Striatal Nitric Oxide Signaling Regulates the Neuronal Activity of Midbrain Dopamine Neurons In Vivo

Anthony R. West and Anthony A. Grace

Departments of Neuroscience and Psychiatry, Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15260


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

West, Anthony R. and Anthony A. Grace. Striatal Nitric Oxide Signaling Regulates the Neuronal Activity of Midbrain Dopamine Neurons In Vivo. J. Neurophysiol. 83: 1796-1808, 2000. A major component of the cortical regulation of the nigrostriatal dopamine (DA) system is known to occur via activation of striatal efferent systems projecting to the substantia nigra. The potential intermediary role of striatal nitric oxide synthase (NOS)-containing interneurons in modulating the efferent regulation of DA neuron activity was examined using single-unit recordings of DA neurons performed concurrently with striatal microdialysis in anesthetized rats. The response of DA neurons recorded in the substantia nigra to intrastriatal artificial cerebrospinal fluid (ACSF) or drug infusion was examined in terms of mean firing rate, percent of spikes fired in bursts, cells/track, and response to electrical stimulation of the orbital prefrontal cortex (oPFC) and striatum. Intrastriatal infusion of NOS substrate concurrently with intermittent periods of striatal and cortical stimulation increased the mean DA cell population firing rate as compared with ACSF controls. This effect was reproduced via intrastriatal infusion of a NO generator. Infusion of either a NOS inhibitor or NO chelator via reverse microdialysis did not affect basal firing rate but increased the percentage of DA neurons responding to striatal stimulation with an initial inhibition followed by a rebound excitation (IE response) from 40 to 74%. NO scavenger infusion also markedly decreased the stimulation intensity required to elicit an IE response to electrical stimulation of the striatum. In single neurons in which the effects of electrical stimulation were observed before and after drug delivery, NO antagonist infusion was observed to decrease the onset latency and extend the duration of the initial inhibitory phase induced by either oPFC or striatal stimulation. This is the first report showing that striatal NO tone regulates the basal activity and responsiveness of DA neurons to cortical and striatal inputs. These studies also indicate that striatal NO signaling may play an important role in the integration of information transmitted to basal ganglia output centers via corticostriatal and striatal efferent pathways.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A substantial body of data implicating dysfunction of striatal output pathways and their postsynaptic targets in Parkinson's disease and other movement disorders (see Carlsson and Carlsson 1990 for review) has led to extensive research into the regulation of these systems in normal and disease states. Multiple neuroanatomic and electrophysiological studies provide evidence that corticostriatal pathways originating in the prefrontal cortex (PFC) may influence basal ganglia function via an interaction with striatonigral projection neurons (Deniau et al. 1996; Gariano and Groves 1988; Jones et al. 1977; Nakamura et al. 1979b; Smith and Bolam 1990; Somogyi et al. 1981). The PFC also may influence striatal output via the modulation of pallidonigral and substantia nigra pars reticulata inhibitory pathways (Deniau et al. 1996; Grofova 1975; Nakamura et al. 1979a) known to innervate midbrain dopamine (DA) neurons (see Haber and Fudge 1997 for review). Although stimulation of striatal GABAergic output pathways has been shown to influence the activity of neurons in the substantia nigra pars reticulata and pars compacta via mono- and polysynaptic connections, the signaling mechanisms involved in striatal processing of cortical information and their subsequent postsynaptic influence on downstream basal ganglia nuclei remain poorly understood.

It is known that the primary targets of corticostriatal glutamatergic terminals are the spiny GABAergic projection neurons (see Smith and Bolam 1990). However, studies also have demonstrated that cortical afferents form synapses on the cell body and proximal dendrites of somatostatin/neuropeptide Y-containing interneurons (Kerkarian et al. 1986; Kubota et al. 1988; Salin et al. 1990; Vincent and Johansson 1983; Vuillet et al. 1989a,b) that are known to colocalize nitric oxide synthase (NOS) immunoreactivity (Figueredo-Cardenas et al. 1996). It is thought that the primary signaling pathway of brain NOS involves a glutamatergic activation of N-methyl-D-aspartate (NMDA) receptors that subsequently stimulates nitric oxide (NO) synthesis via a calcium/calmodulin-dependent mechanism (Garthwaite 1991; Jones et al. 1994; Marin et al. 1992, 1993). Of the several distinct NOS isoforms, the neuronal (type 1) isoform is localized in moderate levels in the rat striatum (Bredt et al. 1991; Kharazia et al. 1994), suggesting a role for NO in regulating striatal function. Given the heterogeneous distribution of striatal NOS interneurons and their extensive plexus of axonal processes (Kharazia et al. 1994), NO generated as a consequence of activation of corticostriatal pathways may be involved in regulating the activity of striatal local circuit and projection neurons and/or their respective afferents inputs.

Although the influence of NO on striatal neuronal activity remains to be thoroughly characterized, evidence has accumulated suggesting that NO signaling may mediate and/or regulate multiple aspects of striatal neurotransmission. A role for striatal NO in modulating inhibitory GABAergic striatonigral systems first was reported by Greengard and colleagues in a study demonstrating that sodium nitroprusside activates guanylyl cyclase in striatonigral terminals leading to the enhancement of protein kinase G (PKG)-dependent phosphorylation of dopamine and cyclic AmP-regulated phosphoprotein (DARPP-32) (Tsou et al. 1993). The NO/cGMP pathway recently has been shown to be critically involved in the induction of long-term depression of excitatory postsynaptic potentials in striatal spiny neurons produced after high-frequency stimulation of corticostriatal pathways (Calabresi et al. 1999). When taken together with studies showing that NOS interneurons receive direct synaptic input from both glutamatergic and DAergic terminals (Fujiyama and Masuko 1996; Vuillet et al. 1989a) and facilitate the concurrent release of DA and glutamate by a NO-dependent process (West and Galloway 1997b), these observations suggest that NO may participate in the integration of convergent motor information within striatal networks. This is supported by evidence that striatal NO generation induced via corticostriatal afferent stimulation produces a rapid increase in the incidence of dye coupling between striatal spiny neurons, probably via increasing gap junction permeability (O'Donnell and Grace 1997).

