Tonic and Synaptically Evoked Presynaptic Inhibition of Sensory Input to the Rat Olfactory Bulb Via GABAB Heteroreceptors

Vassiliki Aroniadou-Anderjaska, Fu-Ming Zhou, Catherine A. Priest, Matthew Ennis, and Michael T. Shipley

Department of Anatomy and Neurobiology and Program in Neuroscience, University of Maryland School of Medicine, Baltimore, Maryland 21201


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ABSTRACT
INTRODUCTION
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Aroniadou-Anderjaska, Vassiliki, Fu-Ming Zhou, Catherine A. Priest, Matthew Ennis, and Michael T. Shipley. Tonic and Synaptically Evoked Presynaptic Inhibition of Sensory Input to the Rat Olfactory Bulb Via GABAB Heteroreceptors. J. Neurophysiol. 84: 1194-1203, 2000. Olfactory receptor neurons of the nasal epithelium send their axons, via the olfactory nerve (ON), to the glomeruli of the olfactory bulb (OB), where the axon terminals form glutamatergic synapses with the apical dendrites of mitral and tufted cells, the output cells of the OB, and with juxtaglomerular (JG) interneurons. Many JG cells are GABAergic. Here we show that, despite the absence of conventional synapses, GABA released from JG cells activates GABAB receptors on ON terminals and inhibits glutamate release both tonically and in response to ON stimulation. Field potential recordings and current-source density analysis, as well as intracellular and whole cell recording techniques were used in rat OB slices. Baclofen (2-5 µM), a GABAB agonist, completely suppressed ON-evoked synaptic responses of both mitral/tufted cells and JG cells, with no evidence for postsynaptic effects. Baclofen (0.5-1 µM) also reversed paired-pulse depression (PPD) of mitral/tufted cell responses to paired-pulse facilitation (PPF), and reduced depression of JG cell excitatory postsynaptic currents (EPSCs) during repetitive ON stimulation. These results suggest that baclofen reduced the probability of glutamate release from ON terminals. The GABAB antagonists CGP35348 or CGP55845A increased mitral/tufted cell responses evoked by single-pulse ON stimulation, suggesting that glutamate release from ON terminals is tonically suppressed via GABAB receptors. The same antagonists reduced PPD of ON-evoked mitral/tufted cell responses at interstimulus intervals 50-400 ms. This finding suggests that a single ON impulse evokes sufficient GABA release, presumably from JG cells, to activate GABAB receptors on ON terminals. Thus GABAB heteroreceptors on ON terminals are activated by ambient levels of extrasynaptic GABA, and by ON input to the OB. The time course of ON-evoked, GABAB presynaptic inhibition suggests that neurotransmission to M/T cells and JG cells will be significantly suppressed when ON impulses arrive in glomeruli at 2.5-20 Hz. GABAB receptor-mediated presynaptic inhibition of sensory input to the OB may play an important role in shaping the activation pattern of the OB glomeruli during olfactory coding.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Olfactory sensory input is transduced by olfactory receptor neurons (ORNs) in the nasal epithelium and relayed to the glomeruli of the main olfactory bulb (OB) via the olfactory nerve (ON). Mammalian ORNs are thought to express a single odorant receptor (Buck and Axel 1991; Chess et al. 1994; Kishimoto et al. 1994; Zhao et al. 1998). A cohort of ORNs expressing the same odorant receptor projects to two (or a few) topographically fixed glomeruli in the olfactory bulb (Mombaerts et al. 1996; Ressler et al. 1994; Vassar et al. 1994). Odorant receptors do not appear to be specific to an odor but rather to molecular features that may be present in many odorants (Johnson et al. 1998). Thus a given odor activates several cohorts of ORNs (those that respond to the molecular features of the odorant) and evokes a specific pattern of glomerular activity (Buck 1996; Friedrich and Korsching 1997; Johnson and Leon 1996; Johnson et al. 1998; Laurent 1996). A key issue in olfaction is how the brain computes odors from patterns of glomerular activity. While the glomeruli are the initial stage in this computation (Shepherd 1994), little is known about the intraglomerular synaptic mechanisms that regulate glomerular activity.

