Local and Propagated Vascular Responses Evoked by Focal Synaptic Activity in Cerebellar Cortex

Costantino Iadecola1, Guang Yang1, Timothy J. Ebner2, and Gang Chen2

1 Laboratory of Cerebrovascular Biology and Stroke, Department of Neurology and 2 Department of Neurosurgery, University of Minnesota Medical School, Minneapolis, Minnesota 55455

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Iadecola, Costantino, Guang Yang, Timothy J. Ebner, and Gang Chen. Local and propagated vascular responses evoked by focal synaptic activity in cerebellar cortex. J. Neurophysiol. 78: 651-659, 1997. We investigated the local and remote vascular changes evoked by activation of the cerebellar parallel fibers (PFs). The PFs were stimulated (25-150 µA, 30 Hz) in halothane-anesthetized rats equipped with a cranial window. The changes in arteriolar and venular diameter produced by PF stimulation were measured with the use of a videomicroscopy system. Cerebellar blood flow (BFcrb) was monitored by laser Doppler flowmetry and the field potentials evoked by PF stimulation were recorded with the use of microelectrodes. PF stimulation increased the diameter of local arterioles (+26 ± 1%, mean ± SE) in the activated folium (n = 10, P < 0.05). The vasodilation was greatest in smaller arterioles (16.5 ± 0.8 µm), was graded with the intensity of stimulation, and was less marked than the vasodilation produced by hypercapnia in comparably sized vessels (+58 ± 5%, CO2 pressure = 50-60 mmHg, n = 8). In addition, the vasodilation was greatest along the horizontal beam of activated PFs and was reduced in arterioles located away from the stimulated site in a rostrocaudal direction. The increases in vascular diameter were associated with increases in BFcrb in the activated area (+55 ± 4%, n = 5). PF stimulation increased vascular diameter (+10 ± 0.5%, n = 10) also in larger arterioles (30-40 µm) located in the folium adjacent to that in which the PFs were stimulated. Higher-order branches of these arterioles supplied the activated area. No field potentials were evoked by PF stimulation in the area where these upstream vessels were located. The data suggest that increased synaptic activity in the PF system produces a "local" hemodynamic response mediated by synaptic release of vasoactive agents and a "remote" response that is propagated to upstream arterioles from vessels residing in the activated folium. These propagated vascular responses are important in the coordination of segmental vascular resistance that is required to increase flow effectively during functional brain hyperemia.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

There is considerable evidence that neural activity is one of the major factors regulating cerebral perfusion (see Edvinsson et al. 1993 and Heistad and Kontos 1983 for reviews). Thus, if neural activity is enhanced, either focally or globally, cerebral blood flow increases in proportion to the intensity of the activation (Ginsberg et al. 1987; Greenberg et al. 1979; Iadecola et al. 1983; Nilsson et al. 1978; Ueki et al. 1992). The increases in cerebral blood flow produced by functional brain activation are localized to the activated regions. For example, stimulation of the somatosensory or visual pathways produces increases in cerebral blood flow in brain regions involved in somatosensory or visual processing (Fox and Raichle 1984; Frostig et al. 1990; Ginsberg et al. 1987; Grinvald et al. 1986; Malonek and Grinvald 1996; Ueki et al. 1992). The close relationship between flow and neural activity has provided a useful strategy for functional brain mapping using the changes in flow as an index of brain work (e.g., Belliveau et al. 1991; Fox and Raichle 1984; Le Doux et al. 1983; Lueck et al. 1989; Ogawa et al. 1992; Olesen 1971; see Raichle 1994 for a review).

The vascular mechanisms by which synaptic activity increases cerebral blood flow are not fully understood. The leading hypothesis, introduced more than a century ago (Roy and Sherrington 1890), is that active neurons release vasoactive substances that reach local vessels by simple diffusion and mediate vasodilation resulting in an increase in local flow (see Iadecola 1993 and Lou et al. 1987 for reviews). However, hemodynamic considerations indicate that vasodilation restricted to microvessels at the site of activation is not sufficient to increase flow because vascular resistance is controlled by larger pial arterioles upstream (Heistad and Kontos 1983; Segal 1992). Rather, the local vascular changes elicited by increased synaptic activity have to occur in concert with dilatation of parent arteries upstream to increase flow effectively (see Duling et al. 1987 and Segal 1992 for reviews). Coordinated changes in proximal and distal vascular resistance are well known to occur in other vascular beds during functional activation (Duling et al. 1987; Segal 1992). However, despite the large number of studies on the control of the cerebral microcirculation by neural activity, little has been learned about such coordinated vascular responses in the brain and their participation in cerebrovascular regulation (Iadecola 1993; Woolsey and Rovainen 1991). One problem has been the lack of a suitable model system in which temporally defined and spatially restricted increases in synaptic activity could be elicited in a brain region with a relatively simple vascular supply.