The current studies were undertaken to examine the potential intermediary role of striatal nitrergic interneurons in regulating the activity of nigral DA neurons by cortical and striatal efferent pathways. Given that NO has been shown to facilitate DAergic and GABAergic transmission in the striatum, augment coupling between striatal neurons, and activate multiple second-messenger cascades in striatonigral pathways (see preceding text), we hypothesized that intrastriatal infusions of agents known to disrupt NO signaling would significantly affect information flow through the striatum, possibly increasing the frequency of observed inhibitory responses of DA cells to stimulation of the striatum and orbital PFC (oPFC). This alteration also should be reflected by changes in the basal activity of DA neurons, in that augmentation of striatal NO signaling via local infusions of NOS substrate or NO generators should increase the firing rate of DA cells and attenuate the influence of inhibitory feedback pathways on DA neuronal activity. To test the preceding hypotheses, the responses evoked by electrical stimulation of the dorsal striatum and oPFC were characterized, and the contribution of striatal NO signaling to these responses was determined via discrete pharmacological manipulations of striatal NO levels. The influence of these striatal NO manipulations on the regulation of DA neuron activity was evaluated in population studies of DA cells from multiple-electrode tracks within the substantia nigra and from single DA neurons in which the effects of electrical stimulation were observed before and after drug delivery.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

NG-hydroxy-L-arginine (H-ARG), hydroxylamine (HA), and Dulbecco's phosphate-buffered saline were purchased from Sigma Chemical (St. Louis, MO). 7-nitroindazole sodium (7-NI) was purchased from Calbiochem (La Jolla, CA). Carboxy PT-10 potassium (CPT-10) was purchased from Research Biochemicals (Natick, MA). D-glucose was purchased from Fisher Scientific (Springfield, NJ). All other reagents were of the highest grade commercially available.

Subjects and surgery

Recordings were obtained from male Sprague-Dawley rats (Hilltop, Scottdale, PA) weighing 275-450 g. Before experimentation, animals were housed two per cage under conditions of constant temperature (21-23°C) and maintained on a 12:12 h light/dark cycle with food and water available ad libitum. All animal procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and adhere to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Before surgery, animals were anesthetized deeply with chloral hydrate (400 mg/kg ip) and placed in a stereotaxic apparatus (Kopf). The level of anesthesia was verified periodically via the hindlimb compression reflex and maintained using supplemental administration of chloral hydrate (80 mg/ml) via a lateral tail vein (~0.2 ml/0.5 h). Temperature was monitored using a rectal probe and maintained at 37°C with a heating pad (Fintronics VL-20F; New Haven, CT).

Extracellular recording and identification of dopaminergic neurons

Extracellular recording microelectrodes were manufactured from 2.0 mm OD borosilicate glass capillary tubing (WPI, New York) using a vertical micropipette puller (Narishige, Tokyo) filled with 2 M NaCl containing 2% Pontamine Sky Blue and broken back against a glass rod to ~1 µm tip diameter. The in vitro impedance of the microelectrodes ranged from 5 to 10 MOmega as measured at 135 Hz using a Winston Electronics BL-1000 impedance meter. After drilling a burr hole (~2-3 mm diam) overlying the substantia nigra, the dura was resected and the electrode was lowered into the substantia nigra pars compacta (coordinates: 2.6-3.2 mm anterior from lambda, 2.20-2.60 mm lateral from the midline, 6.5-8.5 mm ventral from brain surface) with a hydraulic microdrive (Kopf Model 640). DA neurons were identified using established electrophysiological criteria described by Grace and Bunney (1983). Specific DA cell waveform characteristics include an initial segment-somatodendritic break in the initial positive phase, a biphasic (positive-negative) or triphasic (positive-negative-positive) waveform having a duration of 2.0-4.0 ms, and emission of a characteristic low-pitched sound from the audio monitor. Additionally, DA cells generally fired action potentials in a slow irregular pattern that on occasion alternated with bursts of spike activity (2-10 spikes) with a progressive increase in interspike intervals and decrease in spike amplitudes during a burst (Grace and Bunney 1983).

Electrical stimulation

In each experiment, a twisted-pair bipolar stimulating electrode (Plastics One) was implanted into the oPFC (coordinates: 3.7-4.7 mm anterior to bregma, 0.2-2.3 mm lateral to midline, 2.5-4.0 mm ventral to brain surface). An additional stimulating electrode was attached to a microdialysis probe (see following text) and implanted into the dorsal striatum (coordinates: 0.8 posterior to 1.2 mm anterior to bregma, 2.0-5.0 mm lateral to midline, 4.0-5.0 mm ventral to brain surface). Both stimulating electrodes were implanted ipsilateral to the recording electrode. Single pulses of electrical stimuli were generated using a Grass stimulator and photoelectric constant current/stimulus isolation unit (Grass Instrument, Quincy, MA; model S88). The striatum and oPFC were stimulated using current intensities of 0.1-0.8 and 0.25-1 mA, respectively for ~150-300 s with single pulses (75-150) having a duration of 250 µs and delivered at a frequency of 0.6 Hz.