In the glomeruli, ON terminals form excitatory, glutamatergic synapses with the apical dendrites of mitral/tufted (M/T) cells (Aroniadou-Anderjaska et al. 1997; Berkowicz et al. 1994; Ennis et al. 1996), the output cells of the OB, and with juxtaglomerular (JG) interneurons (Bardoni et al. 1996; Keller et al. 1998; Kosaka et al. 1997; Pinching and Powell 1971b), many of which are GABAergic (Gall et al. 1987; Kosaka et al. 1985; Mugnaini et al. 1984; Ribak et al. 1977a). Intraglomerular inhibitory circuits are thought to play an important role in regulating glomerular activity, but physiological evidence in support of this view is very limited. The reciprocal synapses of GABAergic JG cells with M/T cells (Hinds 1970; Pinching and Powell 1971b; Ribak et al. 1977a) suggest that activity in the glomerular dendritic tufts of M/T cells, evoked by sensory input, may be attenuated via both feed-forward and feedback inhibitory mechanisms. In addition, recent evidence suggests that olfactory nerve input to the glomeruli may be presynaptically inhibited via GABAB receptors. Thus in the rat olfactory bulb, baclofen, a GABAB receptor agonist, blocks ON-evoked spiking of mitral cells (Nickell et al. 1994) and optically recorded synaptic responses in the glomerular layer (GL), presumably generated by JG cells (Keller et al. 1998). Although it could not be ruled out that these effects were produced by a postsynaptic mechanism, a recent electron microscopy (EM) study demonstrating the presence of GABAB receptors on ON terminals (Bonino et al. 1999) supports the contention that the effects of baclofen were mediated, at least in part, presynaptically (Keller et al. 1998; Nickell et al. 1994). The purpose of the present study was to investigate the role that GABAB receptors on ON terminals play in the function of the glomeruli.


    METHODS
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INTRODUCTION
METHODS
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Slices of the main olfactory bulb (OB) were prepared from Wistar rats, 17-22 days old. Details of the slice preparation have been described previously (Aroniadou-Anderjaska et al. 1997). Briefly, the rats were anesthetized with chloral hydrate (400 mg/kg body wt) followed by whole-body immersion in ice-cold water. The brain with the two bulbs was removed and glued to the stage of a Vibroslicer. Some of the experiments required the lateral olfactory tract (LOT) to be preserved in the slices (Fig. 1A). To accomplish this, the brain was positioned so that the bulbs were in approximately the same horizontal plane with the most ventral part of the forebrain. Slices, 450-500 µm thick, were cut and transferred to an interface chamber, maintained at 33°C, for field potential and intracellular recordings, while whole cell recordings were performed in submerged slices at room temperature. The slices were perfused with artificial cerebrospinal fluid (in mM: 124 NaCl, 26 NaHCO3, 1.2 NaH2PO4, 3 KCl, 1.3 MgSO4, 2.5 CaCl2, and 10 glucose) at a rate of 1 ml/min in the interface chamber, and 3 ml/min in the submerged conditions. In some experiments, medium with nominally zero concentration of Mg2+ was used; this medium did not include MgSO4.



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Fig. 1. A: a photograph of an olfactory bulb (OB) slice preserving the lateral olfactory tract (LOT). Stimulation was applied to the olfactory nerve layer (ONL), or to both ONL and LOT, alternately. CF, centrifugal fibers. B: schematic diagram of the basic OB circuitry. ON, olfactory nerve; GL, glomerular layer; EPL, external plexiform layer; IPL, internal plexiform layer; GCL, granule cell layer; MC, mitral cell; GC, granule cell; TC, tufted cell; JG, juxtaglomerular cell.

Experiments were initiated 1-2 h after the slices were placed in the chamber. Field potentials were recorded in the GL and, in some experiments, in the granule cell layer (GCL; see basic OB circuitry in Fig. 1), using glass pipettes (0.5-2 MOmega ) filled with 2 N NaCl. They were filtered (3 kHz low-pass), and digitized on-line at 15 kHz. Extracellular stimulation was applied to the ON or the LOT (Fig. 1A) using a dipolar stainless steel electrode (50 µm diam). Stimulus pulses were 100 µs duration and were applied at 0.05 Hz. Unless indicated otherwise in the results, stimulus intensity was adjusted to evoke a field potential of 1.5-2.5 mV in the GL by ON stimulation (10-60 µA), or in the GCL by LOT stimulation (80-150 µA). This amplitude range was 40-70% of the maximum peak amplitude of ON-evoked GL field potentials, and 60-80% of the maximum amplitude of LOT-evoked GCL field potentials.

One set of experiments required recordings of laminar field potential profiles, which were analyzed with the current source density (CSD) method as described previously (Aroniadou-Anderjaska et al. 1999a). Briefly, the approximation formula for one-dimensional CSD (Freeman and Nicholson 1975) was used. The validity of one-dimensional CSD in the main OB, along the axis perpendicular to the laminae, has been shown (Aroniadou-Anderjaska et al. 1999a). Spatial resolution and differentiation grid were 100 and 200 µm, respectively. Conductivity gradients across laminae have no significant influence on the general features of the laminar CSD distribution (Martinez 1982), and therefore they were considered negligible.

Conventional methods were used for intracellular recordings from mitral cells, using the Axoclamp-2A amplifier (Axon Instruments) in the bridge mode. Glass pipettes (50-90 MOmega ) were filled with potassium acetate (4 M). For whole cell recordings from juxtaglomerular cells, patch pipettes were filled with (in mM) 135 KCl, 10 HEPES, 2 Mg-ATP, 0.2 Na-GTP, and 0.5 EGTA; pH and osmolarity were adjusted to 7.3 and 280 mOsm, respectively. Electrical signals were recorded using an Axopatch-200B amplifier (Axon Instruments) with the low-pass filter set at 5 kHz. Juxtaglomerular cells were visualized with an Olympus BX50WI upright microscope equipped with a ×60 water immersion lens and DIC optics. The recorded cells were probably the periglomerular type (GABAergic juxtaglomerular cells) because they had small soma size and high-input resistance. All data acquisition and analysis, including the CSD analysis, were performed with the pClamp software (Axon Instruments). In the results, group data are presented as means ± SE.

