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Am J Physiol Regul Integr Comp Physiol 274: R529-R540, 1998;
0363-6119/98 $5.00
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Vol. 274, Issue 2, R529-R540, February 1998

Cerebellar vascular and synaptic responses in normal mice and in transgenics with Purkinje cell dysfunction

Guang Yang1, Rod M. Feddersen2, Fangyi Zhang1, H. Brent Clark3, Alvin J. Beitz4, and Costantino Iadecola1

1 Laboratory of Cerebrovascular Biology and Stroke, Department of Neurology; 2 Department of Laboratory Medicine and Pathology; 3 Departments of Laboratory Medicine and Pathology (Neuropathology) and Neurology, University of Minnesota Medical School, Minneapolis 55455; and 4 Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We used transgenic mice with Purkinje cell dysfunction (PO3 line) to study the role of these neurons in the increase in cerebellar blood flow (BFcrb) produced by stimulation of the cerebellar parallel fibers (PF). Mice (age 8-10 wk) were anesthetized (halothane) and artificially ventilated. Arterial pressure and end-tidal CO2 were monitored continuously. Arterial blood gases were measured. The PF were stimulated electrically (100 µA, 30 Hz; 40 s), and the increases in BFcrb were monitored by a laser-Doppler flow probe. First, we characterized the increases in BFcrb and the field potentials produced by PF stimulation in normal mice. PF stimulation evoked the typical field potentials and increased BFcrb by 60 ± 4% (100 µA, 30 Hz; n = 10). The increases in BFcrb were attenuated by the broad-spectrum glutamate receptor antagonist kynurenate (-84 ± 3%; P < 0.05 analysis of variance; n = 5), by the DL-alpha -amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (-62 ± 6%; P < 0.05; n = 5), and by the nitric oxide synthase inhibitor Nomega -nitro-L-arginine (-46 ± 7%; P < 0.05; n = 5). In PO3 transgenic mice, the increases in BFcrb produced by PF stimulation were reduced (P < 0.001) at every stimulus intensity and frequency tested (residual increase at 100 µA, 30 Hz: 19 ± 2%; n = 6). The field potentials evoked by PF stimulation also were abnormal in that they lacked the late negative wave (n = 6), a finding consistent with lack of depolarization of Purkinje cells. The residual flow response in the transgenics was abolished by Nomega -nitro-L-arginine (n = 5; P > 0.05). Ultrastructural studies showed that the density of PF-Purkinje cell synapses is reduced in PO3 mice, whereas the morphology of molecular layer interneurons (stellate cells) is normal. The findings suggest that Purkinje cells are responsible for a sizable component of the flow response whereas molecular layer interneurons mediate the remainder of the response. The study provides evidence that mouse mutants with spontaneous or genetically engineered cerebellar abnormalities could be useful to study the cellular and molecular correlates of functional hyperemia in the central nervous system.

cerebral circulation; cerebellum; laser-Doppler flowmetry; vasodilation; glutamate

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE PARALLEL FIBER (PF) system of the cerebellar molecular layer is a useful model to study the increases in blood flow produced by neural activity. The PF are axons of granule neurons that are located near the surface of the cerebellar cortex and terminate on Purkinje cells and molecular layer interneurons (see Ref. 26 for a review). The transmitter released from the PF is glutamate, which is thought to activate predominantly dl-alpha -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors on Purkinje cells (20, 27; see Ref. 22 for a review). Electrical stimulation of the PF in rats produces spatially restricted increases in cerebellar blood flow (BFcrb) that are coupled with a local increase in glucose utilization and depend, in part, on nitric oxide (NO) synthesis (2, 15, 16).

Experiments using glutamate receptor antagonists have provided initial evidence that the BFcrb response depends on activation of Purkinje cells and interneurons (2, 15, 16). However, the relative contributions of Purkinje cells and interneurons to the flow response have not been established. The study of the contribution of the different cerebellar cortical neuronal types to the mechanisms of functional hyperemia could benefit from the investigation of transgenic mice in which gene expression is targeted to Purkinje cells using the Purkinje cell protein 2 (PCP-2) promoter (e.g., Refs. 4, 11, 12, 24, 25, 31). Transgenic mice with Purkinje cell-specific expression of the large T antigen, a simian virus 40 (SV40) oncoprotein, develop Purkinje cell degeneration and cerebellar ataxia (11). In another transgenic mouse line, termed PO3, a form of the T antigen lacking the retinoblastoma tumor suppressor protein binding domain was expressed. PO3 mice become ataxic at the end of the second postnatal week in the absence of significant Purkinje cell loss, suggesting that Purkinje cells are dysfunctional (12). Therefore, PO3 mice may provide the opportunity to study the contribution of Purkinje cells to the vascular responses evoked by PF stimulation.

