6), the flow increases were significantly attenuated (-73 ± 3% at 25 V and 10 Hz, P < 0.05; Fig. 2). In contrast to the hyperemia produced by perioral stimulation, the increases in BFcrb produced by topical superfusion of Crus II with the vasodilator adenosine (100 and 1,000 µM, n = 6 mice/group) or by systemic hypercapnia (n = 7 mice/group) were not affected in nNOS+/+ or nNOS-/- mice (Fig. 3; P > 0.05).
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Field Potentials in nNOS-Null Mice
To determine whether the attenuation of functional hyperemia in nNOS-/- mice was due to a reduction in the intensity of neural activation, the field potentials evoked by perioral stimulation were recorded in Crus II. Activation of the Crus II produced the characteristic polyphasic potentials (Fig. 4) (7). These potentials did not differ in amplitude or morphology between nNOS+/+ and nNOS-/- mice (n = 3 mice/group; Fig. 4).
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Effect of Harmaline on BFcrb in nNOS-Null Mice
The CF represent a major input to Purkinje cells and an important determinant of the neural activity produced by Crus II stimulation (7). Therefore, we investigated whether the increase in BFcrb is attenuated in nNOS-null mice. In nNOS+/+ mice (n = 6), harmaline produced a marked and time-dependent increase in BFcrb that reached a plateau 60 min after administration (Fig. 5). The increases in BFcrb were independent of changes in mean arterial pressure (MAP; Fig. 5) or blood gases (Table 1). The increases in BFcrb were profoundly attenuated in nNOS-/- mice (Fig. 5). In nNOS-/- mice, harmaline produced a small reduction in MAP at most time points (P < 0.05). Although the mechanisms of this effect are unknown, such a reduction in MAP is unlikely to contribute to the attenuation of the BFcrb response.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The reduction of functional hyperemia in nNOS-null mice cannot result from differences in arterial pressure or blood gases because these variables were carefully controlled and did not differ between the groups of mice studied. Furthermore, the effect cannot be a consequence of a nonspecific alteration in cerebrovascular reactivity in nNOS-deficient mice because the increase in BFcrb produced by other stimuli, such as systemic hypercapnia or topical application of adenosine, were not affected. Finally, the observation that the field potentials were not altered in nNOS-null mice suggests that differences in the neural activity evoked by the stimulation do not play a role in the attenuation of activity-induced increase in BFcrb. Therefore, the reduction of the BFcrb response in the nNOS-null mice cannot be attributed to changes in systemic variables, nonspecific alterations in vascular reactivity, or differences in the intensity of the neural activity evoked by the stimulation.
Stimulation of the perioral region activates Purkinje cells in the Crus II through two major inputs: PF and CF (33). The transmitter released by these pathways is glutamate (18). Glutamate receptor activation is closely linked to NO synthesis through increases in intracellular calcium that activate nNOS, an enzyme whose activity depends on calcium and calmodulin (3). Glutamate receptor activation releases NO and increases BFcrb (30, 37). It is therefore not surprising that the increases in BFcrb produced by glutamate are attenuated in nNOS-null mice. However, the fact that glutamate still elicits an increase in BFcrb in mice lacking nNOS is of interest as it provides strong evidence that NO is not the only agent involved in the hemodynamic response associated with glutamate receptor activation. The increase in BFcrb evoked by Crus II activation is also not completely abolished in nNOS-deficient mice. Although these residual responses may reflect compensatory mechanisms attempting to take over functions normally subserved by NO, they also suggest that redundant neurovascular signaling pathways are responsible for the increase in flow. Other factors may include adenosine (22), extracellular ions (6), cyclooxygenase 2 reaction products (24), and cytochrome P-450 metabolites (25). However, the role of these factors in the increase in BFcrb produced by Crus II activation has not been explored.
The observation that the increase in BFcrb evoked by harmaline-induced activation of CF is attenuated in nNOS-null mice is also of interest. The CF are responsible for a major component of the neural activity elicited by Crus II activation, and their inactivation attenuates somatosensory-evoked potentials in the Crus II (7). Therefore, the fact that the response to CF excitation is reduced in nNOS-null mice suggests that CF input is an important determinant of the hemodynamic response elicited by Crus II activation. This hypothesis is supported by preliminary observations showing that lesion of the CF attenuates the increase in BFcrb produced by Crus II activation (Y. Zhang and C. Iadecola, unpublished observations).
The neuronal type responsible for the increase in BFcrb during functional hyperemia has been investigated by using mice lacking cyclin D2. These mice have a reduced number of stellate interneurons, one of the major sources of NO in the cerebellar molecular layer (13). It was found that cyclin D2-deficient mice have attenuated BFcrb responses to Crus II activation, whereas the increases in BFcrb produced by nonneural stimuli are preserved (36). However, this study could not completely exclude the possibility that other vasodilators released from stellate interneurons were also involved. Comparison of the data in cyclin D2- and nNOS-null mice indicates that the magnitude of the attenuation in functional hyperemia is similar. Therefore, the present findings support the hypothesis that NO originates from stellate interneurons. This conclusion is also supported by experiments in which cyclin D2-deficient mice were treated with the nNOS-inhibitor 7-nitroindazole, demonstrating no further attenuation of the response (36).
In the cerebral cortex, the increase in CBF produced by activation of the facial whiskers is not attenuated in nNOS-deficient mice (23). In contrast, as shown here, the increase in BFcrb produced by activation of the same somatosensory system in the cerebellum is attenuated in these mice. This finding suggests that, whereas in the cerebral cortex other vasoactive factors are able to compensate completely for the absence of neuronal NO, in the cerebellum such full compensation does not occur and the flow response is attenuated. These observations suggest that, although NO is involved in functional hyperemia both in the cerebral and cerebellar cortex, its role in the vasodilation differs between the two regions. Long-term depression is absent in the cerebellum of nNOS-deficient mice (21). Therefore, nNOS-derived NO seems to be more critical to synaptic signaling and to the associated hemodynamic response in the cerebellum than in other brain regions. Studies of cerebellar functional hyperemia in eNOS-null mice would provide further evidence in support of this hypothesis. In contrast to Crus II activation, the response to hypercapnia is not attenuated in the cerebellum of nNOS-null mice. Therefore, it is likely that compensatory mechanisms took over in the nNOS-deficient mice and reestablished the response. In this respect, the cerebellum resembles the cerebral cortex, a region in which hypercapnic vasodilation is preserved in mice lacking nNOS (16).
The use of transgenic and "knockout" mice to study the physiological role of specific proteins is not devoid of pitfalls (15). For example, the lack of a protein from the early stages of development may induce compensatory changes in related systems aimed at restoring the function exerted by the missing protein. Accordingly, it cannot be assumed that mice genetically engineered to be deficient in selected protein(s) are identical to wild-type mice in all other respects. Thus pharmacological inhibitors remain an important tool that, when complemented with studies using null mice, provide strong evidence in favor of or against the involvement of a specific protein. Therefore, the results of the present study in nNOS-deficient mice, in conjunction with previous studies using pharmacological inhibitors, provide the most compelling evidence to date that nNOS is a major factor in the mechanism of functional hyperemia in the cerebellum.
In conclusion, we demonstrated that the increases in BFcrb produced by glutamate, harmaline-induced excitation of CF, and activation of the Crus II by somatosensory stimuli are selectively attenuated in mice lacking nNOS. Responses to ACh, adenosine, and hypercapnia are not affected. These findings provide the first nonpharmacological evidence that nNOS-derived NO plays a critical role in the hemodynamic responses initiated by glutamatergic synaptic activity in the cerebellar cortex.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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