Cerebellar Parallel Fibers Research Articles

General and variable features of varicosity spacing along unmyelinated axons in the hippocampus and cerebellum.

Along unmyelinated central axons, synapses occur at focal swellings called axonal varicosities (boutons). The mechanisms regulating how frequently synapses and varicosities occur along axons remain poorly understood. Here, to investigate varicosity distribution patterns and the extent to which they may be conserved across different axons, we analyzed varicosity numbers and positions along fluorescently labeled axon branches in hippocampal area CA1 (CA3-to-CA1 "Schaffer collateral" axons) and five other synaptic regions of rat hippocampus and cerebellum. Varicosity spacing varied by region; e.g., 3.7 +/- 0.6 microm (mean +/- SD) for CA3-to-CA1 axons and 5.2 +/- 1.0 microm for cerebellar parallel fibers. Surprisingly, when 56 axons from these different regions were pooled into a single heterogeneous group, a general relationship emerged: the spacing variability (SD) was a constant fraction of the mean spacing, suggesting that varicosities along different axons are distributed in a fundamentally similar, scaled manner. Varicosity spacing was neither regular nor random but followed a pattern consistent with random synaptic distributions and the occurrence of multiple-synapse boutons. A quantitative model reproduced the salient features of the data and distinguished between two proposed mechanisms relating axonal morphogenesis and synaptogenesis.

RIM1alpha is required for presynaptic long-term potentiation.

Two main forms of long-term potentiation (LTP)-a prominent model for the cellular mechanism of learning and memory-have been distinguished in the mammalian brain. One requires activation of postsynaptic NMDA (N-methyl d-aspartate) receptors, whereas the other, called mossy fibre LTP, has a principal presynaptic component. Mossy fibre LTP is expressed in hippocampal mossy fibre synapses, cerebellar parallel fibre synapses and corticothalamic synapses, where it apparently operates by a mechanism that requires activation of protein kinase A. Thus, presynaptic substrates of protein kinase A are probably essential in mediating this form of long-term synaptic plasticity. Studies of knockout mice have shown that the synaptic vesicle protein Rab3A is required for mossy fibre LTP, but the protein kinase A substrates rabphilin, synapsin I and synapsin II are dispensable. Here we report that mossy fibre LTP in the hippocampus and the cerebellum is abolished in mice lacking RIM1alpha, an active zone protein that binds to Rab3A and that is also a protein kinase A substrate. Our results indicate that the long-term increase in neurotransmitter release during mossy fibre LTP may be mediated by a unitary mechanism that involves the GTP-dependent interaction of Rab3A with RIM1alpha at the interface of synaptic vesicles and the active zone.

Activation of α6 GABA A receptors on depolarized cerebellar parallel fibers elicits glutamate release through anion channels

Rat cerebellar synaptosomes labeled with [ 3 H] d-aspartate ( [ 3 H] d-ASP) were exposed in superfusion to muscimol. The GABA A receptor agonist did not affect [ 3 H] d-ASP basal release or the overflow provoked by 15 mM K +; muscimol potentiated the 35 mM K +-evoked overflow of [ 3 H] d-ASP or endogenous glutamate. Membrane potential measured by Rhodamine 6G fluorescence was −65 mV under resting conditions and −32 mV in the presence of 35 mM K +. The membrane potential was not significantly affected by muscimol. The muscimol effect on the K +(35 mM)-evoked [ 3 H] d-ASP overflow was not inhibited by omitting external Ca 2+ or by entrapping BAPTA to chelate cytosolic Ca 2+. Muscimol lost its ability to release glutamate following superfusion with d-aspartate to deplete cytosolic glutamate by heteroexchange suggesting that GABA A receptor activation elicits release of cytosolic glutamate. The non-transportable glutamate carrier blockers dihydrokainate or dl-TBOA did not reduce the muscimol potentiation. This was abolished by the anion channel blockers niflumic acid and NPPB. To conclude, when cerebellar parallel fiber terminals are sufficiently depolarized, activation of α6 GABA A receptors on these terminals mediates glutamate release in addition to that evoked by depolarization. This extra-release does not occur by exocytosis or transporter reversal but involves the opening of anion channels present on parallel fiber terminals.