The parallel fiber (PF) system of the cerebellar cortex has recently been used as a model to investigate the relationship between synaptic activity and blood flow in the CNS (Akgören et al. 1994; Iadecola and Ebner 1993). The PFs are axons of the cerebellar granule neurons that travel from the granule cell layer to the molecular layer, bifurcate, and run in opposite directions on the cerebellar surface (see Ito 1984 for a review). PF terminals make synaptic contacts with Purkinje cell dendrites and molecular layer interneurons (Ito 1984). The transmitter released from the PFs is most likely glutamate (see Ross et al. 1990 for a review). Activation of the PFs produces focal increases in cerebellar blood flow (BFcrb) that are greatest in the activated areas, are associated with an increase in glucose utilization, and are mediated by glutamate receptors through the production of nitric oxide (NO) and adenosine (Akgören et al. 1994; Iadecola et al. 1995, 1996a,b; Li and Iadecola 1994). Therefore the PF system is a useful model for investigating the relationships between synaptic activity and blood flow. In addition, because the vascular supply of the cerebellar molecular layer is relatively simple (Scremin 1995), this model should be well suited for the study of propagated vascular responses.

In the present study, therefore, we used the PF system to investigate the local and remote microvascular changes associated with focal increases in synaptic activity. We found that stimulation of the PFs produces arteriolar dilatation that is maximal at the site of activation. However, PF stimulation produces vasodilation also in upstream arterioles whose branches supply the activated area. Because no evoked neural activity could be detected in the region in which these upstream vessels are located, the evidence suggests that the local vascular responses in the activated region are propagated to parent vessels upstream. The data provide direct demonstration of propagated vascular responses evoked by increased neural activity in the CNS.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Methods for electrical stimulation of the PFs, for recording field potentials, and for monitoring of flow in the cerebellar cortex have been described previously (Iadecola et al. 1995, 1996a,b; Li and Iadecola 1994) and are summarized below.

General surgical procedures

Studies were performed on 76 male Sprague-Dawley rats (Sasco, Omaha, NE) weighing 275-300 g. Rats 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 both femoral arteries, in the left femoral vein, and in the trachea. Animals were then placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA; model 1404) mounted on a vibration-free table (TMC, Peabody, MA) and artificially ventilated with a oxygen-nitrogen mixture with the use of a mechanical ventilator (Harvard Apparatus, South Natick, MA; model 638). The oxygen concentration in the mixture was adjusted to obtain an arterial oxygen pressure of approx 120 mmHg (Table 1). Body temperature was maintained at 37 ± 0.5°C (mean ± SE) with the use of a heating lamp thermostatically controlled by a rectal probe (YSI, Yellow Springs, OH; model 73A-TA). One of the arterial catheters was used for continuous recording of arterial pressure and heart rate on a chart recorder (Grass, Quincy, MA; model 716P), whereas the other arterial catheter was used for blood sampling. Arterial CO2 pressure (pCO2), oxygen pressure, and pH were measured at multiple times on 100 µl of blood with the use of a blood gas analyzer (CIBA-Corning, Medfield, MA; model 178). 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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TABLE 1. Arterial pressure and blood gases in the rats in which vascular diameter and cerebellar blood flow were studied