The following drugs were used: R(+) baclofen hydrochloride (referred to as baclofen), a selective GABAB receptor agonist (Research Biochemicals International); CGP-35348 (Olpe et al. 1990) or CGP-55845A (Frostl et al. 1992), specific, competitive GABAB receptor antagonists (gift from NOVARTIS Pharma); 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a kainate/alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist (Research Biochemicals International); D-2-amino-5-phosphonovalerate (APV), an N-methyl-D-aspartate (NMDA) receptor antagonist (Research Biochemicals International); and bicuculline methchloride, a GABAA receptor antagonist (Research Biochemicals International). To prepare stock solutions, all drugs were dissolved in dH2O, except for CNQX, which was dissolved in DMSO (final concentration of DMSO in the slice medium was 0.01%, vol/vol). All drugs were delivered by bath application.


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INTRODUCTION
METHODS
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Pharmacological activation of GABAB receptors

EFFECTS OF BACLOFEN ON ON-EVOKED RESPONSES OF M/T CELLS. In the first series of experiments, our aim was to obtain solid physiological evidence for the presence of functional GABAB receptors on ON terminals. First, we examined the effects of GABAB receptor activation by baclofen on the ON-evoked responses of M/T cells. Single pulses to the ON evoke a two-component field excitatory postsynaptic potential (EPSP) in the GL (Fig. 2A). This field potential reflects predominantly synaptic currents in the apical dendrites of M/T cells and is mediated by glutamate receptors (Aroniadou-Anderjaska et al. 1997, 1999a). The fast component (N1) is mediated by kainate/AMPA receptors, whereas most of the slow component (N2) depends on NMDA receptor activation. Bath application of 5 µM baclofen reduced both the N1 and N2 components of the field EPSP (Fig. 2B). The peak amplitude of N1 was reduced by 79.6 ± 5.6% (mean ± SE, n = 10). These effects were reversed after washing-out baclofen.



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Fig. 2. Baclofen suppresses ON-evoked field excitatory postsynaptic potentials (EPSPs) of mitral/tufted (M/T) cells. A: the field EPSP generated by the glomerular dendritic tufts of M/T cells in response to ON stimulation consists of a fast component (N1), which is blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM), and a slow component (N2), most of which is blocked by alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (APV; 50 µM). B: baclofen (5 µM) reversibly suppressed the ON-evoked glomerular field EPSPs. C: in slices where the GL was surgically isolated from the deeper layers, baclofen (5 µM) also reduced the ON-evoked field EPSP. Traces are averages of 5-10 sweeps.

In slices where the GL was surgically isolated from the deeper OB layers (see Aroniadou-Anderjaska et al. 1997), baclofen also reduced the glomerular field EPSP (77.7 ± 2.8% reduction in N1 peak amplitude, n = 3, Fig. 2C). Thus baclofen acted primarily, or exclusively, in the GL.

Baclofen also suppressed completely the EPSPs recorded intracellularly from mitral cells (Fig. 3). This was associated with a small change in the resting membrane potential, from -63.6 ± 2.6 mV, in control conditions, to -65.1 ± 2.5 mV after addition of baclofen (n = 7, P < 0.05, paired t-test). Input resistance was not affected significantly (P > 0.05). The suppression of synaptic transmission by baclofen was very effective, as it was not overcome substantially by increasing the intensity of ON stimulation (Fig. 3). All of the effects of baclofen were largely reversed by the specific GABAB receptor antagonists CGP-35348 (0.5-1 mM; Fig. 3) or CGP-55845A (10 µM).



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Fig. 3. ON-evoked responses recorded intracellularly from mitral cells are completely suppressed by baclofen. The cell shown (resting membrane potential -64 mV) generated a prolonged EPSP in response to 20 µA stimulation. The GL field potential (FP), recorded simultaneously, is shown above each trace of the intracellular response. When the stimulus intensity was increased to 40 µA, the EPSP was preceded by a fast prepotential, which gave rise to an action potential following further increase of stimulation intensity (70 µA). Baclofen (5 µM) significantly reduced or blocked the field and cell responses even at the higher stimulus intensities. The effects of baclofen were largely reversed by CGP-35348 (500 µM). Traces are averages of 5 sweeps.

SITE OF BACLOFEN ACTION. The dramatic effect of baclofen on ON-evoked responses of M/T cells suggested that baclofen may act on the ON terminals and inhibit glutamate release. The apparently minor postsynaptic effects on mitral cells were consistent with this possibility; however, if GABAB receptors on mitral cells were present exclusively on the apical dendritic tufts, postsynaptic effects of baclofen may be difficult to detect with somatic recordings.