Therefore, in the present study the vascular responses evoked from PF stimulation were studied in PO3 transgenics and in nontransgenic littermates. We found that PF stimulation in anesthetized mice with controlled arterial pressure and blood gases produces increases in BFcrb that have characteristics similar to those reported in rats (2, 15, 16). In PO3 mice, the flow response evoked by PF stimulation was reduced and the late negative wave of the field potential, reflecting Purkinje cell depolarization, was absent. Ultrastructural studies showed that the number of PF-Purkinje cell synapses are reduced in PO3 mice, whereas the morphology of stellate interneurons is relatively normal. The findings suggest that Purkinje cells are responsible for a sizable component of the flow response, whereas molecular layer interneurons may mediate the remainder of the response.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

General Surgical Procedures

Studies were performed in 60 C57B6 mice (Jackson Laboratories, Bar Harbor, ME; age 8-10 wk), 31 PO3 transgenic mice, and 30 nontransgenic littermates weighing 25-30 g. PO3 transgenics and littermates (FVB/N background) were studied at an age (8-10 wk) when there is behavioral evidence of ataxia in the absence of significant Purkinje cell loss (12). Techniques for cerebrovascular studies in mice with controlled arterial pressure and blood gases were similar to those previously described (7, 35). 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 (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). Body temperature was maintained at 37 ± 0.5°C with the use of 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.

Artificial Ventilation and Monitoring of Arterial Blood Gases

Mice were artificially ventilated with an oxygen-nitrogen mixture by a mechanical ventilator (SAR-830; CWI, Ardmore, PA). The inspiration time was set at 0.1 s, the respiratory rate at 120 breaths/min, and the inspiratory flow at approx 250 ml/min. The oxygen concentration in the mixture was adjusted to obtain an arterial PO2 of 130-140 mmHg (Table 1). End-tidal CO2 was continuously monitored using a CO2 analyzer (Capstar-100, CWI). The sample flow rate of the CO2 analyzer was set at 10 ml/min. Great care was taken to accurately monitor arterial PCO2 and PO2, critical variables for studies of the cerebral circulation (14). In preliminary studies (n = 4), we established the relationship between end-tidal CO2 and arterial PCO2, measured by a blood gas analyzer (model 178; CIBA-Corning, Medfield, MA). The relationship was linear between PCO2 values of 24 and 59 mmHg (r2=0.96; P < 0.001; n = 21). We observed, however, that the slope of the relationship between arterial PCO2 and end-tidal CO2 depends on the respiratory dead space, which, in turn, is influenced by the body weight and length of the tracheal tube. Consequently, care was taken to keep the weight of the mice within a narrow range and to use the same tracheal tube in all studies. Throughout the experiment end-tidal CO2 was maintained at 2.6-2.7%, which corresponds to a PCO2 of 33-35 mmHg (Table 1). In experiments in which BFcrb was monitored, arterial blood gases were measured both before the responses to PF stimulation were first tested and at the end of the experiment, ~3 h later (Table 1).

                              
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  		Table 1.   Arterial pressure and blood gases in normal mice in which the effect of PF stimulation on cerebellar blood flow was studied

Stimulation of the PF and Monitoring of Field Potentials

Techniques for stimulation of PF and recording of the field potentials in mice were similar to those used in rats. As described in detail elsewhere (15, 16), a small hole (3 × 3 mm) was drilled in the interparietal bone. The dura was carefully removed and the cerebellar vermis exposed (lobule VI). The cranial window so produced was continuously superfused with Ringer at a rate of 0.33 ml/min (16). As in previous studies, solutions were equilibrated with 95% O2 and 5% CO2 (pH 7.3-7.4) and warmed to 37°C (15, 16). The PF were activated electrically using monopolar tungsten microelectrodes (resistance 1 MOmega ) inserted into the molecular layer (15, 16). 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). A silver wire attached to the occipital muscles served as ground.

Field potentials were recorded by glass micropipettes (tip diameter 5-10 µm) filled with 2 M NaCl (resistance 2-5 MOmega ) and inserted for 20-30 µm into the molecular layer. The signal from the micropipettes was amplified (Grass, microelectrode amplifier, model 7P5), displayed on an oscilloscope, and digitized using a computerized data acquisition system (MacAdios IIjr; GW Instruments, Sommerville, MA). In studies of field potentials, PF were stimulated at the rate of 1/s (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) (15, 16). Field potentials and BFcrb were recorded in separate groups of mice.

Monitoring of Blood Flow in Cerebellar Cortex

As described in detail elsewhere (15, 16) BFcrb was monitored using 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. Changes in BFcrb were calculated as percentage of the baseline value determined at the end of the experiment.