Stimulation of the PFs and monitoring of field potentials

As described in detail elsewhere (Iadecola et al. 1995, 1996a), a small hole (3 × 3 mm) was drilled in the interparietal bone. The dura was carefully removed and the cerebellar vermis was exposed (lobule VI). The cranial window so produced was continuously superfused with Ringer solution at a rate of 0.33 ml/min (Iadecola et al. 1996a). To minimize upstream spreading of the vasoactive metabolites generated by neural activity, the Ringer solution was allowed to flow in a rostrocaudal direction. As in previous studies, solutions were equilibrated with 95% O2-5% CO2 (pH 7.3-7.4) and warmed to 37°C (Iadecola et al. 1995, 1996a). The PFs were activated electrically with the use of monopolar tungsten microelectrodes (resistance 1 MOmega ) inserted into the molecular layer (Iadecola et al. 1995, 1996a). Stimuli were negative square waves (pulse duration 0.3 ms) delivered from a stimulator (Grass, model S88) through a stimulus isolation unit (Grass, model PSIU6). A silver wire attached to the occipital muscles served as ground. The cerebellar cortex, unlike the cerebral cortex, does not generate waves of spreading depression easily (see Iadecola et al. 1996a for references). This property of the cerebellar cortex is advantageous for cerebrovascular studies because it eliminates the concern of the long-lasting cerebrovascular effects of the spreading depression produced by insertion of the stimulating electrode.

Field potentials were recorded by glass micropipettes (tip diameter 5-10 µm) filled with 2 M NaCl (resistance 2-5 MOmega ) and inserted at a depth of approx 30 µm. The signal from the micropipettes was amplified (Grass, microelectrode amplifier, model 7P5), displayed on an oscilloscope, and digitized with the use of a computerized data acquisition system (GW Instruments, Sommerville, MA; MacAdios IIjr). In studies of field potentials, PFs were stimulated at the rate of one per second (100 µA, pulse duration 0.3 ms). In each trial, 10 traces were acquired, averaged, and stored for off-line analysis (Superscope software, GW Instruments) (Iadecola et al. 1995, 1996a). Field potentials, BFcrb, and pial vascular diameter were recorded in separate groups of rats.

Monitoring of blood flow in cerebellar cortex

As described in detail elsewhere (Iadecola et al. 1995, 1996a), BFcrb was monitored with the use of a Vasamedic laser Doppler flowmeter (model BPM 403A; Saint Paul, MN). 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 was amplified (Grass, DC amplifier, model 7P1) and displayed on the polygraph. To avoid pulsatile variations in the flow signal, a long time constant was used (5 s). Changes in BFcrb were calculated as percentage of the baseline value determined at the end of the experiment.

Measurement of pial vascular diameter

Pial vessel diameter was measured with the use of a microscope (Nikon) positioned directly over the cranial window and equipped with an objective (×20) with a long working distance. Illumination was provided by a fiberoptic light source with a green filter and a heat filter. Images were captured by a video camera (Panasonic) and displayed on a monitor. Vascular diameter was measured in real time directly from the monitor with the use of a calibrated video microscaler (Optech, Trenton, NJ) (Wang et al. 1994). Cerebral arterioles could easily be distinguished from venules on the basis of fast red blood cell flow, thicker walls, and bright red blood color. However, in some studies the type of vessel studied was confirmed at the end of the experiments by intra-arterial infusion of ink (see also below).

Vascular anatomy of the rostral cerebellum

For determination of the vascular anatomy of the region of the cerebellum in which the PFs were stimulated, black drawing ink (Pelikan AG, Hanover, Germany) was injected into the aorta to fill the arterial system (n = 4) (Scremin 1995). The branches of the basilar artery vascularizing the dorsal aspect of the rostral cerebellum were identified with the use of a surgical microscope. A composite drawing describing the vascular supply of this region of the cerebellum was then constructed (Fig. 1).


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FIG. 1. Drawing illustrating arteries vascularizing rostromedial cerebellum. Course of arteries was determined in rats perfused intra-arterially with black ink. Superior cerebellar artery (SCA) is branch of basilar artery that gives rise to medial and lateral SCA (MSCA and LSCA, respectively). Rostral vermis (lobule 5 and 6) is vascularized by MSCA through several branches that run in rostrocaudal direction perpendicular to venous arches that outline folia. Lobule 6 has 3 folia. Stimulating electrode was placed in intermediate folium of lobule 6. AICA, anteroinferior cerebellar artery; BAS, basilar artery.

Experimental protocol

After the cranial window was drilled and the superfusion with Ringer solution was started, the arterial blood gases were adjusted. The microelectrode for stimulation of the PFs was placed on the middle folium of lobule 6 of the cerebellar vermis (Fig. 1). After the placement of the electrode, the preparation was allowed to stabilize for 30 min. The experiments commenced after arterial blood gases were in a steady state.