GABA(B) receptor activation enhances mGluR-mediated responses at cerebellar excitatory synapses.

Metabotropic gamma-aminobutyric acid type B (GABAB) and glutamate receptors (mGluRs) are postsynaptically co-expressed at cerebellar parallel fiber (PF)-Purkinje cell (PC) excitatory synapses, but their functional interactions are unclear. We found that mGluR1 agonist-induced currents and [Ca2+]i increases in PCs were enhanced following co-activation of GABAB receptors. A GABAB antagonist and a G-protein uncoupler suppressed these effects. Low-concentration baclofen, a GABAB agonist, augmented mGluR1-mediated excitatory synaptic current produced by stimulating PFs. These results indicate that postsynaptic GABAB receptors functionally interact with mGluR1 and enhance mGluR1-mediated excitatory transmission at PF-PC synapses. The interaction between the two types of metabotropic receptors provides a likely mechanism for regulating cerebellar synaptic plasticity.

Pressure-induced depression of synaptic transmission in the cerebellar parallel fibre synapse involves suppression of presynaptic N-type Ca2+ channels.

High pressure induces CNS hyperexcitability while markedly depressing synaptic transmitter release. We studied the effect of pressure (up to 10.1 MPa) on the parallel fibre (PF) synaptic response in biplanar cerebellar slices of adult guinea pigs. Pressure mildly reduced the PF volley amplitude and to a greater extent depressed the excitatory field postsynaptic potential (fPSP). The depression of the PF volley was noted even at supramaximal stimulus intensities, indicating an effect of pressure on the amplitude of the action potential in each axon. Low concentrations of TTX mimicked the effects of pressure on the PF volley without affecting the fPSP. Application omega-conotoxin GVIA (omega-CgTx) reduced the synaptic efficacy by 34.3+/-2.7%. However, in the presence of omega-CgTx the synaptic depression at pressure was significantly reduced. Reduced Ca2+ entry by application of Cd2+ or low [Ca2+]o did not have a similar influence on the effects of pressure. Application of omega-AGA IVA, omega-AGA TK and Funnel-web spider toxin did not affect the synaptic response in concentrations that usually block P-type Ca2+ channels, whilst the N/P/Q-type blocker omega-conotoxin MVIIC reduced the response to 52.7+/-5.0% indicating the involvement of Q-type channels and R-type channels in the non-N-type fraction of Ca2+ entry. The results demonstrate that N-type Ca2+ channels play a crucial role in the induction of PF synaptic depression at pressure. This finding suggests a coherent mechanism for the induction of CNS hyperexcitability at pressure.

CAMP-Dependent Long-Term Potentiation of Nitric Oxide Release from Cerebellar Parallel Fibers in Rats

