Dendrodendritic Recurrent Excitation in Mitral Cells of the Rat Olfactory Bulb

Vassiliki Aroniadou-Anderjaska, Matthew Ennis, and Michael T. Shipley

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Aroniadou-Anderjaska, Vassiliki, Matthew Ennis, and Michael T. Shipley. Dendrodendritic Recurrent Excitation in Mitral Cells of the Rat Olfactory Bulb. J. Neurophysiol. 82: 489-494, 1999. Most neuronal interactions within the olfactory bulb network are mediated by dendrodendritic synapses. Dendritic transmitter release potentially could affect the parent dendrite as well as local neuronal elements that have receptors for the released transmitter. Here we report that under conditions that facilitate N-methyl-D-aspartate (NMDA) receptor activity (reduced GABAA inhibition and extracellular Mg2+), a single action potential evoked by brief intracellular current pulses in mitral cells is followed by a prolonged depolarization, which is blocked by an NMDA receptor antagonist. This depolarization also is evoked by a presumed calcium spike in the presence of tetrodotoxin. A similar NMDA-receptor-dependent prolonged depolarization is elicited by stimulation of the lateral olfactory tract at current intensities subthreshold for antidromic activation of the recorded neuron. These observations suggest that glutamate released from the dendrites of mitral cells excites the same and neighboring mitral cell dendrites. Further evidence suggests that both the apical and lateral dendrites of mitral cells participate in this recurrent excitation. These dendrodendritic interactions may play a role in the prolonged, NMDA-receptor-dependent depolarization of mitral/tufted cells evoked by olfactory nerve stimulation.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Olfactory sensory input enters the brain at the glomeruli of the main olfactory bulb (MOB), where the axon terminals of olfactory receptor neurons synapse with the apical dendrites of mitral and tufted (M/T) cells, the output cells of the MOB, and with juxtaglomerular (JG) interneurons (see Fig. 1; for a review Shipley et al. 1996). Via reciprocal dendrodendritic synapses (Pinching and Powell 1971; Shepherd 1972; White 1972), M/T cells excite JG cells in the glomerular layer (GL) and granule cells in the external plexiform layer (EPL) and receive feedback inhibition from these interneurons. There are no anatomically identified excitatory synapses onto mitral cell dendrites apart from the synapses with olfactory nerve (ON) terminals (Kosaka et al. 1997; Pinching and Powell 1971; White 1972). However, there is physiological evidence, in the turtle olfactory bulb, that these dendrites are excited by an amino acid they release (Nicoll and Jahr 1982). It now is known that the dendrites of M/T cells release glutamate at their synapses with bulbar interneurons (Aroniadou-Anderjaska et al. 1999; Bardoni et al. 1996; Isaacson and Strowbridge 1998; Schoppa et al. 1998; Wellis and Kauer 1994). The present study investigated whether glutamate released from mitral cell dendrites also excites the parent and neighboring mitral cells in the mammalian MOB.



