Section of Neurobiology, Yale Medical School, New Haven, Connecticut 06510
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ABSTRACT |
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Jia, Changping, Wei R. Chen, and Gordon M. Shepherd. Synaptic organization and neurotransmitters in the rat accessory olfactory bulb. J. Neurophysiol. 81: 345-355, 1999. The accessory olfactory bulb (AOB) is the first relay station in the vomeronasal system and may play a critical role in processing pheromone signals. The AOB shows similar but less distinct lamination compared with the main olfactory bulb (MOB). In this study, synaptic organization of the AOB was analyzed in slice preparations from adult rats by using both field potential and patch-clamp recordings. Stimulation of the vomeronasal nerve (VN) evoked field potentials that showed characteristic patterns in different layers of the AOB. Current source density (CSD) analysis of the field potentials revealed spatiotemporally separated loci of inward current (sinks) that represented sequential activation of different neuronal components: VN activity (period I), synaptic excitation of mitral cell apical dendrites (period II), and activation of granule cells by mitral cell basal dendrites (period III). Stimulation of the lateral olfactory tract also evoked field potentials in the AOB, which indicated antidromic activation of the mitral cells (period I and II) followed by activation of granule cells (period III). Whole cell patch recordings from mitral and granule cells of the AOB supported that mitral cells are excited by VN terminals and subsequently activate granule cells through dendrodendritic synapses. Both CSD analysis and patch recordings provided evidence that glutamate is the neurotransmitter at the vomeronasal receptor neuron; mitral cell synapses and both NMDA and non-NMDA receptors are involved. We also demonstrated electrophysiologically that reciprocal interaction between mitral and granule cells in the AOB is through the dendrodendritic reciprocal synapses. The neurotransmitter at the mitral-to-granule synapses is glutamate and at the granule-to-mitral synapse is -aminobutyric acid. The synaptic interactions among receptor cell terminals, mitral cells, and granule cells in the AOB are therefore similar to those in the MOB, suggesting that processing of chemosensory information in the AOB shares similarities with that in the MOB.
The vomeronasal system is a chemosensory pathway which is parallel to but separated from the main olfactory system. Chemosensory receptor neurons of the vomeronasal system are located in the sensory epithelium of the vomeronasal organ. They are believed to detect nonvolatile odors or pheromones associated primarily with social and reproductive behaviors (for reviews see Halpern 1987 Slice preparation
The method for slice preparation was adapted from Ennis et al. (1996) Field potential recording and CSD analysis
The laminar organization of the AOB (Fig. 1) was clearly visible under a dissecting microscope. For the coronal sections of the AOB, one stimulating electrode was placed on the VN bundles and another on the lateral olfactory tract (LOT). For the parasagittal sections, the stimulating electrode was placed either in the anterior part or in the posterior part of the VN layer. A square pulse (0.2 ms, 20-1,000 µA, negative current) was delivered through the stimulating electrodes. Recording of electrically evoked field potentials was made with glass microelectrodes filled with 2% Chicago sky-blue in 0.5 M sodium acetate solution to allow marking of the recording sites. Field potentials were recorded with Axoclamp-2A and 2B amplifiers (Axon Instruments) and were digitized through an ITC-16 computer interface (Instutech).
FIELD POTENTIAL ANALYSIS.
The early analysis of the field potentials in the in vivo olfactory bulb was based on two basic assumptions. (1) The bulbar layers approximated to spherical symmetry. (2) There was synchronous activation of bulbar neuronal populations (mitral and granule cells). Under these conditions, the potentials recorded within the bulb largely reflect the potentials generated by the primary radial current flow through a cone around the activated element (Rall and Shepherd 1968 CSD ANALYSIS.
CSD analysis is a method used to determine the net extracellular current flow into and out of tissue volumes (Haberly and Shepherd 1973
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Meredith 1991
; Wysocki 1979
). Odor information detected by the vomeronasal receptor neurons is conveyed through the accessory olfactory bulb (AOB) to amygdala and then to hypothalamus (Kevetter and Winans 1981
; Scalia and Winans 1975
).