Drug administration via reverse dialysis

Concentric microdialysis probes (Harvard Apparatus) having 3 mm of exposed membrane (225 µM diam, 6,000 d permeability) were attached to twisted-pair bipolar electrodes using Quick Gel cyanoacrylate adhesive (Duro), such that the tips of the dialysis probes extended 2.0-2.5 mm beyond the tips of the electrodes and were implanted into the dorsal striatum as described in the preceding text. After implantation, probes were perfused with artificial cerebral spinal fluid (ACSF) containing (in mM) 136.9 NaCl, 2.7 KCl, 0.5 MgCl2, 0.9 CaCl2, 1.47 KPO4, 8.1 Na2PO4 (Dulbecco's phosphate-buffered saline), and 10.0 D-glucose at a rate of 2 µl/min using a BAS Baby Bee microperfusion pump (Bioanalytical Systems, Lafayette, IN). Experimentation began ~1.5-2.0 h after probe/stimulating electrode implantation. In population studies, the substantia nigra first was located electrophysiologically. Next either drug or ACSF vehicle (control) was infused intrastriatally for 1 h, and the sampling of an additional five to seven electrode tracks for the presence of spontaneously firing DA cells was commenced as described in the preceding text. In within-subjects experiments, after isolating a DA cell and recording basal firing activity: the effects of separate periods of striatal and oPFC stimulation were observed and drugs were infused intrastriatally via reverse dialysis and stimulation was repeated in the presence of drug. It is estimated that the time elapsed between the preparation of the NO generator and antagonist solutions and the beginning of their infusion into the brain was 8-10 min (taking into account the syringe loading period and dead space in the microdialysis inlet tubing). To ensure that drug was being delivered into the striatum before and during the electrical stimulation periods, the dialysis tubing dead space (11 µl) and perfusate flow rate (2 µl/min) were taken into account, and syringes containing drug were switched 5.5 min before initiating basal firing rate assessment. Drug was allowed to diffuse intrastriatally for 5-60 min, and multiple periods of striatal and cortical stimulation were delivered at 10-min intervals. All drugs were soluble in ACSF. Effective doses of the NO generators and scavenger, NOS substrate, and NOS inhibitor were derived from previous studies (Park et al. 1998; Southam and Garthwaite 1991; Wallace et al. 1991; West and Galloway 1996, 1997a,b, 1998).

Data collection and analysis

Extracellular electrode potentials were passed through a high-input impedance headstage amplifier connected to a preamplifier-window discriminator (Fintronics WDR 420, New Haven, CT) and a Grass AM-8 audio monitor and displayed on a Hitachi V-134 storage ocilloscope. Data also were digitized using a Neurocorder DR-390 A/D converter (Neurodata, New York) and stored on VHS videotapes and analyzed off-line using Neuroscope software applications developed within our laboratory and using an Intel-based PC with a data-acquisition board interface (Microstar Laboratories, Bellevue, WA).

Previous studies have demonstrated that manipulations of efferent pathways regulating DA neurotransmission can alter multiple dependent measures of DA cell firing activity including basal firing rate, percent of spikes fired in bursts, and number of spontaneously active DA cells encountered per electrode track (Braszko et al. 1981; Bunney and Grace 1978; Grace and Bunney 1985; Shim et al. 1996). In the current population studies, we examined the influence of striatal NO manipulations on basal DA cell activity (recorded before electrical stimulation) using the preceding dependent measures. In these studies, the substantia nigra was examined systematically for the presence of spontaneously firing DA cells during intrastriatal infusion of ACSF, NO generators, or NO antagonists in the following manner: the recording electrode was lowered three to seven times through a rectangular area in the substantia nigra starting from 2.2 mm lateral to the midline and either 2.6 or 2.8 mm anterior to the lambdoid suture. Individual electrode tracks were separated by 200 µm as described by Bunney and Grace (1978). After identifying (as described in the preceding text) and isolating a DA cell, 60-300 s of basal spike activity was recorded before initiation of striatal stimulation (described in the preceding text). Approximately 1-2 min after striatal stimulation, electrical stimulation of the oPFC was commenced. To enable the examination of a large population of DA cells and minimize the number of animals used in the study, this process was repeated for each DA cell encountered over the course of the three to seven electrode tracks sampled. Analysis of burst firing was performed as described previously by Grace and Bunney (1984). Differences in DA cell population firing characteristics between controls and groups receiving pharmacological manipulations were determined using a one-way ANOVA with Dunnett's post hoc test.

Additional studies were performed on individual DA cells (1 per rat) to determine the effects of striatal NO manipulations on the evoked activity of single DA neurons before and after drug administration. In this within-subject design, the basal activity and response of single DA neurons to electrical stimulation of the striatum and oPFC was recorded as described above during control conditions (i.e., intrastriatal ACSF infusion) and again after intrastriatal drug delivery via the dialysis probe (described in the following text).

In all studies, peristimulus time histograms (PSTH) were constructed (1.4-ms bins) to enable the analysis of spike response patterns after electrical stimulation. The PSTHs generated from population studies were analyzed qualitatively and responsive DA cells were categorized based on the primary pattern of activity elicited. Similar to previous reports (Overton et al. 1996; Tong et al. 1995, 1996), observed responses could be classified as having an initial inhibition that often was followed by a rebound excitation (IE response) or exhibiting an initial excitation (E response). The percentage of the total cells encountered in three to seven electrode tracks from control and treatment groups exhibiting these response profiles and those that were nonresponsive (NR) was determined. The effects of drug infusions on the proportion of DA cells responding and the prevalence of the IE and E response patterns were assessed via comparisons between control and drug treatment groups using a Fisher exact test. In some experiments, the probability of eliciting the IE pattern in response to striatal and oPFC stimulation was assessed as a function of increasing stimulation intensities (0.1-1.0 mA) delivered in five separate stimulation periods (with intervals of >1 min) as described for the preceding population studies. In within-subject studies, stimulus-induced changes in response pattern were assessed by comparing the mean count of events per bin during a 100-ms prestimulus baseline period with the mean number of events per bin during the poststimulus interval. The onset of a stimulus-induced effect was defined as the first of five consecutive poststimulus bins (3 ms) that differed from prestimulus baseline by >50%. The criterion for offset of a stimulus-induced effect was defined as the first of five consecutive poststimulus bins that did not differ from baseline by >50%. When possible, multiple stimulation trials (5 min intervals) were performed 5-45 min after the initiation of striatal drug infusion, and the single postdrug stimulation trial exhibiting the most robust response was compared with the predrug control stimulation trial.