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Fig. 1. A single spike evoked in mitral cells by depolarizing current pulses triggers a N-methyl-D-aspartate (NMDA)-receptor-dependent excitatory postsynaptic potential (EPSP). A: schematic diagram of the basic main olfactory bulb (MOB) circuitry. B: photograph of the slice preserving the lateral olfactory tract (LOT). ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; IPL, internal plexiform layer; GCL, granule cell layer; MC, mitral cell; GC, granule cell; TC, tufted cell; JGC, juxtaglomerular cell; CFF, centrifugal fibers. C and D show 2 different cells. Recordings are in Mg2+-free medium, bicuculline methylchloride (BMCl; 10 µM) and 6-cyano-7-nitroqunioxaline-2,3-dione (CNQX; 10 µM). C, top: a single spike evoked by a 0.8 nA, 5 ms depolarizing pulse was followed by a prolonged depolarization. Occasional spikes with slightly varying latency were triggered close to the peak of this depolarization (their small size is due to averaging). When the cell was hyperpolarized so that the same current pulse could not bring the cell to spike threshold, the postpulse depolarization also was blocked (inset). Bottom: synaptic response of the same cell to ON stimulation, and the simultaneously recorded glomerular field EPSP. Hyperpolarization blocked the ON-evoked spike but not the EPSP (inset). Bath-applied D-2-amino-5-phosphonovalerate (APV; 50 µM) reversibly blocked both the postpulse depolarization and the ON-evoked responses. Traces are averages of 5-10 sweeps. D: a train of action potentials evoked by a 0.5 nA, 50 ms current pulse was followed by the prolonged depolarization. Action potentials and the postpulse depolarization were blocked by 1 µM TTX in the bath. After addition of tetraethylammonium (TEA; 5 mM) to the medium, a higher intensity (1 nA) current pulse evoked a prolonged, presumably Ca++ spike and caused the reappearance of a postpulse depolarization. Traces are single sweeps.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Wistar rats, 15- to 22-days old were used. The MOB slice preparation has been described previously (Aroniadou-Anderjaska et al. 1997). Briefly, the rats were anesthetized with chloral hydrate (400 mg/kg body wt). The brain with the two bulbs was removed and glued to the stage of a Vibroslicer. To preserve the lateral olfactory tract (LOT) in the slices, the brain was positioned so that the bulbs were in approximately the same horizontal plane with the most basal part of the brain. Slices, 450- to 500-µm thick, were cut and transferred to an interface chamber, maintained at 33°C. The slices were perfused with artificial cerebrospinal fluid [which contained (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. Zero Mg2+ medium did not include MgSO4.

Conventional methods were used for intracellular recordings in the bridge mode. Glass pipettes (50-90 MOmega ) were filled with potassium acetate (4 M). For field potential recordings, pipettes (0.5-2 MOmega ) were filled with 2 N NaCl. Extracellular stimulation (10-100 µA, 100-µs duration) was applied with a dipolar stainless steel electrode.

Current source density (CSD) analysis of laminar field potential profiles was performed as previously described (Aroniadou-Anderjaska et al. 1999). Briefly, the approximation formula for one-dimensional CSD (Freeman and Nicholson 1975) was used. The validity of one-dimensional CSD in the MOB (along the axis perpendicular to the laminae) has been shown (Aroniadou-Anderjaska et al. 1999). The spatial resolution and differentiation grid were 100 and 200 µm, respectively. Conductivity gradients across laminae were considered negligible (Martinez 1982).

The following drugs (Research Biochemicals International) were used: bicuculline methchloride (BMCl), a GABAA receptor antagonist; D-2-amino-5-phosphonovalerate (APV), a N-methyl-D-aspartate (NMDA) receptor antagonist; 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a kainate/AMPA receptor antagonist; tetrodotoxin (TTX), a sodium channel blocker; and tetraethylammonium (TEA), a potassium channel blocker. Drugs were delivered by bath application. In some experiments, APV was applied locally by diffusion from a broken glass pipette (30-50 µm tip diameter).


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INTRODUCTION
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RESULTS
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Intracellular recordings were obtained from 27 mitral cells in the presence of BMCl, to block GABAA inhibition and in nominally zero concentration of extracellular Mg2+ to facilitate activation of NMDA receptors. In these conditions, the resting membrane potential of mitral cells ranged from -50 to -60 mV, and spontaneous activity was high. To avoid contamination of evoked responses with spontaneous spikes, current was injected to maintain the membrane potential at -70 to -75 mV. Thus in all cells shown in the figures, the membrane potential is -70 to -75 mV unless indicated otherwise. The kainate/AMPA receptor antagonist, CNQX, also was used in most of the recordings to reduce spontaneous activity and to minimize deterioration of the slice that could result from prolonged exposure to BMCl in the absence of Mg2+.