, 1994
; Lloyd-Thomas and Keverne 1982
). The laminar pattern of the AOB is similar to but less distinct than that of the main olfactory bulb (MOB). The output neurons of the AOB, the mitral cells, have apical dendrites that enter the glomerular layer to receive synaptic input from the vomeronasal nerve (VN) terminals. Analysis of the field potentials in the in vivo AOB suggested that mitral cells receive excitatory synaptic input from the VN terminals in the glomerular layer (MacLeod and Reinhardt 1983; Reinhardt et al. 1983
). It was speculated that the neurotransmitter at the synapses between VN terminals and mitral cell apical dendrites in the glomerular layer is glutamate, as in the glomerular layer of the MOB. However, electrophysiological evidence is still controversial. For example, broad-spectrum excitatory amino acid antagonist kynurenate does not block the synaptic event in the glomerular layer (Kaba and Keverne 1992
). In extracellular recordings from the AOB, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or D-2-amino-5-phosphonovaleric acid (APV) reduces the VN-evoked responses in only some of AOB cells tested (Dudley and Moss 1995
).
). It is not clear, however, whether the dendrodendritic interactions are involved in the mitral cell inhibition in the AOB and which neurotransmitter(s) is involved (Hayashi et al. 1993
; Reinhardt et al. 1983
).
-aminobutyric acid (GABA). Preliminary results were reported elsewhere in abstract form (Jia and Shepherd 1997
).
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
, Nickell et al. (1996)
, and Chen and Shepherd (1997)
. Male and female young adult rats (20-38 days old, Sprague-Dawley strain) were anesthetized with an overdose of ketamine and decapitated. The AOB together with the MOB was quickly dissected out from the skull and placed in ice-cold artificial cerebrospinal fluid. The AOB slices were sectioned at 300 to 400 µm thickness with a vibratome in coronal planes. In some experiments, parasagittal sections of the AOB were also made to compare the anterior and posterior subdivisions. The slices were incubated in a holding chamber and then transferred into recording chambers for either field potential or whole cell recordings at room temperature (25-27°C). The composition of the artificial cerebrospinal fluid used in slice preparation and recording was as follows (in mM): 124 NaCl, 3 KCl, 0 or 1.3 MgSO4, 2 CaCl2, 26 NaHCO3, 1.25 NaH2PO4, and 10 glucose. The artificial cerebrospinal fluid was saturated with 95% O2-5% CO2 to maintain pH at 7.4.
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FIG. 1.
Nissl stained coronal section of the accessory olfactory bulb (AOB), showing the vomeronasal nerve layer (VNL), glomerular layer (GL), external plexiform layer (EPL), internal plexiform layer/lateral olfactory tract (IPL/LOT), and granule cell layer (GRL). Mitral cells are distributed in the external plexiform layer. Stimulating electrodes were placed on the vomeronasal nerve bundles (S1) and lateral olfactory tract (S2). Bar: 100 µm.
). Subsequent analysis of the field potentials in partially activated bulb showed a similar pattern and amplitudes comparable with those recorded under uniform activation of the whole bulb (Shepherd and Haberly 1970
). This indicated that the potential recorded within the activated region of the bulb is still reflective of the primary radial current flow around the activated elements with small contributions by the secondary current flow through the nonactivated area as long as the synchronously activated radial elements are relatively large and the recordings are made within the activated area.
for generation of sufficient synchronous radial current to apply the potential divider model developed in the in vivo MOB (Rall and Shepherd 1968
) to the field potentials in the in vitro AOB slice. This in turn provides the basis for carrying out CSD analysis of these field potentials in the AOB slice preparation.
; Ketchum and Haberly 1993
; Mitzdorf 1985
). This method was used in neural tissues in vivo as well as in slice preparations to reveal the locations of excitatory and inhibitory synaptic activities on the basis of spatiotemporal distribution of sinks (inward membrane current) and corresponding sources (outward membrane current). The validity of one-dimensional CSD analysis in the AOB was tested in the mouse (Kaba and Kawasaki 1996
; Kaba and Keverne 1992
). One-dimensional CSD analysis was made on the field potentials recorded in a slice preparation of the AOB. Stimulation was applied to VN and LOT, and the field potentials were recorded every 25 or 50 µm along the axis perpendicular to the layers of the AOB. The one-dimensional CSD distribution was calculated according to the formula
where Im is the net current along the z-axis, which is perpendicular to the lamination of the AOB, óz is the conductivity tensor of the tissue, and ø is the field potential. The second spatial derivative of the field potential was approximated by the formula
where h is the sampling interval (50 µm in this study).