Light and Electron Microscopy

Procedures for electron microscopy were similar to those previously described by our laboratory (6). Mice were anesthetized with Avertin (0.2 ml of a 2.5% solution ip) and perfused transcardially with phosphate-buffered saline (PBS) followed by 10-20 ml of 2% paraformaldehyde and 0.2% glutaraldehyde in PBS. Cerebella were removed, postfixed in the same fixative, and cut sagittally (thickness 70 µm) using a vibratome. Sections were dehydrated in an ascending series of ethanols, embedded in Polybed resin (Polysciences, Warrington, PA) and polymerized between dimethyldichlorosilane-coated slides. After polymerization for 1 day at 40°C and 2 days at 60°C, the slides were separated and tissue sections were examined by light microscopy. Cerebellar folia from the vermis and paravermis of PO3 and normal littermates were circumscribed with a diamond scribe and excised. These regions were mounted onto Polybed blocks, trimmed, sectioned (thickness 90-110 nm), stained with uranyl acetate and lead citrate, and examined with a Jeol 1200-EXII transmission electron microscope. Representative sections from three PO3 mice and two nontransgenic littermates were examined. Approximately 25 sections (5 from each mouse) and at least 30 fields/section were examined. For light microscopy, sections (7 µm) from paraffin-embedded cerebella of transgenic and nontransgenic mice were cut on a microtome and stained with hematoxylin and eosin using conventional procedures (see Ref. 12).

Experimental Protocol

The superfusion with Ringer solution was started and the end-tidal CO2 was set at 2.6-2.7%. The microelectrode for stimulation of the PF was placed on the intermediate folium of lobule IV. After the placement of the electrode, the preparation was allowed to stabilize for 30 min. The experiments commenced when the hemodynamic and respiratory parameters were in a steady state.

Effect of PF stimulation on BFcrb. The cranial window was superfused with Ringer. The stimulating electrode and the light-Doppler flowmetry probe were placed on the cerebellar cortex, and the preparation was allowed to stabilize. The PF were stimulated for periods of 40 s, and the corresponding BFcrb increases were recorded. In experiments in which the relationship between stimulus intensity and magnitude of the flow response was established (n = 5), the intensity of the stimulus was varied (25-150 µA) while the frequency was maintained at 30 Hz. In experiments in which the relationship between stimulus frequency and flow response was established (n = 5), the PF were stimulated with increasing stimulus frequencies (10-50 Hz) while the stimulus intensity was maintained at 100 µA.

Spatial distribution of the increases in BFcrb produced by PF stimulation. In these experiments the area of increased BFcrb was mapped on the cerebellar cortex. The lateral edge of the flow probe was placed 0.2 mm lateral to the stimulating electrode. The PF were stimulated for 40 s (100 µA, 30 Hz), and the changes in BFcrb were recorded. In some experiments (n = 5), the flow probe was moved horizontally in 0.2-mm steps and the change in BFcrb elicited by PF stimulation was recorded. In other experiments (n = 5), the probe was moved vertically (rostrocaudally) in 0.2-mm steps. The changes in BFcrb provoked by PF stimulation were recorded at each site.

Effect of kynurenate or 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline on the increases in BFcrb produced by PF stimulation. The cerebellar cortex was superfused with normal Ringer, and baseline flow responses to PF stimulation were obtained. The superfusion solution was then changed to Ringer containing the broad-spectrum glutamate receptor antagonist kynurenate (5 mM; n = 5) or the AMPA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX; 100 µM; n = 5). The concentration of the inhibitors and the duration of superfusion were selected based on previous studies (2, 10, 16) and on the results of the electrophysiological studies described below. Solutions were prepared daily and used fresh. PF were stimulated (100 µA, 30 Hz; 40 s) after 20-30 min of superfusion. The superfusion solution was then changed back to normal Ringer, and the response was tested again 30 min later. In other mice (n = 5), the NO synthase (NOS) inhibitor Nomega -nitro-L-arginine (L-NNA; 1 mM) was studied. Responses were tested after 45-60 min of superfusion, when the effect of L-NNA on NOS activity is maximal (15).

Mapping of field potentials produced by PF stimulation. In these experiments (n = 5), the spatial distribution of the field potentials evoked by PF stimulation was studied. The stimulating electrode was positioned at a fixed site and the recording micropipette was moved along the major axis or across the stimulated folium in 100-µm steps. At each step the PF were stimulated and field potentials recorded.

Effect of agents that block synaptic transmission on field potentials. Superfusion of the cerebellar cortex with Ringer containing 12 mM Mg2+ and 0 mM Ca2+ (nominal) is thought to block synaptic transmission by preventing synaptic release from PF terminals (10, 23). First, the field potentials evoked by PF stimulation were obtained during superfusion with normal Ringer. Then the superfusion solution was switched to Ringer containing 12 mM Mg2+ and 0 mM Ca2+ (n = 6). In other experiments the effect of Ringer containing kynurenate (5 mM; n = 5) or NBQX (100 µM; n = 5) was studied. Solutions were applied until the desired effect on the field potentials was obtained, usually 20 min. After the effect on field potentials was tested, the superfusion solution was switched back to normal Ringer and the field potentials were recorded again 20-30 min later.