To determine whether baclofen acted presynaptically, first we examined how baclofen affects responses to paired-pulse stimulation of the ON. When two identical stimuli are delivered at various interstimulus intervals (ISI), the first pulse may produce depression (paired-pulse depression, PPD) or facilitation (PPF) of the response to the second pulse. An important factor determining whether PPD or PPF is produced is the probability of transmitter release in response to the first (conditioning) pulse (Debanne et al. 1996; Manabe et al. 1993; Thomson et al. 1993). The present paired-pulse experiments were carried out in the presence of APV (50-100 µM) to suppress the long-lasting N2 component of the field potential, and thus allow a more accurate measurement of changes in the kainate/AMPA component. Prior to addition of baclofen, paired-pulse stimulation of the ON produced depression of the second (test) response at ISIs starting from 10 ms. The longest ISI at which PPD was still present ranged from 400 to 700 ms (542.8 ± 43.5 ms, n = 8). Baclofen (0.5-1 µM) reduced disproportionately the conditioning versus test responses, resulting in the reversal of PPD to PPF (n = 4, Fig. 4, A and B). The effects of baclofen were significant at all ISIs tested, i.e., 100, 200, and 300 ms (P < 0.05, paired t-test). Increasing the stimulus intensity to bring the conditioning response magnitude closer to the control, still produced PPF (Fig. 4Ac). The effects of baclofen were reversible (Fig. 4Ad). These results suggest that baclofen reduces the probability of glutamate release from ON terminals. However, postsynaptic effects of baclofen could also contribute to these results.



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Fig. 4. Baclofen reverses paired-pulse depression (PPD) of the ON-evoked field EPSP to paired-pulse facilitation (PPF). A: in a-d, 3 traces are superimposed, and each trace is an average of 5 sweeps. The slice medium includes APV (50 µM). PPD of the field EPSP (a), reversed to PPF (b) in the presence of 1 µM baclofen (interstimulus intervals 100, 200, and 300 ms). PPF persisted when the stimulus intensity was increased (c) to produce a response of an amplitude comparable to the control. PPF was no longer present after the response had recovered partially, following wash of baclofen (d). B: group data from 4 slices showing the amplitude of the test response relative to the conditioning response, before and after baclofen. Error bars are SE. * Statistically significant (P < 0.05).

To assess the relative contribution of potential postsynaptic effects of baclofen to the suppression of the ON to M/T cell transmission, we examined the effects of baclofen on synaptic responses in the apical dendrites of M/T cells, evoked via two independent pathways. Thus we stimulated alternately the ON and the LOT and analyzed the evoked glomerular field potentials by the CSD method. Antidromic activation of M/T cells, by LOT stimulation, evokes a sink (inward membrane currents) in the GL, which is mediated primarily by NMDA receptors (Aroniadou-Anderjaska et al. 1999a). The apical dendrites of mitral cells have the ability to back-propagate action potentials (Chen et al. 1997), which trigger glutamate release from the distal dendritic tufts; glutamate acts back on the parent and/or neighboring M/T cell dendrites and generates this glomerular sink (Aroniadou-Anderjaska et al. 1999b). To record this NMDA sink we performed experiments in the presence of CNQX and zero Mg2+. Baclofen (up to 50 µM) completely suppressed the ON-evoked glomerular sink but had virtually no effect on the current sink evoked by LOT stimulation (n = 5, Fig. 5B). Thus baclofen blocks ON-evoked synaptic responses of M/T cell dendrites, without causing any significant change in the response of the same dendrites to another glutamatergic input. These results indicate that baclofen can block ON input to M/T cells, by a presynaptic action alone.



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Fig. 5. Baclofen blocks ON-evoked synaptic responses of M/T cell dendrites, without affecting the response of the same dendrites to another glutamatergic input. A and B are from the same experiment. A: field potential in the GL evoked by ON or LOT stimulation in Mg2+-free medium and in the presence of CNQX (10 µM). Baclofen (50 µM) suppressed the ON-evoked field potential, but did not affect the LOT-evoked field potential. The effect of baclofen was partly reversed by CGP-55845A (10 µM). Both ON- and LOT-evoked responses were blocked reversibly by APV (50 µM). Traces are averages of 5 sweeps. B: corresponding CSD distribution of N-methyl-D-aspartate (NMDA) receptor-mediated currents in the GL, evoked by ON or LOT stimulation. Baclofen (50 µM) completely suppressed the ON-evoked currents but did not affect significantly the currents evoked by antidromic stimulation of M/T cells. Number of mm to the left show distances from the surface of the slice.