EFFECT OF PF STIMULATION ON PIAL VASCULAR DIAMETER. The microscope was positioned on the vessel of interest and its resting diameter was measured. Vessels were selected on the basis of size and distance from the stimulating electrode (see below). PFs were stimulated with continuous trains of stimuli at 30 Hz (pulse duration 0.3 ms) and with an intensity of 100 µA. The evoked increase in pial arteriolar diameter was observed in real time on the video monitor. Once the vasodilation reached a steady state, usually after 20 s of stimulation, the vascular diameter was measured and the stimulation was stopped. Vascular diameter was measured again after the vasodilation had subsided to document that the vessel had returned to baseline. The effect of PF stimulation on the diameter of arterioles of sizes ranging from 15 to 40 µm was studied in 10 rats. In other experiments (n = 9), the relationship between stimulus intensity and vasodilation was investigated. At each stimulation the intensity of the stimulus was varied (25-150 µA) while the stimulus frequency was maintained at 30 Hz. The resting vascular diameter in the arterioles studied was stable throughout the experimental period (Fig. 1A; P > 0.05, n = 25).

SPATIAL DISTRIBUTION OF THE INCREASES IN PIAL VESSEL DIAMETER. In these experiments (n = 25), arterioles located at increasing distances from the stimulating electrode were studied. In the "horizontal" group, the arterioles were located at increasing lateral distances (0.1-0.9 mm) from the electrode. In the "vertical" group, the arterioles were located at increasing distances (0.1-0.9 mm) in the rostrocaudal plane. All the arterioles studied were located within the stimulated folium.

EFFECT OF HYPERCAPNIA ON PIAL VASCULAR DIAMETER. In these experiments (n = 8), the effect of hypercapnia on pial vascular diameter was studied. Arterioles of different sizes were studied (15-40 µm). First, resting vascular diameter was determined. Hypercapnia was then produced by increasing the concentration of CO2 in the respiratory mixture until a pCO2 of 50-60 mmHg was reached (Iadecola et al. 1995). The increase in vascular diameter was measured after the vasodilation had reached a steady state.

UPSTREAM VASCULAR RESPONSES EVOKED FROM PF STIMULATION. In these experiments (n = 10), the effect of PF stimulation on arteriolar diameter was mapped at different sites upstream along the course of the vessel. First, the origin, course, and branching pattern of one of the arterioles overlying the stimulated folium and the folium immediately rostral to it were determined under the microscope and drawn on paper. Then the PFs were stimulated and the associated vascular diameter changes were measured at different points along the course of the selected arteriole. A cartoon representing the branching pattern and reactivity of each segment of the vessel was then constructed from the data collected in each rat.

FIELD POTENTIALS AND CHANGES IN BFcrb. In separate rats (n = 5), the middle folium of lobule 6 was stimulated and field potentials were recorded in the stimulated folium and in the folium rostral to it. In other rats (n = 5), the middle folium of lobule 6 was stimulated and BFcrb was monitored at the site of stimulation and in the folium rostral to it. In these experiments the stimulus intensity producing the largest change in flow was used (100 µA).

Data analysis

Data in text, tables, and figures are presented as means ± SE unless otherwise indicated. Two-group comparisons were evaluated by the two-tailed Student's t-test. Multiple comparisons were evaluated by the analysis of variance and Tukey's test (Systat, Evanston, IL). Differences were considered significant for probability values <0.05.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Arterial supply of the rostral cerebellum and lobule 6

The distribution of the arterioles supplying lobule 6 was studied in four rats in which the arterial system was visualized by ink injection. Data on the arteriolar supply of the stimulated region were also collected from rats in which the spatial distribution of the vasodilation was studied (see below). The arterial supply of the rostral cerebellum originates from the superior cerebellar artery, a branch of the basilar artery (Fig. 1). The superior cerebellar artery wraps around the caudal part of the cerebral peduncles and divides into a lateral and a medial branch. The lateral branch supplies the lateral aspect of the cerebellar hemisphere and the flocculus. The medial branch runs deeply into the sulcus between the cerebellum and the cerebral hemisphere, reaches the rostral vermis, and divides into several branches. These branches run in a rostrocaudal direction perpendicular to the superficial venous arches that demarcate the folia and supply lobule 5 and the three folia of lobule 6. Therefore the arterioles that provide the blood supply to the rostral vermis are branches of the medial superior cerebellar artery.