Histology

At the end of each experiment, the site at which recordings were performed was marked via iontophoretic ejection of Pontamine Sky Blue dye from the tip of the recording electrode (-30µA constant current for 20-30 min). After dye injection, the animal was decapitated and the brain was fixed in formalin for >= 1 wk. The brains were then immersed in phosphate-buffered sucrose solution (25%) until saturated. Brains were sectioned into 40-µM coronal slices, mounted, and stained with cresyl violet to enable histological determination of stimulating and recording electrode sites.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Electrode and microdialysis probe placement

All stimulating electrode tips implanted into the cortex were confirmed to lie in the medial, ventral, or lateral oPFC between 3.7 and 4.7 mm anterior to bregma, 0.2 and 2.3 mm lateral to the midline, and 2.2 and 4.3 mm ventral to dural surface (Paxinos and Watson 1986). All dialysis probe/stimulating electrode tips implanted into the striatum were verified to lie between 0.8 mm posterior and 1.2 mm anterior to bregma, 1.8 and 5.0 mm lateral to the midline, and 3.4 and 7.7 mm ventral to dural surface (Paxinos and Watson 1986). All recording electrode tracks were confirmed to extend through the substantia nigra between 2.5 and 3.9 mm anterior to the interaural line, 1.5 and 3.2 mm lateral to the midline, and 6.3 and 8.2 mm ventral to the surface of the brain (Paxinos and Watson 1986).

Response patterns of nigral dopamine neurons to stimulation of the dorsal striatum and orbital PFC

In the current study, 363 electrophysiologically identified DA neurons were recorded from 61 animals undergoing striatal microdialysis. Additionally, 267 and 251 of the preceding recorded DA neurons were subjected to striatal and oPFC stimulation, respectively. Under control conditions (intrastriatal ACSF infusion), stimulation of the dorsal striatum at 800 µA produced responses in 68% of nigral DA cells tested (32/47). After striatal stimulation, electrical stimulation of the oPFC at 1000 µA produced responses in 80% of these same DA neurons (38/47). The response patterns of DA neurons evoked during electrical stimulation of both striatum and oPFC could be classified as IE or E. Under the conditions of the current study, the IE pattern was the most frequently observed response to electrical stimulation of the both the striatum and oPFC and was elicited in 40 and 64% of DA neurons, respectively (Table 1, Fig. 1). The E response pattern was observed in 28 and 17% of DA neurons recorded during striatal and oPFC stimulation, respectively (Table 1, Fig. 1).


                              
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Table 1. Observed frequency of IE and E responses of DA cells to electrical stimulation of the PFC and striatum during intrastriatal ACSF infusion



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Fig. 1. Peristimulus time interval histograms depicting the responses of dopamine (DA) neurons to electrical stimulation of the striatum and orbital prefrontal cortex (oPFC). In each case, stimulation occurred at time 0. A: initial inhibition/rebound excitation (I/E) response of a DA cell after electrical stimulation of the striatum (top) and oPFC (bottom). B: initial excitation (E) induced in an individual DA cell in response to electrical stimulation of the striatum (top) and oPFC (bottom). Each histogram represents the spike distribution (1.4-ms bins) across ~100-200 sweeps. up-arrow , onset of the initial inhibition; *, initial excitatory response.

Effects of concurrent intermittent electrical stimulation of the striatum and oPFC and striatal nitric oxide manipulations on dopamine neuron activity

The number of spontaneously active nigral DA cells encountered per electrode track was increased after 2- to 4-min periods of intermittent electrical stimulation of the striatum and oPFC (F = 3.92, P < 0.001, Fig. 2A). Specific comparisons between the nonstimulated ACSF control group (n = 6 rats; mean = 0.76 cells/track) and the ACSF stimulation control group (n = 7 rats; mean = 1.53 cells/track) revealed that the number of active DA cells doubled as a result of intermittent stimulation (P < 0.001, t-test). Intrastriatal infusion of either the NO generators H-ARG (2 mM, n = 6 rats; mean = 1.75 cells/track) and HA (2 mM, n = 5 rats; mean = 1.31 cells/track), the NOS inhibitor 7-NI at 100 (n = 7 rats; mean = 1.71 cells/track) and 300 µM (n = 6 rats; mean = 1.38 cells/track) concentrations, or the NO scavenger CPT-10 (1 mM, n = 6 rats; mean = 1.06 cells/track) did not have significant effects on the number of active DA cells per electrode track as compared with the ACSF stimulation control group (F = 1.64, P = 0.15).



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Fig. 2. Effects of intrastriatally infused nitric oxide (NO) generators and antagonists and intermittent electrical stimulation on DA neuronal activity. Mean ± SE number of active cells per electrode track (A), population firing rate (B), and percentage of spikes fired in bursts (C) of DA cells recorded in the substantia nigra was determined in rats receiving intrastriatal artificial cerebrospinal fluid (ACSF) or drug infusion. Intermittent electrical stimulation of the striatum and oPFC elevated the number of spontaneously active nigral DA cells encountered from 0.76 ± 0.12 to 1.53 ± 0.17 cells per track (#P < 0.05, ANOVA/Dunnet's test). No significant differences in cells/track were observed between the ACSF stimulation control and NO generator or antagonist groups (P > 0.5). Intermittent stimulation also decreased the basal firing rate of DA cells in control animals from 4.74 ± 0.4 to 3.29 ± 0.2 (#P < 0.05, ANOVA/Dunnet's test). When infused during periods of intermittent striatal and oPFC stimulation, the NO generators, NG-hydroxy-L-arginine (H-ARG) and hydroxylamine (HA) increased the mean population DA cell firing rate from 3.29 ± 0.2 (control) to 4.34 ± 0.2 and 4.67 ± 0.2 spikes/s respectively (*P < 0.05, ANOVA/Dunnet's test). Similar treatment with NO antagonists did not affect basal DA cell firing rate (P > 0.05, ANOVA/Dunnet's test). Intermittent stimulation also decreased the percent of spikes fired in bursts in control animals from 23.0 ± 5.4 to 6.7 ± 1.8 (#P < 0.05, ANOVA/Dunnet's test). When infused during periods of intermittent striatal and oPFC stimulation, carboxy PT-10 (CPT-10) elevated the percentage of spikes fired in bursts from 6.68 ± 1.75 (control) to 18.20 ± 4.41% (*P < 0.05, ANOVA/Dunnet's test). Similar treatment with both doses of the NO synthase (NOS) inhibitor and the NO generators did not significantly affect DA cell population burst firing (P > 0.05, ANOVA/Dunnet's test).