A final test to determine whether baclofen can completely suppress synaptic transmission from the ON, without any postsynaptic effects, was to investigate the effects of baclofen on ON-evoked synaptic currents of JG cells. Any potential postsynaptic effects of baclofen on JG cells should be measurable because, due to their small size, JG cells can be voltage clamped effectively. AMPA/kainate excitatory postsynaptic currents (EPSCs) were isolated by adding APV (50 µM) and bicuculline (10 µM) to the medium. Addition of baclofen (2 µM) completely suppressed the ON-evoked EPSCs (Fig. 6A, n = 4); this effect was not associated with any measurable change in input resistance (2.3 ± 0.2 GOmega , n = 4) or holding current (-70 mV). Thus baclofen can completely suppress synaptic transmission from the ON to JG cells by a presynaptic action alone. Lower concentrations of baclofen (0.5-1 µM) that reduced but did not completely suppress the ON-evoked synaptic currents, reduced the depression of these currents during repetitive (5 Hz) stimulation (n = 4, Fig. 6B). This is consistent with the interpretation that baclofen reduces the probability of glutamate release from the ON terminals.



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Fig. 6. Baclofen blocks responses of juxtaglomerular cells to ON stimulation. A: ON-evoked synaptic current from a voltage-clamped (Vh = -70 mV) juxtaglomerular cell. The slice medium contains APV (50 µM) and bicuculline (10 µM). Bath-applied baclofen abolished the synaptic current but had no effect on input resistance or holding current. B: depression of the synaptic current during repetitive ON stimulation (5 Hz) was reduced by low concentrations of baclofen.

Activation of GABAB receptors by endogenous GABA

To investigate whether GABAB receptors on ON terminals play a role in the function of the OB, we next examined whether these receptors are activated by endogenous GABA. The origin of endogenous GABA in the glomeruli is a population of JG cells, the GABAergic periglomerular (PG) cells (Gall et al. 1987; Kosaka et al. 1985; Mugnaini et al. 1984; Ribak et al. 1977a). PG cells receive excitatory input from the ON (present data and Bardoni et al. 1996; Heyward et al. 1997; Keller et al. 1998) and from the dendrites of M/T cells (Bardoni et al. 1996). Ultrastructural studies have revealed symmetrical synapses from PG cells to the dendrites of M/T cells and other PG cells, but no evidence has been found for synapses from PG cells onto ON terminals (Hinds 1970; Pinching and Powell 1971b; Ribak et al. 1977b).

To determine whether, despite the lack of anatomical synapses, GABA released from PG cells gains access to GABAB receptors on ON terminals, we investigated the effects of the GABAB antagonists, CGP-35348 (0.5-1 mM) and CGP-55845A (10 µM), on glomerular field EPSPs evoked by paired-pulse ON stimulation. The GABAB antagonists increased both the conditioning and the test responses. The effect on the test responses was significantly greater, resulting in reduction of PPD, or reversal of PPD to PPF. An example is shown in Fig. 7A, and group data from 10 slices where CGP-35348 was used are shown in Fig. 7B. The reduction in PPD by CGP-35348 was significant at interstimulus intervals from 50 to 400 ms (P < 0.02, paired t-test). These results suggest that GABA released from PG cells in response to the conditioning pulse of the ON activates GABAB receptors on ON terminals and thus reduces the M/T cell response to the test pulse.



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Fig. 7. GABAB receptors on ON terminals are activated both tonically and in response to ON stimulation: GABAB antagonists increase both conditioning and test responses of M/T cells to paired-pulse stimulation of the ON, while reducing PPD of test responses. In all experiments, APV is included in the medium. A: responses to paired-pulse stimulation of the ON (interstimulus intervals 50, 100, 200, 300, and 400 ms) before and after application of CGP-35348 (1 mM). The GABAB antagonist increased the amplitude of the conditioning response and reduced or blocked PPD. Each trace is an average of 5 sweeps, and 5 traces are superimposed. B: group data (n = 10) of the effects of CGP-35348 (500 µM to 1 mM) on PPD of the ON-evoked glomerular field EPSP. The reduction of PPD was statistically significant (*P < 0.05) at interstimulus intervals from 50 to 400 ms. C: time course of the effects of CGP-55845A (10 µM) on conditioning and test responses (interstimulus interval 100 ms). Group data from 6 slices. Error bars in B and C are SE.

The responses to the conditioning pulses, and responses to single-pulse ON stimulation, were also increased by the GABAB antagonists (Fig. 7, A and C). The peak amplitude of N1 increased by 30%, from 2.0 ± 0.13 mV, in control medium, to 2.6 ± 0.18 mV, after application of either GABAB antagonist (n = 19, P < 0.0004, paired t-test). Figure 7C shows the time course of the effect of CGP-55845A on conditioning and test responses evoked at an interstimulus interval of 100 ms (group data from 6 slices). The latency to peak amplitude of N1 ranges from 9 to 12 ms, whereas ON-evoked activation of GABAB receptors on ON terminals takes effect after 30 ms post-ON stimulation (Fig. 7B). Therefore the increase of the conditioning responses by the GABAB antagonists cannot be due to the same mechanism as the test responses, i.e., blockade of polysynaptically mediated activation of GABAB receptors; rather, this increase suggests that the GABAB antagonists blocked tonic activation of GABAB receptors. Thus taken together, these results show that GABAB receptors on ON terminals are tonically activated by ambient concentrations of extracellular GABA, and are further activated by GABA release from PG cells in response to a single stimulus pulse applied to the ON.