Whole cell patch recording
The patch pipettes were pulled from thick-wall glass capillaries and had a resistance of 8-15 M when filled with a solution composed of 110 mM K gluconate, 2.0 mM MgCl2, 1.0 mM CaCl2, 10 mM ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 2.0 mM Na2-ATP, 0.3 mM Na3-GTP (pH adjusted to 7.2-7.4 and osmolarity to 300 with KOH and sucrose, respectively). An Olympus BX50WI infrared DIC microscope equipped with ×40 water-immersion objective and a charge-coupled device camera were used to visually identify the neurons to be recorded and to guide the patch electrodes. Whole cell recording was made in current-clamp mode with Axoclamp 2A and 2B amplifiers. Membrane potentials were digitized with an ITC-16 interface and analyzed with Axodata and Axograph on a Mac II computer.
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RESULTS |
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Anatomy of the rat AOB
The laminar organization of the AOB is similar to that of the MOB (Fig. 1). In coronal sections of the AOB, the most superficial is the VN layer, which is formed by incoming axons of the vomeronasal receptor neurons. Beneath the VN layer is the glomerular layer where VN terminals make synapses with dendrites of mitral and periglomerular cells in the AOB. The external plexiform layer is situated beneath the glomerular layer. Mitral cell somata of the AOB are distributed diffusely in the external plexiform layer rather than arranged in a single layer as in the MOB. Apical dendrites of the mitral cells enter the glomerular layer to make synapses with the VN terminals. Mitral cell basal dendrites spread in the external plexiform layer. Axons of the mitral cells project out of the AOB through the LOT. The most interior layer is the granule cell layer in which numerous small inhibitory granule cells are located. These granule cells have peripheral dendrites projecting into the external plexiform layer. The internal plexiform layer is located between the external plexiform layer and granule cell layer.
Analysis of field potentials in the AOB slice
VN SIMULATION.
A single shock to the VN layer evoked highly reproducible field potentials in the AOB slice. The field potentials showed a series of changes in amplitudes and polarities when the recording electrode was shifted vertically among the different layers of the AOB. A typical experiment is shown in Fig. 2A. In the glomerular layer, VN stimulation evoked three sequential negative deflections. These three waves were used as the basis for dividing the responses into three periods or components (Fig. 2, A and B), as in the analysis of evoked potentials in the MOB (Rall and Shepherd 1968; Waldow et al. 1981
).
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100 Hz) stimulation and was very stable. CSD analysis showed in the period I a brief source followed by a brief large sink restricted to the VN layer and glomerular layer (S1, Fig. 2B). This suggested that period I was a presynaptic component generated by population action potentials of the VN fibers. The calculated conduction velocity of the VN was 0.15 m/s, which was much slower than that observed in the in vivo rabbit AOB (MacLeod and Reinhardt 1983).
; Takami and Graziadei 1991
). This dipole was probably generated by synchronous activation of mitral cells. The large sink in the glomerular layer was indicative of excitation or depolarization of the mitral cell dendritic tufts in the glomerular layer.
LOT STIMULATION.