The effects of 2- to 4-min periods of intermittent electrical stimulation of the striatum and oPFC on the basal firing rate of DA neurons also was determined for cells in the preceding control and treatment groups. Overall, significant differences in mean firing rate were observed between the ACSF stimulation control, ACSF nonstimulation control, and NO treatment groups (F = 5.58, P < 0.0001, Fig. 2B). Post hoc comparisons between the ACSF nonstimulation and stimulation control group mean firing rates revealed that intermittent electrical stimulation decreased DA cell spike discharge (nonstimulation control: n = 6 rats, 28 cells, mean = 4.74 spikes/s; stimulation control: n = 7 rats, 56 cells, mean = 3.29 spikes/s; P < 0.05). Additionally, post hoc comparisons revealed that the mean firing rates of DA neurons from the H-ARG (n = 6 rats; 53 cells; mean = 4.34 spikes/s) and HA (n = 5 rats; 32 cells; mean = 4.67 spikes/s) infusion groups were significantly greater than that of the ACSF stimulation control group (P < 0.05). Significant differences in cell firing rate were not observed between the ACSF stimulation control group and the 7-NI 100 µM (n = 7 rats; 45 cells; mean = 3.04 spikes/s), 7-NI 300 µM (n = 6 rats; 40 cells; mean = 3.61 spikes/s; P > 0.05), or CPT-10 (n = 6 rats; 31 cells; mean =3.97 spikes/s) groups (P > 0.05).

The percentage of spikes fired in bursts also was calculated from cells recorded in the preceding control and treatment groups. Significant differences in the percentage of spikes fired in bursts were observed between the ACSF nonstimulation control group and the stimulation control and NO treatment groups (F = 3.24, P < 0.01, Fig. 2C). Comparisons between the ACSF nonstimulation control (23.0% spikes fired in bursts) and stimulation control groups (6.7% spikes fired in bursts) revealed that intermittent electrical stimulation of the striatum and oPFC significantly decreased the overall percentage of spikes fired in bursts (P < 0.001, t-test). Intrastriatal infusion of H-ARG or HA during electrical stimulation did not significantly alter the percentage of spikes fired in bursts (H-ARG = 14.7%; HA = 16.0%) compared with the ACSF stimulation control group (P > 0.05). A significant elevation in burst firing was observed during intrastriatal CPT-10 infusion (18.2%, P < 0.05, compared with the ACSF stimulation control group; t-test); however, this effect was not observed during infusion of 7-NI (100 µM, 9.5%) or 7-NI (300 µM, 11.4%, P > 0.05).

Effects of striatal NOS inhibition and NO generation on the response patterns of nigral dopamine cells

The initial response of DA cells to electrical stimulation of the striatum and oPFC during ACSF and NO antagonist and generator infusion was assessed, and elicited patterns of activity (IE or E) were categorized, as described in METHODS. Both striatal and cortical stimulation elicited a response from the majority of DA cells tested in ACSF stimulation control and drug treatment groups (Fig. 3). In all groups, the IE response was the most frequently observed response pattern to electrical stimulation of both the striatum and oPFC (Table 2). Intrastriatal infusion of H-ARG, HA, or the low dose of 7-NI (100 µM) did not alter the proportion of DA cells responding to striatal or cortical stimulation (P > 0.05, Fisher exact test) as compared with ACSF stimulation control. Although intrastriatal infusion of either the high dose (300 µM) of 7-NI or CPT-10 did not influence the proportion of DA cells responding to cortical stimulation (P > 0.05), both of these treatments significantly increased the proportion of DA cells responding to striatal stimulation from ~70% (ACSF control) to 97% (P < 0.01). Additionally, comparison of the proportions of striatal evoked IE responses between ACSF stimulation control and 7-NI (300 µM) and CPT-10 groups (Table 2) revealed that an increase in the prevalence of the IE response pattern occurred during intrastriatal infusion of the high dose of the NOS inhibitor and NO chelator (P < 0.01, Fisher exact test).



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Fig. 3. Effects of striatal NO manipulations on the proportion of DA cells responding to striatal and cortical stimulation. Left: intrastriatal infusion of either 7-nitroindazole (7-NI; 300 µM) or CPT-10 increased the proportion of DA cells responding to striatal stimulation from 70 (control) to ~97% (*P < 0.01, Fisher exact test). Right: same manipulations did not significantly affect the proportion of cells responding to oPFC stimulation (P > 0.05, Fisher exact test).


                              
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Table 2. Observed frequency of IE and E responses of DA cells to electrical stimulation of the PFC and striatum: effects of striatal NO manipulations

Effects of intrastriatal NO scavenger infusion on the current intensity-dependent response of dopamine cells to electrical stimulation

The probability of eliciting an IE pattern in response to electrical stimulation of the striatum increased as a function of current intensity (single pulses delivered during 5 separate periods of electrical stimulation per brain region). Under control conditions (intrastriatal ACSF infusion), DA neurons rarely responded to striatal stimulation with the IE pattern at low current intensities (0/25 cells at 100 µA, 2/25 cells at 250 µA, Figs. 4A and 5). After intrastriatal infusion of the NO chelator CPT-10 (1 mM), the probability of eliciting an IE pattern in response to striatal stimulation with current intensities of 250 µA increased from 0.08 (control) to 0.52 (11/21 cells, P < 0.05, Fisher exact test). Similar CPT-10-induced increases in DA cell responsiveness to striatal stimulation were observed when stimulus intensities were increased to 0.4, 0.6, and 1.0 mA.



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Fig. 4. Effects of intrastriatal NO scavenger infusion and current intensity on the probability of eliciting an initial inhibition/excitation (IE) response pattern to electrical stimulation of the striatum and oPFC. A: intrastriatal infusion of CPT-10 (1 mM) increased the responsiveness of DA neurons to striatal stimulation (0.6 Hz, 250 µs) at low current intensities (250 µA) without changing the threshold of response. CPT-10 administration also increased the probability of eliciting an IE pattern in response to striatal stimulation at high intensities (0.4-0.8 mA). B: intrastriatal infusion of the NO chelator did not alter the probability of eliciting an IE response to cortical stimulation (0.25-1.0 mA).