M/T to granule cell transmission is not affected by baclofen or CGP-35348

The lateral dendrites of M/T cells release glutamate onto the dendrites of granule cells producing a negative field EPSP in the external plexiform layer (EPL) and a corresponding positive field potential in the GCL (Rall and Shepherd 1968). The EPL field EPSP evoked by ON stimulation influences to some extent the fast, kainate/AMPA component of the GL field EPSP, increasing its amplitude and broadening its time course (Aroniadou-Anderjaska et al. 1997, 1999a). Thus if there were GABAB receptors on the lateral dendrites of M/T cells, then the effects of the GABAB antagonists on the GL field EPSP (Fig. 7) could, in part, be due to a change in the EPL field EPSP. To investigate this possibility we delivered stimulus pulses alternately to the LOT and to the ON, while recording granule cell field responses in the GCL. Baclofen (up to 50 µM) had no effect on the LOT-evoked field potential, although it completely suppressed the ON-evoked GCL field potential (n = 3, Fig. 8A). Similarly, CGP-35348 had no effect on PPD of the LOT-evoked, GCL field potential, although it reduced PPD of the ON-evoked GCL field potential (ISIs 50, 100, 150, and 200 ms; n = 4, Fig. 8B). Thus baclofen and CGP-35348 do not affect transmission from M/T to granule cells; the effects of these drugs on the ON-evoked GCL field potential were due to activation of GABAB receptors on the ON terminals. These results allow us to conclude that the effects of the GABAB antagonists on the ON-evoked, GL field EPSP (Fig. 7) are solely due to an action in the GL.



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Fig. 8. Baclofen or CGP-35348 do not affect transmission from M/T cells to granule cells. A and B are 2 different slices. A: field potential recorded in the GCL in response to ON or LOT stimulation. When baclofen (50 µM) blocked the ON-evoked response, the response evoked by LOT stimulation was unaffected. B: CGP-35348 (1 mM) reduced the depression of the GCL field potential during paired-pulse ON stimulation, but did not affect depression of the GCL field potential in response to paired-pulse stimulation of the LOT. Each trace is an average of 5 sweeps. In the paired-pulse traces, 4 traces are superimposed.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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In the present study, it was found that 1) baclofen suppresses synaptic transmission from the ON to M/T cells and JG cells by reducing glutamate release from the ON axon terminals, and 2) GABAB receptors on ON terminals are activated by endogenous GABA, both tonically and in response to ON stimulation.

Evidence for functional GABAB receptors on ON terminals

Functional GABAB receptors are heteromeric complexes of the GABABR1 subunit, which exists in two splice variants, R1a and R1b, and the GABABR2 subunit (Kaupmann et al. 1998; Kuner et al. 1999). Combination of the GABABR1 with the GABABR2 appears to be essential for transport to the membrane and full functionality of the receptor (Couve et al. 1998; Kaupmann et al. 1998; White et al. 1998). In the rat OB, the glomeruli have the highest concentration of GABAB receptors as determined by radioligand autoradiographic studies (Bowery et al. 1987; Chu et al. 1990) and by immunohistochemical localization of the GABABR1a/b subunit (Margeta-Mitrovic et al. 1999). Combined electron microscopy and immunohistochemical staining for GABABR1 revealed that the dense labeling of the glomeruli is due to the presence of GABAB receptors on ON terminals and in the somata of periglomerular cells (Bonino et al. 1999). In addition, ORNs in the nasal epithelium label uniformly with antiserum to GABABR1 (Aroniadou-Anderjaska et al. 1999c), suggesting that all ORNs express GABAB receptors, which may be transported to their axon terminals. Although it remains to be determined whether ORNs also express the GABABR2 subunit, the functionality of GABAB receptors on the axon terminals of the ORNs was suggested by previous studies (Keller et al. 1998; Nickell et al. 1994), and is clearly demonstrated by the present data. Thus it has been shown that baclofen blocks ON-evoked mitral cell spiking (Nickell et al. 1994) and optical signals generated by JG cells (Keller et al. 1998). It was suggested that the dramatic effects of baclofen may imply, at least in part, a presynaptic action (Keller et al. 1998; Nickell et al. 1994). The present study provides solid support of this view, and further shows that baclofen blocks ON input to the OB by a presynaptic action alone. Thus ON-evoked synaptic responses of both glomerular targets of the ON, the M/T cells, and JG cells, were completely suppressed by baclofen, with no concomitant postsynaptic changes in input resistance or holding current. The small hyperpolarization of mitral cells following application of baclofen could be due to blockade of tonic activation of these cells by glutamate release from ON terminals. Additional evidence that baclofen can block ON transmission by acting exclusively on ON terminals comes from the experiment where an NMDA current sink was evoked in the apical dendrites of M/T cells via two independent pathways (ON and LOT stimulation), and only the ON-evoked sink was suppressed by baclofen (Fig. 5).