Nitric Oxide (NO) is released from parallel fibers (PFs) after PF stimulation. NO-cGMP signaling is essential for long-term depression (LTD) in cerebellar PF-Purkinje cell synapses, which also exhibit presynaptic long-term potentiation (LTP) after tetanic PF stimulation. This LTP is dependent on cAMP but not NO-cGMP signaling. In this study, we analyzed long-term changes of NO release from PFs in rat cerebellar slices using electrochemical NO probes. Repetitive PF stimulation at 10 Hz for 2 sec elicited a transient increase in NO concentration (2.2 +/- 0.1 nM; mean +/- SEM; n = 116). This NO release exhibited long-term potentiation (LTPNO) by 36 +/- 3% (n = 15) after tetanic PF stimulation. Induction of LTPNO was not affected by Glu receptor antagonists. NO release from PFs was also potentiated by L-Arg (ARG) (100 microM), forskolin (50 microM), and 8-bromo-cAMP (Br-cAMP) (1 mM) but not by 1,9-dideoxyforskolin (50 microM), a biologically inactive analog of forskolin. The potentiation induced by forskolin was significantly suppressed by H89 (10 microM), a blocker of cAMP-dependent protein kinase. The potentiation induced by forskolin, but not that induced by Arg, interfered with LTPNO. H89 (10 microM) and KT5720 (1 microM), another blocker of cAMP-dependent protein kinase, but not KT5823 (300 nM), a blocker of cGMP-dependent protein kinase, significantly suppressed LTPNO. These data indicate that neural NO release is under activity-dependent control, just as synaptic transmitter release is. LTPNO might play a role in cross talk between presynaptic and postsynaptic plasticity by facilitating NO-cGMP-dependent postsynaptic LTD after induction of cAMP-dependent presynaptic LTP and LTPNO.

Modification of activity-dependent increases of cerebral blood flow by excitatory synaptic activity and spikes in rat cerebellar cortex.

1. Mechanisms of activity-dependent increases in cerebral blood flow (CBF) were examined in rat cerebellar cortex using the laser Doppler flow technique and extracellular recordings of single unit activity and field potentials. 2. Stimulation of the monosynaptic climbing fibre system evoked long-lasting complex spikes in Purkinje cells, and extracellular field potentials with a characteristic profile that indicated contributions from both passive and active membrane mechanisms. The concomitant CBF increases were reproducible at fairly short intervals, and suggest that both synaptic activity and spikes may contribute to increased CBF. 3. Stimulation of the disynaptic parallel fibre system inhibited the spiking activity in Purkinje cells, while the postsynaptic activity increased as indicated by the simultaneously recorded field potential. Nevertheless, CBF always increased. The inhibition of spike firing activity was partly dependent on GABAergic transmission, but may also relate to the intrinsic membrane properties of Purkinje cells. 4. The CBF increases evoked by parallel or climbing fibre stimulation were highly correlated to the sum of neural activities, i.e. the negativity of field potentials multiplied by the stimulus frequency. This suggests a robust link between extracellular current flow and activity-dependent increases in CBF. 5. AMPA receptor blockade attenuated CBF increases and field potential amplitudes, while NMDA receptor antagonism did not. This is consistent with the idea that the CBF responses are of neuronal origin. 6. This study has shown that activity-dependent CBF increases evoked by stimulation of cerebellar parallel fibres are dependent on synaptic excitation, including excitation of inhibitory interneurones, whereas the net activity of Purkinje cells, the principal neurones of the cerebellar cortex, is unimportant for the vascular response. For the climbing fibre system, not only synaptic activity but also the generation of complex spikes from Purkinje cells contribute to the increases in CBF. The strong correlation between CBF and field potential amplitudes suggests that extracellular ion fluxes contribute to the coupling of brain activity to blood flow.

Impaired Cerebellar Long-Term Potentiation in Type I Adenylyl Cyclase Mutant Mice

Activation of adenylyl cyclase and the consequent production of cAMP is a process that has been shown to be central to invertebrate model systems of information storage. In the vertebrate brain, it has been suggested that a presynaptic cascade involving Ca influx, cAMP production, and subsequent activation of cAMP-dependent protein kinase is necessary for induction of long-term potentiation (LTP) at the cerebellar parallel fiber–Purkinje cell synapse. We have used mutant mice in which the major Ca-sensitive adenylyl cyclase isoform of cerebellar cortex (type I) is deleted to show that this results in an ∼65% reduction in cerebellar Ca-sensitive cyclase activity and a nearly complete blockade of cerebellar LTP assessed using granule cell–Purkinje cell pairs in culture. This blockade is not accompanied by alterations in a number of basal electrophysiological parameters and may be bypassed by application of an exogenous cAMP analog, suggesting that it results specifically from deletion of the type I adenylyl cyclase.