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Fig. 5. Effects of intrastriatal CPT-10 infusion on the probability of eliciting an IE pattern in a single DA neuron in response to electrical stimulation of the striatum. Separate trials (A-E) of single-pulse electrical stimulation of the striatum were administered during CPT-10 infusion and stimulus current intensities were increased after each trial (100-800 µA) as indicated. Each histogram represents the spike distribution over ~100 sweeps (same cell).

In contrast to observations with striatal stimulation, intrastriatal infusion of CPT-10 did not increase the probability that DA cells would respond to stimulation of the oPFC with an IE pattern (P > 0.05, Fisher exact test, Fig. 4B). At lower current intensities (250 µA), however, a trend for an increase in DA cell responsiveness was observed after intrastriatal CPT-10 administration (ACSF control = 2/22 cells, CPT-10 = 8/20 cells, P = 0.08, Fisher exact test).

Effects of striatal NO antagonists on the response of single dopamine cells to electrical stimulation of the orbital PFC and striatum

The response patterns of individual DA cells to electrical stimulation of the striatum and oPFC before and after intrastriatal infusion of 7-NI (300 µM) and CPT-10 (1 mM), respectively, were examined (Figs. 6 and 7). The mean basal firing rates for the 7-NI (300 µM) and CPT-10 (1 mM) groups were 4.16 ± 0.55 (mean ± SE; n = 6 cells/rats) and 5.03 ± 0.59 spikes/s (n = 10 cells/rats), respectively. Comparisons of the initial inhibitory periods elicited by electrical stimulation before and after CPT-10 infusion (Table 3) revealed that removal of striatal NO tone decreased the mean onset latency of observed IE responses induced by striatal stimulation (P < 0.05). Of the eight DA cells tested, four cells also responded to oPFC stimulation with an IE pattern during ACSF infusion. After intrastriatal CPT-10 infusion, all eight cells exhibited the IE pattern in response to oPFC stimulation, and a trend toward a decrease in the mean onset latency of the elicited response was observed (P = 0.09, comparisons between pre- and postdrug onset latencies could be made only for the 4 cells responding to oPFC stimulation during ACSF infusion). In addition to its influence on mean onset latency, intrastriatal CPT-10 infusion increased the mean duration of IE responses elicited by oPFC stimulation (P < 0.05).



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Fig. 6. Effects of intrastriatal 7-NI infusion on the responses of single DA neurons to electrical stimulation of the striatum (A) and oPFC (B). Top: predrug ACSF control. Bottom: 7-NI (300 µM) infused for 25-30 min. Each histogram represents the spike distribution over ~100 sweeps. Arrows indicate the onset of the initial inhibition. Asterisks in A indicate the initial and secondary excitatory responses. Large arrow indicates the drug-induced blockade of the secondary excitatory response in the control condition. Asterisks in B indicate the rebound excitatory period that often follows the initial inhibition-induced by electrical stimulation.



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Fig. 7. Effects of intrastriatal CPT-10 infusion on the responses of single DA neurons to electrical stimulation of striatum (A) and oPFC (B). Top: predrug ACSF control. Bottom: response of the same DA cell after intrastriatal CPT-10 (1 mM) infusion (45 min). Each histogram represents the spike distribution over ~100 sweeps. Arrows indicate the onset of the initial inhibition. Asterisks indicates an excitatory response that is attenuated after drug infusion (indicated by large arrow).


                              
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Table 3. Mean onset latencies and duration of initial inhibitory periods elicited by striatal and PFC stimulation before and after (15-45 min) intrastriatal CPT-10 infusion


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NO is known to mediate numerous neuronal processes and act as a secondary neural messenger in multiple brain regions including the striatum (Bredt et al. 1990; Garthwaite et al. 1988; see Garthwaite and Boulton 1995 for review). The current study examined the influence of striatal NO signaling on the basal neuronal activity of midbrain DA neurons and the response of these neurons to electrical stimulation of the striatum and oPFC. Our data suggest that NO, generated after activation of corticostriatal afferents or intrinsic excitatory pathways, regulates a component of the striatal information integration and output. Thus, the manipulation of tonic striatal NO levels exerted a significant impact on multiple dependent measures of DA cell activity. For example, augmentation of striatal NO signaling via local NO substrate or generator infusion produced consistent increases in the mean population firing rate of DA neurons. Moreover, intrastriatal infusion of either the NOS inhibitor (higher dose) or the NO scavenger enhanced the proportion of nigral DA neurons exhibiting an IE pattern in response to electrical stimulation of the striatum. Pharmacological disruption of striatal NO tone also was observed to increase the probability of eliciting an IE pattern in response to low levels of striatal stimulation. NO antagonists were shown further to decrease the onset latency and increase the duration of the initial inhibitory phase of the IE pattern response to electrical stimulation. Therefore NO was found to exert effects that altered the balance of the input-output relationship of the striatum in a way that produced a functionally significant impact on its target neurons.

Responses to electrical stimulation of the striatum and orbital PFC

Although the observed patterns of spike discharge of individual DA cells produced in response to electrical stimulation of the striatum and oPFC were often complex, these response patterns could be subdivided into two classes: either an initial inhibition (IE response) or activation (E response, sometimes with multiple components) of spike activity usually occurring within a variable range of onset latencies (1.5-100 ms). Consistent with previous studies (Collingridge and Davies 1981; Dray et al. 1976; Gariano and Groves 1988; Grace and Bunney 1985; Nakamura et al. 1979a; Overton et al. 1996; Precht and Yoshida 1971; Tepper et al. 1990; Tong et al. 1995, 1996; Yoshida and Precht 1971), single-pulse electrical stimulation of the striatum and oPFC elicited response patterns in the majority of DA neurons examined, with the IE response having the highest incidence. The consistency of the observed responses to both striatal and oPFC stimulation demonstrates that the integrity of the striatonigral pathway was not significantly compromised by implantation of the microdialysis probe/stimulating electrode into the striatum (see Moore et al. 1998) or by the microdialysis procedure.