Effect of PF stimulation on the diameter of local vessels

Stimulation of the PFs increased the diameter of arterioles within the stimulated folium (Figs. 2 and 3). The magnitude of the increase depended on the intensity of the stimulus (Fig. 2). The response was well developed at 25 µA, became more pronounced at 50 and 75 µA, and reached a maximum at 100 µA (Fig. 2). The vasodilation was greater in arterioles 10-20 µm diam than in larger vessels (Fig. 3).


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FIG. 2. A: time course of resting diameter of arterioles of different size throughout experimental period. Resting diameter of arterioles studied remained stable over 3-h experimental period (P > 0.05, analysis of variance and Tukey's test). B: effect of stimulus intensity on vasodilation produced by parallel fiber (PF) stimulation. Magnitude of response increased steadily between 25 and 100 µA. Decline of response was observed at 150 µA (P < 0.05, analysis of variance and Tukey's test).


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FIG. 3. Effect of PF stimulation on diameter of arterioles in cerebellar cortex. Resting diameter is indicated in each bar. PF stimulation increases arteriolar diameter. Smaller vessels are more reactive than larger ones(P < 0.05, analysis of variance and Tukey's test).

We next studied the spatial distribution of the increases in vascular diameter within the stimulated folium (Fig. 4). In these experiments, arterioles 20.9 ± 0.15 µm diam were studied. In arterioles located up to 1 mm away from the stimulated site in a horizontal direction, the magnitude of the vasodilation did not decrease with distance (Fig. 4). However, in arterioles located away from the stimulated site in a vertical (rostrocaudal) direction, the response decreased as a function of the distance. These observations are in agreement with previous data demonstrating that the increase in blood flow is greatest along the beam of active PFs (Iadecola et al. 1996a).


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FIG. 4. Relationship between horizontal and vertical distance from stimulating electrode and magnitude of increase in arteriolar diameter produced by PF stimulation (100 µA, 30 Hz). These data were obtained from 25 arterioles of average diameter 20.9 ± 0.5 (SE) µm. Vasodilation does not decrease in arterioles located at increasing horizontal distances from stimulation site (top). However, in vertical direction (rostrocaudal), magnitude of response decreased with distance, an effect that reaches statistical significance at 0.7 mm from stimulating electrode (P < 0.05, analysis of variance and Tukey's test). At such distance, however, vasodilatation is still present, albeit attenuated by ~50%.

Effect of hypercapnia on the diameter of cerebellar vessels

For comparison, we also studied the effect of hypercapnia on the vascular diameter in the microvessels of lobule 6. Hypercapnia (pCO2 = 50-60 mmHg) increased vascular diameter in all vessels studied (Fig. 5). The magnitude of the vasodilation depended on the size of the vessel, the smaller arterioles responding more vigorously than the larger ones (Fig. 5). Therefore the increases in vascular diameter produced by hypercapnia are more pronounced than those evoked by PF stimulation in comparably sized vessels.


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FIG. 5. Effect of arterial hypercapnia on arteriolar and venular diameter in cerebellar cortex. Hypercapnia [CO2 pressure (pCO2) = 54.0 ± 1.1 mmHg] increased arteriolar diameter. Response was more pronounced in smaller arterioles (P < 0.05, analysis of variance and Tukey's test).

Upstream vascular and electrical responses evoked by PF stimulation

In these studies we investigated whether the vascular response initiated by PF stimulation is propagated upstream to parent arteries whose branches supply the stimulated folium. As described above, lobule 6 is supplied by arteries that are first-order branches of the medial division of the superior cerebellar artery (Fig. 1). These branches divide, giving rise to daughter arteries (2nd order) that, in turn, divide again (3rd and 4th order). Therefore the stimulated folium is vascularized by third- to fourth-order branches of the medial superior cerebral artery. A schematic reconstruction of the branching pattern is provided in Fig. 6. The PFs in the middle folium of lobule 6 were stimulated and the evoked diameter changes were measured in arterioles at the stimulation site and in progressively lower-order parent branches until the main branch from the medial superior cerebellar artery was reached. As expected, the largest changes in diameter were observed in higher-order branches crossing the beam of activated PFs (Fig. 6). However, increases in diameter were also observed in upstream parent arteries located in the folium rostral to that in which the beam of PFs was activated (Fig. 6).