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

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 microA, 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 microA, 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 N omega-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 microA, 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 N omega-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.

Local and propagated vascular responses evoked by focal synaptic activity in cerebellar cortex.

We investigated the local and remote vascular changes evoked by activation of the cerebellar parallel fibers (PFs). The PFs were stimulated (25-150 microA, 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 microm), 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 microm) 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.

Branching of cerebellar parallel fibres can assist the convergence of mossy fibre input sequences that are temporally and spatially dispersed

It is suggested that bifurcation of parallel fibres in the cerebellar cortex assists the spatiotemporal convergence of temporally dispersed and asomatotopic inputs to granule cells. This increases the number of combinations of inputs which can be compared for the purpose of sequence recognition.

Obligatory role of NO in glutamate-dependent hyperemia evoked from cerebellar parallel fibers

Electrical stimulation of cerebellar parallel fibers (PF) increases cerebellar blood flow (BFcrb), a response that is attenuated by glutamate receptor antagonists and NO synthase (NOS) inhibitors. We investigated whether administration of NO donors could counteract attenuation by NOS inhibitors of vasodilation produced by PF stimulation. In halothane-anesthetized rats the cerebellar cortex was exposed and superfused with Ringer solution. PF were stimulated with microelectrodes (100 microA, 30 Hz), and BFcrb was recorded by a laser-Doppler probe. During Ringer superfusion, PF stimulation increased BFcrb by 56 +/- 7% and hypercapnia by 72 +/- 5% (n = 5). Superfusion with the nonselective NOS inhibitor N-nitro-L-arginine (L-NNA, 1 mM) reduced resting BFcrb and attenuated the response to PF stimulation (-47 +/- 5%) and hypercapnia (-46 +/- 7%; PCO2 = 50-60 mmHg). After L-NNA, superfusion with the NO donors 3-morpholinosydnonimine (100 microM, n = 5) or S-nitroso-N-acetyl-penicillamine (5 microM, n = 5) reestablished resting BFcrb (P > 0.05 vs. before L-NNA) and reversed L-NNA-induced attenuation of the response to hypercapnia (P > 0.05 vs. before L-NNA) but not PF stimulation (P > 0.05 vs. after L-NNA). Similar results were obtained when NOS activity was inhibited with the inhibitor of neuronal NOS 7-nitroindazole (50 mg/kg i.p.). Like NO donors, the guanosine 3',5'-cyclic monophosphate analog 8-bromoguanosine 3',5'-cyclic monophosphate (n = 5), administered after L-NNA, restored resting BFcrb and counteracted inhibition of the response to hypercapnia but not PF stimulation. In contrast to NO donors and 8-bromoguanosine 3',5'-cyclic monophosphate, the NO-independent vasodilator papaverine (100 microM, n = 5) had no effect on attenuation of responses to PF stimulation or hypercapnia. Thus NO donors are unable to reverse the effect of NOS inhibition on vasodilation produced by PF stimulation. The data support the hypothesis that the vascular response to PF stimulation, at variance with hypercapnia, requires NOS activation and NO production. Thus NO plays an obligatory role in vasodilation produced by increased functional activity in cerebellar cortex.

LTP of no release from cerebellar parallel fibers under excessive arginine

LTP of no release from cerebellar parallel fibers under excessive arginine

301 LTP of no release from cerebellar parallel fibers under excessive arginine

301 LTP of no release from cerebellar parallel fibers under excessive arginine

Synaptic responses between a cerebellar purkinje cell and parallel fibres by temporal pattern stimulation

Synaptic responses between a cerebellar purkinje cell and parallel fibres by temporal pattern stimulation

329 Synaptic responses between a cerebellar purkinje cell and parallel fibres by temporal pattern stimulation

329 Synaptic responses between a cerebellar purkinje cell and parallel fibres by temporal pattern stimulation

Glutamate microinjections in cerebellar cortex reproduce cerebrovascular effects of parallel fiber stimulation.