In addition to the observed stimulus-induced inhibitory and excitatory patterns of activity, intermittent electrical stimulation of the striatum and oPFC decreased the mean firing rate and burst firing of DA cells and increased the number of spontaneously active DA cells recorded under control conditions. Because the stimulation paradigm used in the current study involved both striatal and oPFC stimulation in the same animals, it is not possible to determine the precise contribution of cortical and striatal efferents to the observed changes in basal DA neuron activity. However, the stimulation-induced decrease in firing rate and burst firing could have resulted from activation of mono- and/or polysynaptic GABAergic pathways known to innervate both the pars compacta and pars reticulata subdivisions of the substantia nigra (Deniau et al. 1996; Jones et al. 1977; Smith and Bolam 1990; Somogyi et al. 1981). Additionally, excitatory cortical inputs may regulate DA cell activity via the direct and indirect activation of inhibitory neurons in the globus pallidus and substantia nigra pars reticulata (Deniau et al. 1996; Grofova 1975; Nakamura et al. 1979a).

Although the precise basal ganglia circuitry involved in regulating DA cell activity remains to be fully characterized, it is likely that the PFC regulates the neuronal activity of DA cells under nonstimulated conditions because ibotenic acid lesions of this region have been shown to increase the basal firing rate of DA cells in the substantia nigra (Shim et al. 1996). This same study found a significant reduction in the number of spontaneously active DA cells in the ventral tegmentum after lesions of the medial PFC, raising the possibility that the observed increase in active DA cells in the current study may have resulted from increased excitatory drive from cortical inputs (Goswell and Sedgwick 1973; Kornhuber et al. 1984; Naito and Kita 1994). Alternatively, the increase in spontaneously active DA cells observed here may have resulted from a suppression of the above-mentioned pathways involved in inhibiting DA cell activity. In any case, the observed increase in active cells/track after intermittent electrical stimulation of the oPFC and striatum provides further evidence for the existence of a subpopulation of nonspontaneously spiking or "silent" DA cells in the substantia nigra (Bunney and Grace 1978; Chiodo and Bunney 1983; Harden and Grace 1995; White and Wang 1983). In contrast to our results and those cited in the preceding text, a recent study comparing the relative numbers of antidromically activated nigrostriatal DA cells in controls and animals in which DA neuronal spike discharge was pharmacologically suppressed was unable to find evidence of nonspontaneously spiking DA neurons (Dai and Tepper 1998). Given the above-cited studies combined with our results showing that striatal and oPFC stimulation increases the number of DA cells/track, however, it is likely that the inability to detect nonspontaneously active DA cells in the preceding study resulted from confounds associated with alterations in the spontaneous activity of the sampled DA neurons induced by the antidromic stimulation procedure.

Influence of striatal NO on stimulation-induced modulation of basal firing activity

A variety of evidence shows that it is likely that NO produced by NOS-containing interneurons mediates some of the actions of the excitatory corticostriatal afferent pathways involved in controlling striatal output (see INTRODUCTION). The role of striatal NO signaling in regulating basal DA cell activity was determined in the current study in controls and in animals receiving intrastriatal infusions of NO antagonists or generators (recorded in between intermittent periods of striatal and cortical stimulation). Intrastriatal infusion of NO antagonists did not affect the stimulation-induced decrease in DA cell firing rate. When infused into the striatum during intermittent periods of electrical stimulation, however, the NO generators significantly increased the mean firing rate of nigral DA neurons as compared with ACSF stimulation controls. These results suggest that a stimulation-induced activation of striatal NO production did not mediate the inhibitory effect of intermittent stimulation on DA cell firing rate. On the contrary, increasing striatal NO signaling appeared to reverse the inhibitory influence of an opposing regulatory circuit (see preceding text, Fig. 8). This NO-mediated activation of DA cell firing may contribute to the facilitatory effect of NO generators on striatal DA release reported in multiple in vivo microdialysis experiments (Guevara-Guzman et al. 1994; Nakahara et al. 1994; Spatz et al. 1995; Strasser et al. 1994; West and Galloway 1996, 1997a,b, 1998).



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Fig. 8. Model of NO-mediated activation of striatonigral feedback pathways regulating DA cell activity. In the proposed model, NO can activate a network of striatal output neurons (A), which make synaptic connections (solid arrow) with inhibitory GABAergic neurons in the substantia nigra pars reticulata. These cells in the reticulata innervate DA cells in the pars compacta (Grace and Bunney 1979, 1985) and are proposed to be inhibited after NO activation of this indirect striatonigral pathway, resulting in a disinhibition of DA cell activity. B: an additional network of striatal projection cells that are not activated by NO also may regulate DA cell activity via direct or indirect multisynaptic inhibitory inputs to the substantia nigra pars compacta (dotted arrow). Removal of striatal NO tone after local infusions of NO antagonists is proposed to decrease the activity of indirect excitatory pathway and result in an increase in the influence of the striatonigral inhibitory pathway during striatal stimulation.

A potential role for tonic NO in the modulation of striatal output pathways involved in regulating the mode of firing of DA cells also is supported by the current study. Thus, infusion of the NO scavenger increased the percentage of spikes fired in bursts in DA cells recorded under basal conditions in animals receiving intermittent electrical stimulation of the striatum and oPFC. Given that both doses of 7-NI failed to alter burst firing, the preferential influence of the NO scavenger on DA cell burst firing was not anticipated. It is possible that the NO scavenger was more effective in reducing NO tone than the NOS inhibitor. This seems unlikely, however, given that the higher dose of 7-NI was equally effective in enhancing the proportion of DA cells responding to striatal stimulation. Additional studies aimed at characterizing the mechanisms of CPT-10 and NO effector pathways involved in regulating striatal cell activity may enable the resolution of these issues.

Role of striatal NO in modulating the responsiveness of dopamine cells to electrical stimulation

In addition to its influence on basal DA neuron activity, the current population studies reveal that striatal NO tone regulates the proportion of DA neurons responding to electrical stimulation of the striatum. Thus intrastriatal infusion of both the high dose of 7-NI (300 µM) and the NO scavenger CPT-10 significantly enhanced the proportion of DA cells responding to striatal stimulation (800 µA) from 70.0% under control conditions to ~97.0%. Also, both the 7-NI (300µM) and CPT-10 treatment groups exhibited an increased incidence of the initial inhibition (IE) firing pattern in response to electrical stimulation of the striatum. These findings were replicated in further studies in which current intensities were varied and the effects of intrastriatal ACSF and CPT-10 infusions on the probability of eliciting an IE pattern in response to striatal stimulation were examined. Additionally within-subjects studies revealed that NO antagonist infusion decreased the onset latency of the initial inhibitory period induced by striatal stimulation.