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FIG. 6. Changes in diameter produced by PF stimulation in representative pial arteriole and its higher-order branches. Black dot in A: stimulated site. Vasodilation is greatest in regions surrounding stimulated site (A). However, sizable increases in vascular diameter are also observed in larger vessels located in folium (B) rostral to that in which PFs were stimulated. As illustrated in Fig. 7, no evoked potentials could be recorded in B after stimulation of PFs in A. These observations suggest that increases in vascular diameter in upstream parent arterioles are not mediated by increased neural activity spreading from stimulated site.

To determine whether the changes in vascular diameter corresponded to an increase in tissue perfusion, BFcrb was monitored by laser Doppler probe at the site of stimulation and in the folium rostral to it. As illustrated in Fig. 7, the largest flow increase was observed at the level of the activated beam of PFs. However, increases were also seen in the folium rostral to that in which the PFs were activated (Fig. 7). Temporal analysis of the flow elevations revealed that the time for the signal to reach a plateau, the length of the plateau, and the time for the signal to return to baseline were significantly longer at the remote site (B in Fig. 6) than in the stimulated folium (A in Fig. 6) (Table 2).


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FIG. 7. Effect of PF stimulation on field potentials and cerebellar blood flow (BFcrb) in cerebellar cortex. PFs were stimulated (100 µA) in intermediate folium of lobule 6 (A in Fig. 6). Field potentials and BFcrb were recorded in A and B (see Fig. 6). In stimulated folium (A), PF stimulation produced typical filed potentials (top, oblique right-arrow) and increased BFcrb (bottom). In folium rostral to site of stimulation (B), PF stimulation did not produce field potentials and only an artifact of stimulus can be seen (*). However, increase in BFcrb still occurred at this site. Increase in vascular diameter in parent vessel whose branches supply activated folium was also observed in B (Fig. 6).

 
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TABLE 2. Time course of the increase in BFcrb in the stimulated folium and in the folium rostral to it

We next studied the spatial distribution of field potentials evoked by stimulation of the PFs. PF stimulation (100 µA) produced the typical field potentials characterized by a short-latency, fast component reflecting conduction of action potentials along the PFs, and a longer-latency, slow component reflecting depolarization of Purkinje cells and molecular layer interneurons (Eccles et al. 1966) (Fig. 7). As expected, the typical field potentials were obtained only in a narrow horizontal band (width approx 200 µM) on the stimulated folium (e.g., Elias et al. 1993). No evoked electrical activity was observed on extensive mapping in the folium rostral to that in which the PFs were stimulated (Fig. 7). Therefore, at the relatively low stimulations intensities used in these experiments (100 µA), there was no evidence of activation of the adjacent folium via mossy fiber collaterals (transfolial activation) (Eccles et al. 1966). These findings are in agreement with reports in which voltage-sensitive dyes were used, demonstrating that the synaptic activity evoked by PF stimulation, with intensities comparable with those used in the present study, is restricted to the beam of activated PFs and does not involve the adjacent folium (Elias et al. 1993).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We have demonstrated that electrical stimulation of the cerebellar PFs produces vasodilation in local arterioles supplying the activated folium. The increases in diameter are graded with the intensity of stimulation and are more marked in smaller than in larger vessels. Furthermore, the vasodilation is greatest in the area in which the PFs are activated. Increases in vascular diameter were also observed in parent arterioles that are located rostral to the activated folium. These upstream changes in vascular diameter are associated with an increase in local blood flow. PF stimulation did not evoke any field potentials in the folium in which these remote vascular changes were observed. These data suggest that neural activity, in addition to the well-established local vasodilation, produces vasodilation also in upstream vessels whose higher-order branches supply the activated area.

The findings of the present study cannot be the result of differences in arterial blood pressure or blood gases because these variables were carefully monitored and controlled and did not differ among the experimental groups. Similarly, the changes in vascular diameter cannot be due to instability of the preparation because resting vascular diameter did not change through the duration of the experiments. Therefore the local and remote vascular changes evoked by PF stimulation cannot be attributed to differences in the physiological state of the rats or to instability of the experimental preparation used. It is also unlikely that the increase in flow observed rostral to the stimulated site is due to the poor spatial resolution of the flow probe resulting in detection of flow changes occurring at distant sites. The area of detection of the laser Doppler probe is confined to the region immediately under the probe up to a depth of ~1 mm (Tenland 1982). Therefore it is doubtful that the probe detected changes in flow occurring approx 2 mm away in a rostrocaudal direction. The fact that arteriolar dilatation was observed at the site where the flow increase was observed also supports this conclusion.