Vascular and electrophysiological responses in PO3 mice. In these experiments the effects of PF stimulation were studied in PO3 transgenic and in nontransgenic littermates. Before the experiments, litters were genotyped to determine the presence or absence of the transgene (12). All PO3 mice were ataxic at the time they were studied. We used nontransgenic littermates as controls to assure that no factors other than the presence of the transgene played a role in the differences observed in the response to PF stimulation, for example, age and genetic background. Previous studies have shown that the Purkinje cell abnormality is distributed homogeneously throughout the cerebellum and invariably involves the vermis, the region studied in the present experiments (12). Protocols for experiments in which BFcrb was measured during PF stimulation were identical to those described above (n = 6/group). Hypercapnia was induced by introducing CO2 into the circuit of the ventilator (see Refs. 15 and 16) (Table 2; n = 6/group). After BFcrb reached a steady-state elevation, usually 2-3 min, the CO2 supplementation was discontinued and normocapnia was reestablished.

                              
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  		Table 2.   Arterial pressure and blood gases in PO3 transgenic mice and nontransgenic controls with PF stimulation or hypercapnia

For studies of the field potentials produced by PF stimulation, six PO3 transgenic mice and six nontransgenic littermates were used. First, the relationship between intensity of stimulation and amplitude of the field potential was established. Then, spatial mapping of the activated PF beam was performed at 160 µA, a stimulation intensity that is supramaximal both in nontransgenic and PO3 mice.

Effect of L-NNA or NBQX on vascular response to PF stimulation in PO3 mice. In experiments in which the effect of NOS inhibition was studied, the response to PF stimulation was tested before and 45-60 min after L-NNA (1 mM) superfusion in PO3 mice (n = 5) and in nontransgenic littermates (n = 5). In studies in which the effect of NBQX was examined, the response to PF stimulation was tested before and 30-45 min after NBQX (100 µM) in PO3 mice (n = 5) and in nontransgenic littermates (n = 5).

Data Analysis

Data in text, Tables 1 and 2, and Figs. 2-10 are presented as means ± SE. Multiple comparisons (Figs. 2-4) were evaluated by the analysis of variance and Tukey's test (Systat, Evanston, IL). Two-group comparisons (Figs. 5-10) were evaluated by the two-tailed Student's t-test. Differences were considered significant for probability values less than 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Field Potentials Produced by PF Stimulation in Wild-Type Mice

During Ringer superfusion, stimulation of the PF produced the typical field potentials characterized by a fast polyphasic wave, reflecting conduction of action potentials along the PF, and a slow negative deflection reflecting depolarization of neurons postsynaptically connected to the PF (9). The field potentials evoked by PF stimulation could be recorded over a narrow "beam" ~200 µm wide and 800-1,000 µm long (to one side of the stimulating electrode). Superfusion of the cerebellar cortex with Ringer containing 12 mM Mg2+ and no Ca2+ (n = 6), a treatment that blocks transmitter release from PF terminals (e.g., Refs. 10, 23), abolished the late negative wave, indicating lack of depolarization of Purkinje cells (Fig. 1A). Similarly, kynurenate (5 mM; n = 5) or NBQX (100 µM; n = 5) abolished the late negative wave (Fig. 1, B and C). These results suggest that the pharmacological characteristics and spatial distribution of the field potentials evoked by PF stimulation in mice are similar to those reported in other species.


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  		Fig. 1.   Effect of Ringer containing 12 mM Mg2+ and 0 mM Ca2+ (A), kynurenate (B) or 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) (C) on the field potentials produced by parallel fiber (PF) stimulation. These treatments abolish the late negative wave of the field potentials (arrows), suggesting that they block depolarization of neurons connected to the PF, mainly Purkinje cells. The effect of these inhibitors is reversible because the late negative potential reappears when the superfusion solution is returned to normal Ringer.

Hemodynamic Response Produced by PF Stimulation in Wild-Type Mice

PF stimulation produced increases in BFcrb that were dependent on the frequency (n = 5) and on the intensity (n = 5) of the stimulation (Fig. 2). The increases in BFcrb reached a maximum at 100 µA and 30 Hz (53 ± 1%; n = 5). Mapping of the area of increased flow indicated that the increases in BFcrb are localized in a narrow band oriented along the major axis of the stimulated folium. Accordingly, if the flow probe was moved away from the stimulated site in a rostrocaudal (vertical) direction, the increases in flow declined rapidly as a function of the distance (Fig. 3A). However, when the flow probe was moved horizontally along the stimulated folium, the magnitude of the response was sustained up to 0.8 mm from the stimulating electrode (P > 0.05 from 0.2 mm; analysis of variance and Tukey's test; n = 5; Fig. 3B). Therefore, the area of increased flow overlaps with the beam of active PF.


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  		Fig. 2.   Effect of stimulus parameters on the elevation in cerebellar blood flow (BFcrb) produced by electrical stimulation of the PF in anesthetized and ventilated mice. The increases in BFcrb depend on the stimulus intensity (A) and frequency (B). Maximal responses are observed at 100 µA and 30 Hz (* P < 0.05, analysis of variance and Tukey's test).