Output neurons of the AOB, the mitral cells, project to the amygdala, bed nuclei of stria terminalis, and accessory olfactory tract through the LOT. Stimulation of the LOT at a site just posterior to the AOB antidromically activated mitral cells and the evoked field potentials could be recorded in the AOB. The LOT-evoked field potentials in the AOB slice (Fig. 3A) showed characteristic patterns in amplitudes and polarities similar to the LOT-evoked field potentials in the MOB when recording electrode was shifted in different layers. The LOT-evoked field potentials in the AOB were divided into three periods for the purpose of analysis, as in the MOB (Rall and Shepherd 1968). Period I of the LOT-evoked field potentials was a large negativity in the external plexiform layer and became a positive deflection in glomerular layer. The reversal point was at the level of the superficial external plexiform layer. After period I was period II, which was positive in deep external plexiform layer and negative in the glomerular layer. CSD analysis of the LOT-evoked field potentials revealed a brief sink (S1 in Fig. 3B) in the external plexiform layer and a source in glomerular layer in the periods I and II. The distribution of the period I and II dipole in the external plexiform layer and glomerular layer suggested that it was generated by antidromic activation of the mitral cells. According to the model developed in the MOB (Rall and Shepherd 1968
), periods I and II represented the depolarization and repolarization of the mitral cell soma, respectively.
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Whole cell recording from mitral and granule cells
To make a detailed analysis of synaptic interactions between the VN and mitral cells and between the mitral and granule cells, whole cell patch recording was made from mitral and granule cells.
Neurotransmitters in the AOB
NEUROTRANSMITTER BETWEEN RECEPTOR CELLS AND MITRAL CELLS.
The neurotransmitters at the VN-mitral cell and the mitral-granule cell synapses were analyzed with whole cell recording and field potential analysis. Whole cell recording was made in mitral cells in the external plexiform layer of the AOB. The non-NMDA receptor antagonist CNQX reduced the amplitude of the VN-evoked EPSP in mitral cells and left a small slow component unblocked. This CNQX-resistant slow component of the EPSP was blocked by the NMDA receptor antagonist APV. Perfusion with APV alone had only a small effect on the initial rise phase and amplitude of the EPSP. Combined application of CNQX and APV eliminated the VN-evoked EPSP (n = 17; Fig. 6, A and B). This result suggests that non-NMDA component predominates at this synaptic site. Similar results were obtained by field potential analysis. Periods II and III of VN-evoked field potentials recorded in the glomerular layer were used as an indicator of the postsynaptic response of the synaptic transmission from VN to mitral cells. Perfusion with the non-NMDA receptor antagonist CNQX reduced the amplitude of the periods II and III of the VN-evoked field potentials and left a small, slowly developing, late component (NMDA component) unblocked. NMDA receptor antagonist APV alone only reduced the late component of the period III of the VN-evoked field potential in the AOB (Fig. 6, C-E). CNQX together with APV completely blocked period II and III response (n = 6; Fig. 6E).
NEUROTRANSMITTERS BETWEEN THE MITRAL AND GRANULE CELLS.
Synaptic interactions between mitral and granule cells involve dendrodendritic reciprocal synapses. In Mg2+-free solution, an action potential in a mitral cell was followed by an IPSP. The mitral cell IPSP that followed an action potential could be blocked by bicuculline (n = 18; Fig. 7, A and B). This action potential-evoked IPSP was also blocked by CNQX and APV (n = 12; Fig. 7, C and D). To confirm that the site of action of APV and CNQX was at the dendrodendritic synapses and not at synapses made by the axon collaterals of the mitral cells, TTX was added to the bath solution to block sodium action potentials. A depolarizing pulse large enough to induce calcium spikes in mitral cells evoked an IPSP similar to that seen after sodium action potentials. This IPSP was blocked by CNQX and APV (n = 4; Fig. 7, E and F). In recordings from granule cells, the LOT-evoked EPSP was blocked completely by CNQX and APV (n = 5; Fig. 8A).
The pattern of the field potentials reported here in the slice preparation of the rat AOB in response to VN and LOT shocks was similar to that originally reported in the in vivo MOB for olfactory nerve and LOT shocks (Rall and Shepherd 1968 Neuronal circuit in the AOB
This electrophysiological study of the AOB supports that there are functionally three major neuronal components (the receptor terminals, the mitral cells, and the granule cells) and clarified their synaptic relationships. VN stimulation sequentially activates the three neuronal components.
Dendrodendritic synaptic interaction in the AOB
There are two possible routes through which mitral cells and granule cells interact in the AOB. The main route is in the external plexiform layer through dendrodentritic synapses (Barber et al. 1978 Neurotransmitters in the AOB
Recent studies in the MOB indicate that glutamate is the neurotransmitter at the receptor neuron terminals in the glomerulus (Aroniadou-Anderjaska et al. 1997 We thank Dr. Minghong Ma for helpful discussions and suggestions.