Stimulation of the PFs produces vasodilation in arterioles located in the activated area. The spatial distribution of the vascular response, although not perfectly overlapping, corresponds well to the location of the beam of activated PFs. Although the magnitude of the vasodilatation produced by PF stimulation was comparable with that observed in other brain regions during functional activation (e.g., Ngai et al. 1988), it was smaller than that produced by hypercapnia. This finding is expected considering that pCO2 is, perhaps, the most potent cerebrovasodilator (Heistad and Kontos 1983). However, like the response to PF stimulation, the vasodilatation produced by hypercapnia was most marked in smaller arterioles, reflecting a certain similarity in the microvascular adjustments responsible for two vasodilator responses.

The local vascular changes produced by PF stimulation are likely to be mediated by diffusible agents released during synaptic activity. In previous studies, we and others have determined that the increase in blood flow produced by PF stimulation is mediated by activation of glutamate receptors through NO and adenosine. This conclusion is based on the observation that the increase in BFcrb produced by PF stimulation is blocked by glutamate receptor antagonists and is attenuated by NO synthase inhibitors or by adenosine receptor antagonists (Akgören et al. 1994; Iadecola et al. 1995, 1996a,b; Li and Iadecola 1994). Because the effects of inhibition of NO synthesis and adenosine receptors are additive, it has been suggested that adenosine and NO have independent roles in the vasodilation (Li and Iadecola 1994). Therefore glutamate-receptor-mediated release of NO and adenosine is likely to be responsible for the local hemodynamic response occurring in the area of the activated PFs.

In addition to the local vascular response, PF stimulation produces significant changes in vascular diameter in larger arterioles located in the folium rostral to the stimulated site, whose branches supply the activated area. The mechanisms of these upstream vascular changes are less clear than those responsible for the local response. The vasodilation in upstream vessels cannot be mediated by local release of vasoactive agents, because PF stimulation did not evoke synaptic activity at that site where these vessels were located. It is also unlikely that the remote vascular changes are initiated by upstream diffusion of NO and adenosine from the activated site. First, diffusion in the cerebellar molecular layer is more efficient along the major axis of the PFs than in the direction perpendicular to it (Rice et al. 1993). Therefore diffusion in the direction perpendicular to the activated PFs would be relatively impaired. Second, because the biological half-life of NO and adenosine is short (Phillis 1989; Stamler 1994), these agents are unlikely to reach the folium rostral to that in which the PFs were activated in concentrations sufficient to produce vasodilation. Another possibility is that the vasodilation in parent vessels is mediated through neurogenic mechanisms. This hypothesis implies that stimulation of the PFs activates neurons that project to upstream arterioles, resulting in their dilatation. Cerebral blood vessels are innervated by perivascular nerves arising from cranial autonomic ganglia and, possibly, by intraparenchymal neurons (see Iadecola 1992 for a review). However, autonomic nerves do not participate in the vascular response initiated by PF stimulation (Iadecola et al. 1995) and there are no anatomic data supporting the existence of direct innervation of pial cerebellar arterioles by intrinsic cerebellar neurons (Iadecola 1992). This evidence, therefore, argues against the neurogenic hypothesis for the remote vascular changes occurring during PF stimulation.

The most likely explanation for the upstream vasodilation is that the response is propagated through cellular interactions within the vascular wall. Retrograde propagation of vasodilation has been most extensively studied in the peripheral circulation (see Duling et al. 1987 and Segal 1992 for reviews). Two mechanisms are thought to be responsible for the upstream propagation of vasodilation. One is the retrograde vasodilation originally described by Duling and Berne (1970). The vasodilation produced by topical application of acetylcholine to arterioles is propagated upstream for several millimeters (Segal and Duling 1986). The vasodilation may be propagated through conduction of metabolic or electrical signals via gap junctions between endothelial cells and, possibly, between endothelial cells and smooth muscle cells (Segal and Bény 1992). The other mechanism for retrograde propagation is flow-mediated vasodilation (see Griffith 1996 for a review). Downstream vasodilation during functional activation leads to an increase in blood flow velocity in feeding vessels upstream. The increased shear stress on the endothelium leads to the release of vasoactive factors, resulting in vasodilation of the feeding vessels and amplification of the flow response. In some vascular beds the response is mediated by release of endothelial NO and prostanoids (Griffith 1996). Flow-mediated vasodilation is typically observed in vessels larger that those in which the retrograde vasodilation of Duling and Berne occurs. Therefore it has been proposed that these two mechanisms act on different segments of the vasculature to propagate the vasodilation to progressively larger vessels (Duling et al. 1987; Iadecola 1993). However, flow-mediated vasodilation has recently been documented also in intracerebral arterioles 38-55 µm diam (Ngai and Winn 1995). It would, therefore, seem that both modes of retrograde propagation can operate in similarly sized vessels.