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  		Fig. 3.   Spatial distribution of the increases in BFcrb elicited by PF stimulation in anesthetized and ventilated mice. A: in these experiments, the laser-Doppler probe was first placed on the beam of activated PF (0 mm vertical distance from the stimulating electrode) and then moved away from the stimulating electrode in an anteroposterior direction in 0.2-mm steps. At each probe position the PF were stimulated for 40 s at 100 µA and 30 Hz. Response declines rapidly as the probe is moved away from the stimulated site (* P < 0.05, analysis of variance and Tukey's test). B: probe was first placed at a distance of 0.2 mm from the stimulating electrode (edge to edge), then moved laterally in 0.2-mm steps. The response to PF stimulation is sustained until the flow probe is at a distance of 1 mm from the stimulated site (stim. site) where a significant decline is first observed (* P < 0.05).

We then investigated whether the flow increase evoked from the PF in the mouse depends on activation of glutamate receptors. Superfusion with the broad-spectrum glutamate receptor antagonist kynurenate (5 mM) did not affect resting BFcrb (before kynurenate: 7.5 ± 0.7; 20 min after: 8.2 ± 1.5 perfusion units; P > 0.05; t-test; n = 5). However, kynurenate attenuated the flow response substantially (-84 ± 3%; P < 0.05; Fig. 4A). The attenuation subsided after the superfusion solution was switched back to normal Ringer (Fig. 4A; P > 0.05 from before kynurenate). The AMPA receptor inhibitor NBQX (100 µM) did not influence resting BFcrb (before: 8.4 ± 0.8; 20 min after: 8.9 ± 1.0 perfusion units; P > 0.05; n = 5) but attenuated the response to PF stimulation markedly (-62 ± 6%; n = 5; P < 0.05; Fig. 4B). The attenuation subsided after the superfusion solution was switched back to normal Ringer (Fig. 4B; P > 0.05 from before NBQX). To determine whether NO is involved in the flow response to PF stimulation, the NOS inhibitor L-NNA was used. Superfusion with L-NNA (1 mM) reduced resting flow by 22 ± 8% (P < 0.05; t-test) and attenuated the increase in flow produced by PF stimulation by 46 ± 7% (P < 0.05; n = 5; Fig. 5).


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  		Fig. 4.   A: effect of the broad-spectrum glutamate receptor antagonist kynurenate on the increases in BFcrb produced by PF stimulation in anesthetized and ventilated mice. Kynurenate attenuates the elevations in BFcrb produced by PF stimulation (* P < 0.05; analysis of variance and Tukey's test). Effect of kynurenate is reversed by superfusion with normal Ringer (P > 0.05 from before kynurenate). B: dl-alpha -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor inhibitor NBQX also attenuates the flow response to PF stimulation. The effect of AMPA is reversible and disappears when the superfusion solution is returned to normal Ringer (*P > 0.05 from before NBQX).


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  		Fig. 5.   Effect of the NO synthase (NOS) inhibitor Nomega -nitro-L-arginine (L-NNA) on the elevations in BFcrb produced by PF simulation. L-NNA attenuates the response (*P < 0.05; t-test).

Field Potentials Evoked From the PF in PO3 Transgenic Mice

PF stimulation in PO3 transgenics evoked field potentials that lacked the late negative wave (N2; Fig. 6, A and C). Although the amplitude of the fast phase (P1-N1) was smaller than in controls at stimulation intensities of 60-100 µA (P < 0.05; t-test), the difference was not statistically significant at 160-200 µA (P > 0.05; Fig. 6B). Mapping of the field potentials evoked by PF stimulation showed that the size of the beam of activated PF was generally comparable to that of nontransgenic littermates (Fig. 7). By plotting the latency of the fast phase of the potential as a function of the distance from the stimulating electrode, an index of conduction velocity of the PF can be obtained (8). This analysis demonstrated that, although the velocity of action potential propagation tended to be slower in PO3 transgenic mice, the difference was not statistically significant (P > 0.05; Fig. 6D).


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  		Fig. 6.   Characterization of the electrophysiological responses evoked by PF stimulation in PO3 mice. A: PF stimulation in PO3 mice evokes field potentials that lack the late negative wave of the response (N2). The fast phase (P1-N1) is not substantially affected. B: quantification of the fast phase of the potential (P1-N1) in PO3 mice and nontransgenic (non-Tg) littermates. Notice that at stimulus intensities of 160-200 µA the amplitude of the P1-N1 potential does not differ between PO3 and nontransgenics (P > 0.05; t-test). C: quantification of the late negative wave of the potential (N2). Notice that this potential is absent at all stimulus intensities tested (P < 0.05, from nontransgenics). D: velocity of PF conduction in PO3 mice and nontransgenic littermates. The slope of the relationship between distance from stimulation electrode and latency of the first wave of the potential, a parameter that reflects PF conduction velocity, does not differ between PO3 mice and nontransgenic littermates (P > 0.05).