This work was supported by grants to G. M. Shepherd from the National Institutes of Health, National Aeronautics and Space Administration, and National Institute of Deafness and Other Communications Disorders (Human Brain Project).
Address for reprint requests: G. M. Shepherd, Section of Neurology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Received 11 May 1998; accepted in final form 25 September 1998.
50 to
70 mV. Some of them had a low level of spontaneous activity of 1-4 Hz. In agreement with the field potential analysis, stimulation of the VN evoked a long-lasting EPSP in mitral cells (Fig. 4B). This EPSP could last for >100 ms and may be responsible for the long-lasting nature of the glomerular layer sink S2. This EPSP occurred at the start of period II of the VN-evoked field potentials. The latency of the onset of the EPSP recorded in the mitral cell soma in external plexiform layer was 2-3 ms after the VN action potentials. This latency was relatively stable with different stimulating intensity, indicating a monosynaptic nature. The first action potentials usually occurred in the late period II of the VN-evoked field potentials (Fig. 4, A and B). Superimposed on the long-lasting EPSP was an inhibitory postsynaptic potential (IPSP), which had a delayed onset and occurred in the period III of the VN-evoked field potentials (as indicated by an asterisk in Fig. 4B). The IPSP was associated with decreased firing of action potentials in the mitral cells.
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FIG. 4.
Correlation of time courses of the VN-evoked field potentials in the glomerular layer (A) and the granule cell layer (C) with whole cell recording from a mitral cell (B) and a granule cell (D). A and C are VN-evoked field potentials recorded in the glomerular layer and granule cell layer, respectively, showing the 3 periods (I, II, and III). Mitral cell responds to VN stimulation with an excitatory postsynaptic potential (EPSP), which occurs in the period II of the field potentials. The first action potential (truncated) of the mitral cell occurs in the late period II (B). Note the IPSP (star) superimposed on the EPSP. D: VN-evoked EPSP in a granule cell. The onset of granule cell EPSP is in the period III of the field potentials. Similar results were obtained in 8 animals. GL, glomerular layer; GRL, granule cell layer.
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FIG. 5.
Correlation of the time courses of the LOT-evoked field potentials (A) with whole cell recordings from a mitral cell (B) and a granule cell (C). A: LOT-evoked field potentials recorded in the external plexiform layer. B: LOT stimulation evoked an antidromic action potential (truncated) in the mitral cell which occurred in period I and II. In period III, there is a large inhibitory postsynaptic potential (IPSP). C: LOT-evoked EPSP in granule cell; the onset of the EPSP was in the period III. Similar results were obtained in 7 animals.
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FIG. 6.
Pharmacological properties of the synaptic transmission between vomeronasal receptor cells and mitral cells in the AOB. A: N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovaleric acid (APV) reduces a late component of the VN-evoked long-lasting EPSP in mitral cell. B: Non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduces the amplitude of the EPSP and leaves a small late component unblocked. This CNQX-resistant late component is blocked by the NMDA receptor antagonist APV. C: NMDA receptor antagonist APV reduces the late component of the field potential periods II and III. Non-NMDA receptor antagonist CNQX reduces mainly the early components of the field potentials (D). In the presence of APV and CNQX, the periods II and III are completely blocked (E). Arrows indicate VN stimulation.
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FIG. 7.
Action potentials evoked by injection of depolarizing current are followed by IPSPs (arrows) (A and C). These IPSPs are blocked by 50 µM bicuculline (B) as well as by 50 µM CNQX and 100 µM APV (D). In the presence of 1 µM tetrodotoxin, large depolarization of the mitral cell by current injection is still followed by IPSPs (arrows in E), which are completely blocked by CNQX and APV (F). The dashed lines indicate resting membrane potentials, which are from 55 to
58 mV.
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FIG. 8.