The present data indicate that the focal increases in synaptic activity in the cerebellar molecular layer produce a "local" vascular response, mediated by release of vasoactive agents at the site of activation, and a "remote" response that is propagated upstream, presumably, through intravascular mechanisms. Vasodilatation has to occur both in precapillary arterioles and in larger feeding arteries to increase flow effectively (Segal 1992). This concept is well established in other vascular beds in which the changes in vascular diameter produced by functional activation involve coordinated vasodilation of both precapillary arterioles and larger vessels upstream (Segal 1992). In brain, as in other organs, retrograde vasodilation and flow-mediated vasodilation may be important mechanisms for the retrograde propagation of the vascular responses (Iadecola 1993). A type of retrograde vasodilation resembling that described by Duling and Berne has recently been reported in isolated rat cerebral arterioles (Dietrich et al. 1996). Furthermore, flow-mediated vasodilation has been observed in the rat cerebral circulation in vivo (Fujii et al. 1991) and in isolated intracerebral arterioles comparable in size with those investigated in the present study (Ngai and Winn 1995). Interestingly, in isolated arterioles flow-mediated dilatation was found to be mediated by NO (Ngai and Winn 1995).

The data also suggest that, although there is a general correspondence between the area of activation and the area of vasodilation, the hemodynamic response does not overlap precisely with the region of increased synaptic activity. Thus PF stimulation increases vascular diameter and flow also in areas in which no activation can be detected electrophysiologically (present study; Iadecola et al. 1996a). It would seem, therefore, that neural activity produces hemodynamic changes over an area larger than that in which neural activity is increased. This finding is not surprising, because the distribution of the hemodynamic response is constrained by the microvascular architecture of the region that does not necessarily overlap with the distribution of neural activity. Indeed, a similar conclusion has been reached by a study in which neuronal activity and vascular responses were mapped in the activated somatosensory cortex with the use of optical imaging (Malonek and Grinvald 1996). These observations have important implications for studies in which hemodynamic changes are used to localize brain function. Because flow increases in an area larger than the area in which synaptic activity is enhanced, caution is needed in the interpretation of hemodynamic responses as a reflection of brain activation in functional brain mapping studies (see Malonek and Grinvald 1996 for a discussion).

In conclusion, we have demonstrated that focal increases in synaptic activity produced by PF stimulation elicit increases in arteriolar diameter that are graded with the intensity of stimulation and greatest along the beam of activated PFs. PF stimulation also produces vasodilation in upstream arterioles whose higher-order branches supply the site of activation. This remote vascular response occurs in the absence of evoked local synaptic activity. The evidence suggests that synaptic activity in the PF system evokes a local hemodynamic response mediated by local release of vasoactive factors, as well as a remote response that is propagated upstream from the site of activation through intravascular mechanisms. The findings clearly establish that propagated vasodilation occurs also in the CNS during functional activation. These propagated hemodynamic responses are responsible for the coordination of segmental vascular resistance that is required to increase flow effectively in regions of increased brain work.

    ACKNOWLEDGEMENTS

  The authors thank K. MacEwan for assistance in the preparation of the manuscript.

  This work was supported by National Institute of Neurological Disorders and Stroke Program Project Grant NS-31318. C. Iadecola is an Established Investigator of the American Heart Association.

    FOOTNOTES

  Address for reprint requests: C. Iadecola, Dept. of Neurology, University of Minnesota Medical School, Box 295 UMHC, 420 Delaware St. SE, Minneapolis, MN 55455.

  Received 24 January 1997; accepted in final form 3 April 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society