Taken together, these findings show that NO tone regulates the excitability of striatal output pathways involved in producing the IE response pattern in DA cells during striatal stimulation. This proposed involvement of NO in regulating striatal output neuron excitability is supported by preliminary data from our laboratory showing that intrastriatal NO infusion enhances both the firing rate and the duration of burst firing in striatal neurons (West and Grace 1999). Although it is currently unknown whether NO preferentially regulates the activity of specific striatal output neurons, it is possible that a subpopulation of striatal neurons containing guanylyl cyclase or other NO effector proteins may be responsible for the effects of NO on DA cell activity in the current study. In support of this, it has been demonstrated that a distinct subclass of striatal spiny neurons which project to the substantia nigra localize cGMP-dependent protein kinase (Walaas et al. 1989). These neurons also contain guanylyl cyclase (Ariano 1983) and are activated by NO generators (Tsou et al. 1993). These striatonigral circuits preferentially innervate neurons in the substantia nigra pars reticulata (Deniau et al. 1996; Grofova 1975) but also make direct contacts with DA cell dendrites in the reticulata (Wassef et al. 1981) and compacta (Bolam and Smith 1990). Electrophysiological studies have demonstrated that these circuits have opposing functions and may regulate the activity of nigrostriatal DA cells differentially depending on the level of striatal stimulation (Grace and Bunney 1979, 1985). Thus striatal NO signaling may increase DA cell firing by selectively activating GABAergic inputs to inhibitory GABAergic neurons in the substantia nigra pars reticulata, resulting in a disinhibition of DA cell activity. Removal of striatal NO tone may depress this indirect excitatory pathway and result in an increase in the influence of the mono- and polysynaptic striatal output pathways responsible for producing the initial inhibitory response to striatal stimulation (see Fig. 8). This model is supported by the current findings demonstrating an increase in the incidence of the initial inhibitory response to striatal stimulation and a decrease in the mean onset latency of this response after intrastriatal infusion of NO antagonists. Moreover within-subjects experiments revealed that intrastriatal infusion of NO antagonists often abolished excitatory responses, which in some cases, were masking inhibitory responses to striatal stimulation (see Figs. 6 and 7).

In contrast to striatal stimulation, the probability of eliciting an IE response to oPFC stimulation was not affected by intrastriatal CPT-10 infusion in the population studies. It is possible that the observed lack of effect of the NO antagonists on the responsiveness of DA cells to oPFC stimulation was due to a ceiling effect because ~80% of DA cells responded to stimulation under control conditions. In support of this, within-subjects experiments revealed that intrastriatal CPT-10 infusion decreased the onset latency and increased the duration of the initial inhibitory period evoked by stimulation of the oPFC. These findings suggest that basal NO tone suppresses an aspect of corticostriatal transmission involved in activating striatal output systems. Given that NOS inhibitors have been shown to potentiate striatal neurotransmitter release induced by NMDA (Kendrick et al. 1996; West and Galloway 1997b), it is likely that, under basal conditions, NO exerts an inhibitory influence on striatal NMDA receptor function. Although speculative, it is possible that the observed changes in DA cell responsiveness to oPFC stimulation during CPT-10 infusion may have resulted from a disinhibition of striatal NMDA channel activation. Thus, a lower threshold for NMDA receptor activation resulting from decreased striatal NO tone in turn may increase the strength of the inhibitory influence of striatal efferents activated after electrical stimulation of corticostriatal transmission.

Summary and conclusions

The primary findings of the current study demonstrate that striatal NO signaling has a major impact on the responsiveness of DA neurons to electrical stimulation of the striatum and to some extent, the oPFC. Moreover, it is likely that NO signaling plays an important role in regulating the activity of striatal output neurons. Thus, striatal NOS interneurons may be critically involved in integrating corticostriatal sensorimotor information within striatal networks and synchronizing the activity of functionally related striatonigral subsystems. Given that intrastriatal NO antagonist infusion increased the prevalence of initial inhibitory responses, whereas the NO generating compounds enhanced the firing rate of DA cells, it is plausible that striatal NOS interneurons may regulate the excitability of striatal efferent systems involved in modulating basal ganglia output nuclei largely via the maintenance and control of tonic and phasic DA neurotransmission (Grace 1991; Moore et al. 1999; O'Donnell and Grace 1998). This is supported by microdialysis studies demonstrating that endogenous striatal NO production increases striatal extracellular DA levels (see preceding text). Thus, striatal NO signaling may function to enhance tonic extracellular DA levels and suppress phasic DA activity during conditions of minimal sensory stimulation or behavioral demands (i.e., when the animal is not presented with novel or appetitive stimuli). Under conditions where corticostriatal pathways originating in the oPFC are activated [i.e., during electrical stimulation or the presentation of rewarding stimuli or reinforcement learning (see Schultz et al. 1998 for review)], striatal NO production may promote phasic striatal DA neurotransmission via activation of DA cell firing. This proposed regulatory role of striatal NO would provide the PFC with a mechanism to modulate the firing pattern of midbrain DA neurons and striatal DA neurotransmission differentially depending on the behavioral demands presented by the environment.


    ACKNOWLEDGMENTS

The authors thank N. MacMurdo for excellent technical assistance and B. Lowry for the development of software (Neuroscope) used in data acquisition and analysis. The authors also thank Dr. Holly Moore, H. Jedema, J. A. Rosenkranz, and C. Todd for valuable comments and suggestions regarding this manuscript.

This work was supported by National Institutes of Health Grants MH-45156 and MH-01055 to A. A. Grace and NS-10725 to A. R. West.


    FOOTNOTES

Address for reprint requests: A. R. West, Dept. of Neuroscience, 446 Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 September 1999; accepted in final form 1 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society