The mechanism by which baclofen suppressed transmission from the ON appeared to be a reduction in the probability of glutamate release. In a paired-pulse paradigm, the higher the probability of transmitter release during the conditioning pulse, the smaller the response to the test pulse will be (larger PPD) (Debanne et al. 1996; Manabe et al. 1993; Thomson et al. 1993). Baclofen reversed PPD of the ON-evoked responses of M/T cells to PPF (Fig. 4) and reduced depression of the JG cell EPSCs during repetitive ON stimulation (Fig. 6B), effects that are consistent with a decreased probability of glutamate release from ON terminals. As shown in other CNS areas, this could be effected by a reduction of Ca2+ influx into the terminals (Dolphin 1995; Lambert et al. 1991; Mott and Lewis 1994; Takahashi et al. 1998; Wu and Saggau 1995, 1997), and/or a direct effect on the release process downstream to Ca2+ influx (Dittman and Regehr 1996; Jarolimek and Misgeld 1997; Scanziani et al. 1992). In turtle olfactory nerve terminals, baclofen was recently shown to reduce Ca2+ influx (Wachowiak and Cohen 1999).

The present data do not provide evidence for direct effects of baclofen on JG cells or mitral cells, since there were small or no postsynaptic changes when ON-evoked responses of M/T cells and JG cells were completely suppressed by baclofen. In addition, the lack of any effects of baclofen or CGP-35348 on synaptic transmission from M/T cells to granule cells (Fig. 8) suggests that glutamate release from the lateral dendrites of M/T cells is not modulated by GABAB receptors. Although both JG cells and mitral cells stain with antiserum to GABABR1a/b (Bonino et al. 1999; Margeta-Mitrovic et al. 1999), there are no cells in the rat OB expressing significant levels of the GABABR2 subunit, as determined by in situ hybridization (Kaupmann et al. 1998). Thus JG cells and mitral cells may not have functional GABAB receptors, and the GABAB receptor subunits that these cells express may not be transported to synaptic sites. Consistent with the latter, there is minimal staining for GABABR1a/b in the EPL (Margeta-Mitrovic et al. 1999), where the lateral dendrites of mitral cells receive the vast majority of GABAergic synaptic inputs. In addition, Bonino et al. (1999) did not detect GABABR1 immunoreactivity on dendrites of either JG cells or M/T cells in the glomerular layer. Thus it is possible that in the rat OB only the GABAB receptors on ON terminals are fully functional. This, however, requires further investigation.

Tonic and synaptically evoked activation of GABAB receptors by endogenous GABA

In the present study, GABAB antagonists increased responses of M/T cells evoked by single-pulse ON stimulation, suggesting that ambient levels of extracellular GABA tonically activate GABAB receptors on ON terminals. In addition, GABAB antagonists reduced the depression of test responses of M/T cells to paired-pulse stimulation of the ON, indicating that a single stimulus pulse to the ON (the conditioning pulse) elevates extracellular GABA above tonic levels, to further activate GABAB receptors on ON terminals.

The source of endogenous GABA in the glomeruli of the OB is a subpopulation of JG cells, the PG cells (Gall et al. 1987; Kosaka et al. 1985; Mugnaini et al. 1984; Ribak et al. 1977a). PG cells are activated by ON stimulation (present data and Bardoni et al. 1996; Heyward et al. 1997; Keller et al. 1998); they form dendrodendritic synapses with M/T cells and other JG cells, but they do not form synapses with ON terminals (Hinds 1970; Pinching and Powell 1971b; Ribak et al. 1977a). In most CNS areas, GABAergic neurons do not form synapses with excitatory terminals. However, GABAB heteroreceptors can be activated by low concentrations of extrasynaptic GABA, due to their high affinity for GABA (Sodickson and Bean 1996; Yoon and Rothman 1991). In the hippocampus (Isaacson et al. 1993) and cerebellum (Dittman and Regehr 1997), extrasynaptic GABA reaches sufficiently high levels to activate GABAB heteroreceptors only following high-frequency stimulation of GABAergic neurons. However, tonic activation of GABAB heteroreceptors has been reported in other CNS areas (Emri et al. 1996; Kombian et al. 1996).