Electrical stimulation of cerebellar parallel fibers releases glutamate and increases local blood flow (BFcrb), an effect in part mediated by glutamate-induced nitric oxide (NO) production. We studied whether local microinjection of glutamate into the cerebellar cortex would produce increases in BFcrb comparable to those elicited by parallel fiber stimulation. In halothane-anesthetized rats equipped with a cranial window, glutamate was microinjected into the cerebellar molecular layer, and BFcrb was monitored by laser-Doppler flowmetry. Glutamate microinjections increased BFcrb dose dependently (2-200 pmol in 200 nl) (n = 9) and by 55 +/- 6% at 200 pmol (mean +/- SE). The magnitude and temporal profile of the increases in BFcrb compared favorably with the increase in flow produced by parallel fiber stimulation. The glutamate-induced BFcrb increase was attenuated by superfusion with the Na2+ channel blocker tetrodotoxin (10 microM; -50 +/- 10%; n = 5; P < 0.05; t-test) or by blocking synaptic activity by treatment of the cerebellar cortex with Ringer containing 20 mM Mg2+ and 0 mM Ca2+ (-80 +/- 4%; n = 6; P < 0.05). The glutamate-receptor antagonist kynurenate (10 mM) attenuated the increase in BFcrb by 59 +/- 6% (P < 0.05; n = 5). The relatively selective inhibitor of neuronal NO synthase 7-nitroindazole (100 mg/kg ip) reduced the flow response evoked by microinjection of glutamate (-46 +/- 7%; n = 5; P < 0.05) but not acetylcholine (10 microM; P > 0.05; n = 6). We conclude that glutamate microinjections increase local BFcrb via activation of glutamate receptors. The glutamate-induced vasodilation is mediated, in part, by neurally derived NO. The striking similarities between the vascular responses evoked by parallel fiber stimulation and that produced by microinjection of glutamate support the hypothesis that the increase in BFcrb produced by parallel fiber stimulation is mediated by glutamate release and activation of glutamate receptors. The data also strengthen the hypothesis that glutamate and NO are important mediators in the mechanisms linking synaptic activity to BFcrb in cerebellar cortex.

7-Nitroindazole attenuates vasodilation from cerebellar parallel fiber stimulation but not acetylcholine.

We used the relatively selective inhibitor of neuronal nitric oxide synthase 7-nitroindazole (7-NI) to test the hypothesis that the increases in local cerebellar blood flow (BFcrb) elicited by activation of the cerebellar parallel fibers (PF) are mediated by neuronal production of nitric oxide. In halothane-anesthetized rats, the cerebellar cortex was exposed and superfused with Ringer solution (37 degrees C; pH 7.3-7.4). The PF were stimulated electrically (100 microA, 30 Hz, 40 s), while BFcrb was monitored at the site of stimulation by a laser-Doppler flow probe. In vehicle-treated rats (n = 5), PF stimulation increased BFcrb by 61 +/- 5% (P < 0.05; analysis of variance and Tukey's test). 7-NI attenuated the increase in BFcrb dose dependently (10-100 mg/kg i.p.; n = 5 animals/dose) and by 55 +/- 7% at 100 mg/kg (P < 0.05). The attenuation of the response to PF stimulation was correlated with the degree of inhibition of calcium-dependent brain nitric oxide synthase activity, measured ex vivo by the citrulline assay (n = 21). 7-NI also attenuated the cerebrovasodilation elicited by hypercapnia (PCO2 = 50-60 mmHg) but did not affect the vasodilation evoked by acetylcholine (10 microM; n = 4; P > 0.05; t-test), a response mediated by endothelial nitric oxide synthase. 7-NI did not attenuate the BFcrb increase evoked by the nitric oxide donor S-nitroso-N-acetylpenicillamine (1 mM; n = 5; P > 0.05; t-test). Similarly, 7-NI did not affect resting systemic arterial pressure. These observations suggest that selective inhibition of neuronal nitric oxide synthase by 7-NI attenuates the increases in BFcrb evoked by PF stimulation. The findings provide additional support to the hypothesis that the increase in BFcrb evoked by PF stimulation is mediated, in part, by glutamate-induced activation of neuronal nitric oxide synthase.