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  		Fig. 7.   Spatial distribution of the field potentials evoked by PF stimulation in PO3 mice (n = 6) and nontransgenic littermates (n = 6). Vertical (A) and horizontal (B) distribution of the field potential is slightly smaller in PO3 than in nontransgenic littermates. See Fig. 6A for definition of P1-N1. (* P < 0.05; t-test).

Vascular Effects of PF Stimulation in PO3 Transgenic Mice

Stimulation of the PF in PO3 transgenic mice (n = 6) produced increases in BFcrb that were substantially smaller than those obtained in nontransgenic littermates (n = 6) (Fig. 8). At 100 µA and 30 Hz the increase in BFcrb was 19 ± 2% in PO3 and 58 ± 5% in nontransgenic littermates (P < 0.001; t-test) (Fig. 8). Stimulation with higher frequencies (50 Hz) tended to produce larger increases in BFcrb (Fig. 8B). These, however, were still significantly smaller than the increases in flow observed in nontransgenic littermates (P < 0.001) (Fig. 8B). To rule out the possibility that the reduction in the vascular response to PF stimulation was due to a nonspecific reduction in vascular reactivity in the transgenics, the increase in BFcrb produced by two levels of hypercapnia was tested. As illustrated in Fig. 9, the cerebrovascular reactivity to hypercapnia did not differ between PO3 and nontransgenic mice (P > 0.05; n = 6/group).


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  		Fig. 8.   Elevation in BFcrb produced by PF stimulation in PO3 mice and nontransgenic littermates. Increases in BFcrb are smaller at all stimulus intensities (A) and frequencies (B) studied. (*P < 0.001; t-test).


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  		Fig. 9.   Effect of hypercapnia on BFcrb in PO3 mice and nontransgenic littermates. Elevations in BFcrb produced by two levels of hypercapnia are not different in PO3 and nontransgenic mice (P > 0.05; t-tests). See Table 2 for PCO2 values.

Effect of NBQX or L-NNA on the BFcrb Response to PF Stimulation in PO3 Transgenics

In these studies, we began to investigate the mechanisms of the residual BFcrb response to PF stimulation in PO3 mice. In PO3 mice, the increases in BFcrb produced by PF stimulation (100 µA; 30 Hz) were markedly attenuated by superfusion with NBQX (100 µM; n = 5) (Fig. 10A) or L-NNA (1 mM; n = 5) (Fig. 10B). The residual BFcrb increase after NBQX or L-NNA did not reach statistical significance (P > 0.05 from before stimulation; paired t-test; Fig. 10).


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  		Fig. 10.   Effect of the AMPA receptor antagonist NBQX (A) or the NOS inhibitor L-NNA (B) on the increases in BFcrb produced by PF stimulation (100 µA; 30 Hz) in PO3 mice and nontransgenic littermates. NBQX (100 µM) abolishes the increase in BFcrb in PO3 mice (P > 0.05 from before stimulation; paired t-test). Similarly, L-NNA (1 mM) abolishes the increase in BFcrb produced by PF stimulation in PO3 mice (P > 0.05).

Structure of the Cerebellar Molecular Layer in PO3 Transgenics

The histology of the molecular layer in PO3 transgenics and nontransgenic littermates is presented in Fig. 11. In PO3 mice, Purkinje neurons can be observed in the normal position at the interface between the molecular and granule cell layers, but their cell bodies are smaller than normal. The molecular layer is thinner than that of nontransgenic littermates. However, the molecular layer interneurons are present and have a normal nuclear morphology. The only tissue reaction that is observed consists of Bergmann glia in the Purkinje cell layer. At the electron microscopic level, the density of PF is comparable between transgenic and nontransgenic mice but the synaptic contacts between PF and Purkinje cell dendritic spines are markedly reduced in the transgenics (Fig. 12). However, the morphology and synaptic contacts of stellate cells in the outer molecular layer are comparable between transgenic and nontransgenic mice (at least 10 cells/group) (Fig. 12).


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  		Fig. 11.   Histology of the cerebellar molecular layer (ML) in nontransgenic mice and in PO3 mice at age 2.5 mo. Paraffin-embedded sagittal sections (thickness 7 µm) are shown. Calibration bar = 50 µm. A: in nontransgenics the Purkinje cell layer can be seen at the base of the ML. Arrows in the ML indicate the interneurons, mainly basket and stellate cells. B: Purkinje cells can be observed at the bottom of the ML, but their cell bodies are smaller than normal (arrowheads). The ML is thinner than in nontransgenic littermates. However, the ML interneurons (arrows) are present and have a normal nuclear morphology. The only tissue reaction that is observed consists of Bergmann glia (*, cells with clear vesicular nuclei) in the Purkinje cell layer.