The more effective activation of GABAB heteroreceptors in the OB, compared with the hippocampus or cerebellum, may be due to differences between these structures in the proximity of GABA release sites to GABAB heteroreceptors, and/or the proximity of GABA uptake systems to GABA release sites and GABAB heteroreceptors. GABA transporters are located on GABAergic neurons and glia cells (Hertz 1979). In the OB, the dendrodenritic synapses between PG and M/T cells are partially enclosed by glia processes (Chao et al. 1997; Kasowski et al. 1999). Thus GABA released from PG cells at dendrodendritic synapses with M/T cells would probably have to escape the glia barrier to reach GABAB receptors on ON terminals. Although this cannot be excluded, another possibility is that GABA is released from nonsynaptic dendritic sites of PG neurons that are close to the ON terminals. Consistent with this, synaptic vesicles are present in some PG cell dendrites apposed to olfactory nerve terminals that are immunopositive for GABAB receptors (Bonino et al. 1999). In addition, there are only few glial processes close to axodendritic synapses formed by the ON (Chao et al. 1997; Kasowski et al. 1999), which may facilitate the build-up of extrasynaptic GABA. The glomerular inhibitory interactions suggested by the present and previous findings are summarized schematically in Fig. 9.



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Fig. 9. Schematic representation of GABAB receptor-mediated synaptic interactions in the glomeruli of the OB, as suggested by the present and previous findings. Glutamate released from ON terminals depolarizes the postsynaptic dendrites of JG and M/T cells. Ensuing Ca2+ influx into the dendrites triggers GABA release, probably from vesicles that are scattered on dendrites of JG cells, and are often in apposition to GABAB receptors on ON terminals (Bonino et al. 1999). GABAB receptors are located on both synaptic and extrasynaptic sites on ON terminals (Bonino et al. 1999). The present study suggests that GABA release from JG cell dendrites, and activation of presynaptic GABAB heteroreceptors occur both tonically and in response to ON stimulation. A single ON impulse evokes sufficient GABA release from JG cells to activate the presynaptic GABAB heteroreceptors above the tonic activation levels. Access of GABA to GABAB receptors may be facilitated by the absence of glia from the ON synaptic compartment (Chao et al. 1997; Kasowski et al. 1999). As shown in the schema, GABA is also released in reciprocal synapses between JG cell and M/T cell dendrites, but may not have access to GABAB receptors on ON terminals because 1) these reciprocal dendrodendritic synapses are segregated from the ON synapses (Kasowski et al. 1999; Kosaka et al. 1997), and 2) these synapses are partially enclosed by glia cells (Chao et al. 1997; Kasowski et al. 1999).

Relation to the function of the OB

Olfactory information is thought to be encoded by specific patterns of glomerular activity (Buck 1996; Friedrich and Korsching 1997; Guthrie et al. 1993; Johnson and Leon 1996; Johnson et al. 1998; Laurent 1996; Shepherd 1994; Stewart et al. 1979). The present findings suggest that GABAB receptor-mediated presynaptic inhibition of ON input to the glomeruli may play an important role in determining both spatial and temporal components of glomerular activation. Tonic activation of GABAB receptors on ON terminals may serve to filter out weak ("noise") signals. This may sharpen the spatial pattern of active glomeruli and facilitate detection of the predominant odor. Strong signals are likely to activate more PG cells and evoke stronger inhibition of subsequent ON inputs. This is likely to affect the temporal patterns of glomerular activation in response to repetitive sniffs. During repetitive sniffing, presynaptic GABAB inhibition may adjust the level of glomerular excitation, as a function of sniffing frequency. Our PPD data predict that GABAB presynaptic inhibition will be most effective in inhibiting signals arriving at 100- to 200-ms intervals (5-10 Hz), the dominant frequency during exploratory sniffing (Komisaruk 1970; Macrides and Chorover 1972; Welker 1964). However, a novel odor occurring within such a sniffing bout would activate different glomeruli and, initially, would not be inhibited to the same degree. Thus frequency-dependent GABAB receptor-mediated inhibition of input to the glomeruli may enhance detection of novel stimuli.

The extent to which presynaptic GABAB inhibition influences glomerular excitation may vary dynamically, as centrifugal inputs to the glomeruli may alter PG cell excitability (Pinching and Powell 1971c; Shipley et al. 1996). In addition, there is evidence for anatomical connections between glomeruli (Cajal 1911; Pinching and Powell 1971a). If these connections affect GABA release, then GABAB receptors on ON terminals could mediate interglomerular influences on glomerular activity patterns. Finally, GABAB receptors on ON terminals could play a role in induction of long-term potentiation at the first synapse in the olfactory system (Ennis et al. 1998).


    ACKNOWLEDGMENTS

We thank Dr. Nevin A. Lambert for valuable discussions. We also thank Drs. Asaf Keller, Scott M. Thompson, and Michael Meredith for critical review of the manuscript. We are grateful to Dr. Wolfgang Froestl and Novartis Pharma Inc. for the generous gift of the GABAB antagonists.

This work was supported by National Institutes of Health Grants DC-00347, DC-03195, DC-02173, and NS-36940. F.-M. Zhou was supported in part by a Young Investigator Award from the National Alliance for Research in Schizophrenia and Depression.


    FOOTNOTES

Present address and address for reprint requests: V. Aroniadou-Anderjaska, Dept. of Psychiatry, Uniform Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814 (E-mail: vanderjaska{at}usuhs.mil).

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 February 2000; accepted in final form 24 May 2000.


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ABSTRACT
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