Cyclic AMP Mediates a Presynaptic Form of LTP at Cerebellar Parallel Fiber Synapses

The N-methyl- D-aspartate receptor–independent form of long-term potentiation (LTP) at hippocampal mossy fiber synapses requires presynaptic Ca 2+–dependent activation of adenylyl cyclase. To determine whether this form of LTP might occur at other synapses, we examined cerebellar parallel fibers that, like hippocampal mossy fiber synapses, express high levels of the Ca 2+/calmodulin-sensitive adenylyl cyclase I. Repetitive stimulation of parallel fibers caused a long-lasting increase in synaptic strength that was associated with a decrease in paired-pulse facilitation. Blockade of glutamate receptors did not prevent LTP induction, nor did loading of Purkinje cells with a Ca 2+ chelator. LTP was occluded by forskolin-induced potentiation and blocked by the protein kinase A inhibitor Rp-8-CPT-cAMPS. These findings suggest that parallel fiber synapses express a form of LTP that is dependent on the activation of a presynaptic adenylyl cyclase and is indistinguishable from LTP at hippocampal mossy fiber synapses.

Nitric oxide and adenosine mediate vasodilation during functional activation in cerebellar cortex

Activation of the cerebellar parallel fibers (PF) releases glutamate and leads to depolarization of Purkinje cells and interneurons. These cells, in turn, release GABA. We have studied the role of glutamate, GABA, nitric oxide (NO) and adenosine in the increases in cerebellar cortex blood flow (BF crb) elicited by PF stimulation. In anesthetized rats (halothane 1%) the cerebellar vermis was exposed and the site was superfused with Ringer (37°C, pH 7.4). The PF were stimulated electrically (50–100 μA; 30 Hz) and the increases in BF crb were recorded using a laser-Doppler flowmeter. Field potentials were recorded using glass microelectrodes. During Ringer superfusion, PF stimulation increased BF crb by 58 ± 5% ( P < 0.001; analysis of variance; n = 6). Superfusion with the broad spectrum glutamate receptor antagonist kynurenic acid (Kyn; 5 mM) abolished the negative component of the field potential ( n = 4), a finding reflecting lack of depolarization of Purkinje cells and interneurons, and blocked the increase in BF crb ( P > 0.05 from Ringer; n = 6). In contrast, Kyn did not influence the increase in BF crb evoked by hypercapnia (pCO 2 55.4 ± 1.1 mmHg) or by superfusion with the NO donor SIN-1 (0.1, 1 mM; P > 0.05; n = 6). Superfusion with the adenosine receptor antagonist 8-sulphophenyltheophylline (8-SPT; 100 μM) reduced the elevation in BF crb by 45 ± 4% ( P < 0.05; n = 6) and co-application of 8-SPT and of the NO synthase inhibitor nitro- l-arginine ( l-NA; 1 mM) attenuated the vasodilation further (−82 ± 4% from Ringer; P < 0.01 from 8-SPT alone). The vasodilation elicited by SIN-1 ( n = 6) and the field potentials evoked by PF stimulation ( n = 4) were not affected by 8-SPT or by 8-SPT plus l-NA. The GABA receptor antagonist bicuculline (300 μM) produced the expected changes in the field potentials ( n = 4) but did not affect the vascular response to PF stimulation ( P . 0.05; n = 5). The data suggest that release of glutamate from PF leads to production of NO and adenosine in Purkinje cells and/or interneurons. NO and adenosine may mediate the vasodilation by diffusing toward resistance arterioles and producing smooth muscle relaxation.