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  		Fig. 12.   Electron microscopy of the molecular layer in nontransgenic mice and PO3 littermates. Sections were cut in the sagittal plane. A: asymmetrical synapses between PF (pf) and Purkinje cell spines in the intermediate region of the molecular layer. At least 10-15 synaptic contacts (arrows) can be seen in this representative field. Magnification: ×10,000. B: this section shows PF-Purkinje cell synapses in the same region of the molecular layer in PO3 mice. The number of PF is not reduced. However, remarkably fewer synaptic contacts (arrows) are seen in this representative field. Magnification: ×10,000. C: stellate cell (st) in the outer molecular layer in nontransgenic mice. The indented nucleus with a scanty, watery cytoplasm is characteristic of stellate cells in the outer molecular layer. Open arrows indicate PF synapses on the soma of stellate cells. Solid arrows point to synapses with PF in the superficial molecular layer. Magnification: ×5,000. D: stellate cell in the outer molecular layer in PO3 mice. The morphology of the stellate cells is comparable with that of nontransgenic mice. Magnification: ×6,000. These initial observations indicate that the synaptic contacts between PF and Purkinje cell dendrites are markedly reduced in PO3 mice, whereas the fine structure of stellate cells and their synaptic relationships with PF are not substantially altered.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

We used PO3 transgenic mice to gain additional insights into the cellular basis of the increases in BFcrb produced by PF stimulation. The PO3 line was produced by targeting a mutated form of SV40 large T antigen to Purkinje cells using the PCP-2 promoter (12). The cerebellum of PO3 mice develops normally until the second postnatal week (postnatal day 12), when there is an arrest in the development of Purkinje cells (12). At this time, mice exhibit ataxic behavior when compared with littermate controls. At 2 mo of age, PO3 mice have reduced glucose utilization in cerebellar cortex and deep nuclei, findings that suggest dysfunction of Purkinje cells (12). Histologically, the Purkinje cells are preserved but their dendritic tree appears stunted and simplified. The thickness of the molecular layer is slightly reduced, a finding that probably reflects incomplete maturation of Purkinje cell dendrites. Because the transgene is targeted only to Purkinje cells, the cerebellar cortical dysfunction is principally localized to that cell type. Therefore, PO3 mice could help clarify the contribution of Purkinje cell activity to the hyperemia produced by PF stimulation.

Because the cerebrovascular effects of PF stimulation in the anesthetized mice had not been determined, we first established methods for monitoring BFcrb in the mouse cerebellum. Using this preparation, we found that PF stimulation produces increases in BFcrb levels that are attenuated by the broad-spectrum glutamate receptor antagonist kynurenate, by the AMPA receptor antagonist NBQX, or by the NOS inhibitor L-NNA. These observations suggest that, in mice as in rats (2, 15, 16), the vascular response to PF stimulation is mediated by activation of glutamate receptors, in part through NO production. We found that the area of increased flow is larger than the area of increased neural activity. A similar mismatch also was observed in the rat cerebellum during PF stimulation (16). Studies in which arteriolar diameter was measured during PF stimulation suggest that there is retrograde propagation of the vasodilation, which, in turn, results in a larger area of flow increase (18). Although the mechanisms of the propagation have not been elucidated, they may involve retrograde spread of the vasodilation through mechanisms intrinsic to the vascular wall as well as neurogenic factors (18).

We then investigated the effects of PF stimulation on BFcrb and field potentials in PO3 transgenic mice. We found that the hemodynamic response evoked by PF stimulation was attenuated and that the negative phase of the field potential was absent. The reduction in the vascular response could not be attributed to a nonspecific reduction in vascular reactivity in PO3 mice because the increase in BFcrb produced by hypercapnia, a vasodilator stimulus that is independent of local neuronal circuitry (16), was comparable to that of nontransgenic littermates. Therefore, the reduction in the vascular response to PF stimulation observed in PO3 mice is a consequence of neural dysfunction and not vascular factors.

The morphological substrates of the synaptic and hemodynamic abnormalities observed in PO3 transgenics were then studied. Hematoxylin and eosin-stained sections confirmed that the Purkinje cells had an abnormal structure (cf. Ref. 12) and showed that the number and morphology of molecular layer interneurons were relatively normal. At the ultrastructural level, the density of PF did not differ markedly between transgenic and nontransgenic mice. However, the number of synaptic contacts between PF and Purkinje cell dendrites was reduced in the transgenics. The morphology and synaptic contacts of stellate cells in the outer molecular layer were comparable between transgenic and nontransgenic mice. These observations need to be expanded with more detailed and quantitative studies that are beyond the scope of the present paper. The morphology of the other classes of interneurons, e.g., basket and Golgi cells, needs to be investigated. These initial data suggest that synaptic contacts between PF and Purkinje cells are reduced and that the morphology of stellate cells and their synaptic contacts are relatively preserved.