A: LOT-evoked EPSPs (and action potentials) in a granule cell. The EPSPs are reduced by APV. A small APV-resistant component of the EPSP was eliminated by CNQX. B-D: period III of the LOT-evoked field potentials in external plexiform layer was used as an indicator of synaptic excitation of granule cells by mitral cells. APV reduces only the late component of the period III (B). CNQX reduces the amplitude of the period III (C). Combined application of APV and CNQX completely blocks period III (D). Arrows: LOT stimulation.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). It is also similar to the pattern in the in vivo AOB of the rabbit (MacLeod and Reinhardt 1983; Reinhardt et al. 1983
) and mouse (Kaba and Keverne 1992
). Results from CSD analysis of the field potentials in the AOB slice were similar to those obtained in the in vivo mouse AOB (Kaba and Keverne 1992
). This suggests that the basic neuronal circuit in the AOB shows similarity with the MOB and was well preserved in the slice preparation.
). Another possible route is in the granule cell layer through synapses made by mitral cell axon collaterals onto granule cells. There is evidence in the MOB that mitral cell axon collaterals make synapses with granule cells in the granule cell layer, but it remains to be determined in the AOB (Kishi et al. 1984
). In the experiments in which TTX was used to block sodium action potentials, direct depolarization of the mitral cells was still followed by an IPSP. This indicates that the reciprocal inhibition between mitral and granule cells was generated at a synaptic site electrically close to the soma (at the dendrites through dendrodendritic reciprocal synapses) and rules out the involvement of mitral cell axon collaterals.
; Isaacson and Strowbridge 1998
; Jahr and Nicoll 1980
; Mori and Shepherd 1979
; Rall and Shepherd 1968
; Rall et al. 1966
; Shepherd 1971
, 1972
). This study indicates that dendrodendritic reciprocal synaptic interactions are also present in the AOB and are responsible for the recurrent inhibition of the mitral cells after action potentials.
; Berkowicz et al. 1994
; Chen and Shepherd 1997
; Ennis et al. 1996
). In the AOB, immunocytochemical and in situ hybridization studies showed the presence of NMDA and non-NMDA receptor subunits in the glomerular layer (Petralia et al. 1994a
,b
; Watanabe 1993
). Electron microscopic studies showed type I asymmetrical synapses between VN terminals and mitral cell apical dendrites (Wilson and Raisman 1981
) and glutamate immunoreactivity in the VN terminals (Quaglino et al. 1997
). However, evidence from electrophysiological studies was controversial (Brennan 1994
; Dudley and Moss 1995
; Kaba and Keverne 1992
). In this study, the EPSP in the mitral cells in response to VN stimulation was completely blocked by non-NMDA receptor antagonist CNQX and NMDA receptor antagonist APV. This indicates that glutamate is the neurotransmitter at the VN terminals. In addition, we found that CNQX reduces a large proportion of the early component of the VN-evoked EPSP. This suggests that non-NMDA receptors predominate at this site. Similar findings were reported at the olfactory receptor-mitral cell synapse in the MOB (Berkowicz et al. 1994
; Chen and Shepherd 1997
; Ennis et al. 1996
).
). In whole cell recordings in the presence of TTX, the mitral cell IPSP after a large depolarization was blocked completely by APV and CNQX. Similarly, the granule cell EPSP after antidromic activation of mitral cells was also completely blocked by APV and CNQX. This suggests that the neurotransmitter involved in the mitral-to-granule dendrodendritic synaptic transmission is glutamate, as reported in the MOB (Isaacson and Strowbridge 1998
). These observations were consistent with an in vivo study in which excitatory amino acid antagonists blocked a VN-evoked current sink in external plexiform layer in CSD recording from the mouse AOB (Kaba and Keverne 1992
). A behavioral study suggested that glutamate is involved in the formation of odor memory in the mouse AOB (Brennan 1994
).
, 1994
; Shepherd and Greer 1997
; Wellis and Kauer 1993
; Yokoi et al. 1995
). Our study of the mitral cell IPSP indicates the presence of similar dendrodendritic reciprocal synaptic interactions between the mitral and granule cells in AOB. These dendrodendritic interactions may also play important roles in refining sensory information in the vomeronasal system.
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References
0022-3077/99 $5.00 Copyright ©1999 The American Physiological Society