It is likely that the reduction in the hemodynamic response produced by PF stimulation in PO3 mice is due primarily to disruption of Purkinje cell development and function. This conclusion is supported by the following evidence. First, with the use of the PCP-2 promoter, the transgene is expressed in Purkinje cells and not in other cell types in the cerebellum, such as granule cells, interneurons, or glia (11, 12). Second, Purkinje cells in PO3 mice have an abnormal morphology (12). Third, studies using zebrin II show that, in PO3 mice, some aspects of Purkinje cell development are arrested at the second postnatal week (12). Fourth, the number of PF terminals on Purkinje cells is markedly reduced, a finding consistent with the developmental arrest of the Purkinje cells. Fifth, the lack of the late negative wave of the field potential is consistent with dysfunction of the PF-Purkinje cell synapse. The contribution of stellate cells to the late negative potential (N2) is thought to be small because interneurons, particularly stellate cells, are scattered in the molecular layer and the direction of their dendrites is random (19, 26). Consequently, the currents generated by their depolarization cancel each other out. However, a contribution from the basket cells that are more regularly arranged cannot be ruled out. Sixth, the stellate cells, one type of molecular layer interneuron, have a normal morphology at both the light- and electron-microscopic level. These observations suggest that the expression of the transgene in Purkinje cells leads to a disruption of PF-Purkinje cell interaction and that such disruption results in a reduced flow response to PF stimulation. However, secondary effects deriving from the Purkinje cell abnormality also are likely to occur. Further studies are in progress to better define the molecular, ultrastructural, and neurophysiological correlates of these abnormalities.

The observation that the morphology and synaptic contacts of stellate cells are not markedly abnormal in PO3 mice indicates that interneurons may be responsible for the residual flow response elicited by PF stimulation in PO3 mice. Because molecular layer interneurons contain NOS (3, 30, 32), these cells could contribute to the component of the response that depends on NO (present study; 2, 15, 17, 21). To test this hypothesis, we investigated the effect of NOS inhibition on the residual flow response elicited by PF stimulation in PO3 mice. It was found that the residual response is completely abolished by NOS inhibition. The AMPA receptor antagonist NBQX also abolished the residual BFcrb response. These observations are consistent with the idea that activation of glutamate receptors in NOS-containing interneurons contributes to the NO-dependent component of the response. Although NO could also be generated by PF terminals, this possibility seems less likely in view of the evidence that the increases in BFcrb are mediated by depolarization of neurons postsynaptically connected to the PF (16). Irrespective of the source(s) of NO, the fact that the residual component is NO mediated indicates that Purkinje cells, which do not contain NOS, are not involved in the residual response. This observation adds further support to the hypothesis that Purkinje cells are dysfunctional in PO3 mice and that these neurons constitute an important link between neural activity and blood flow in the cerebellar molecular layer.

The PF system of the cerebellar cortex is emerging as a useful model to study the relationships between synaptic activity and blood flow in the central nervous system (2, 15-18, 21, 33, 34). The cerebellar cortex offers the advantage of having a simpler and better-understood organization than the cerebral cortex, the brain region traditionally used for functional activation studies (see Ref. 16 for a review). Another reason for using the cerebellum to investigate the mechanisms of functional hyperemia is that there are numerous naturally occurring or genetically engineered mouse mutants with specific cellular and molecular abnormalities involving the cerebellum (1, 4, 5, 11, 12, 28; see Refs. 13 and 29 for a review). The results of the present study suggest that mouse mutants could be helpful in unraveling the mechanisms of functional hyperemia in the central nervous system.

In conclusion, we have used PO3 transgenic mice to further elucidate the role of the PF-Purkinje cell synapse in the BFcrb increase produced by PF stimulation. We found that stimulation of the PF in PO3 mice produces attenuated vascular responses associated with field potentials lacking the late negative component. The residual increase in BFcrb in PO3 mice is abolished by NOS inhibition. Morphological data indicate that the PF-Purkinje cell synapses are reduced in PO3 mice whereas stellate cells are relatively intact. The findings suggest that the PF-Purkinje cell synapse mediates a sizable portion of the increase in flow produced by PF stimulation, whereas the PF-interneuron synapse may be responsible for the remainder of the response through NO production. The data also demonstrate that studies of functional hyperemia using the cerebellum as a model are feasible in anesthetized-ventilated mice with careful monitoring of physiological variables. The application of these methods to spontaneous or genetically engineered mouse mutants may provide the opportunity to better define the cellular and molecular mechanisms responsible for functional hyperemia in the central nervous system.

    ACKNOWLEDGEMENTS

The authors thank Karen MacEwan for her help in the preparation of the manuscript.

    FOOTNOTES

This work was supported by the Cerebellar Program Project Grant NS-31318. C. Iadecola is an Established Investigator of the American Heart Association.

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

Received 12 June 1997; accepted in final form 16 October 1997.

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Abstract
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Discussion
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