Synaptic responses to depolarizing current pulses

Single spikes evoked by brief depolarizing current pulses to mitral cells, in BMCl (10 µM) and zero extracellular Mg2+, were followed by a prolonged depolarization (Fig. 1C). The peak amplitude of this depolarization was 13 ± 1.6 mV (mean ± SE, n = 12), and the duration ranged from 200 to >800 ms. The magnitude of the postpulse depolarization was enhanced by longer depolarizing pulses that evoked a train of spikes, and in a few cells (3 of 15), the postpulse depolarization was produced only after multiple spikes (see Fig. 2B). All mitral cells responded to ON stimulation. The ON-evoked responses consisted of a prolonged depolarization, often preceded by a spike. The prolonged depolarization had a time course similar to the field excitatory postsynaptic potential (EPSP) recorded simultaneously in the GL (Fig. 1C). Both the postpulse depolarization and the ON-evoked responses were blocked by the NMDA receptor antagonist APV (50 µM, Fig. 1C). The postpulse depolarization was blocked by APV regardless of the presence or absence (n = 2) of CNQX in the medium.



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Fig. 2. Mitral cells generate a NMDA-receptor-dependent EPSP in response to antidromic stimulation of neighboring mitral/tufted cells. A and B show recordings from 2 different cells in Mg2+-free medium and BMCl (10 µM). A: antidromic activation of mitral/tufted cells by LOT stimulation, subthreshold for the recorded neuron, evoked an EPSP that was reversibly blocked by bath-applied APV (50 µM). B: cell shown did not produce a prolonged depolarization in response to a 5 ms intracellular current pulse. However, a prolonged depolarization was evoked by longer current pulses (40 ms, shown in inset) or LOT stimulation with or without (inset) an antidromic spike. Traces in A and B are averages of 5-10 sweeps.

A prolonged depolarization also was evoked by intracellular current pulses when action potentials were blocked by TTX (1 µM), and Ca2+ spikes were facilitated by TEA (5 mM, n = 3, Fig. 1D). This suggests that M/T cell axon collaterals are not required for generation of the prolonged event.

Taken together these results suggest a recurrent mechanism whereby glutamate released from the dendrites of the activated (parent) mitral cell either directly activates NMDA receptors on the parent cell or excites neighboring cells, which act back on the parent cell. This mechanism may contribute to the prolonged response of mitral cells when the apical dendrites are depolarized by synaptic input from the ON.

Responses to lateral olfactory tract stimulation

To determine if mitral cells are depolarized by glutamate release from the dendrites of neighboring mitral cells, we activated M/T cells antidromically by selective stimulation of the LOT. The LOT and the centrifugal fibers are intermixed below the MCL within the MOB, but the course of the LOT is anatomically distinct caudal to the accessory olfactory bulb. These pathways are preserved in slices and, caudal to the MOB, they become distinct both visually (Fig. 1B) and physiologically. Stimulation of the LOT produces a positive field potential in the granule cell layer, as expected when granule cell dendrites in the EPL are excited by the lateral dendrites of M/T cells; stimulation of the centrifugal pathway produces a negative field potential in the granule cell layer due to direct excitation of granule cell somata and proximal dendrites (data not shown).

The intensity of LOT stimulation was adjusted to be subthreshold for antidromic spikes in the recorded mitral cell. Under these conditions, and in BMCl and zero Mg2+, single pulses applied to the LOT evoked a prolonged depolarization in the recorded cell. This depolarization was blocked by APV (n = 5, Fig. 2A).

A few cells (3 of 15) did not produce a detectable postpulse depolarization when a single spike was evoked by brief depolarizing pulses. However, a prolonged depolarization was produced after multiple spikes evoked by longer depolarizing pulses, or by LOT stimulation (n = 3, Fig. 2B). Thus a single spike does not always evoke sufficient glutamate release to produce a detectable, NMDA-receptor-dependent depolarization in the parent cell. Multiple spiking of the parent cell or simultaneous glutamate release from a population of neighboring M/T cells can be more effective in generating the NMDA depolarization.

Taken together, these observations suggest that glutamate released from mitral cell dendrites can excite neighboring dendrites of mitral cells via NMDA receptors.

Apical versus lateral mitral cell dendrites

To determine whether the apical and/or the lateral dendrites of M/T cells are involved in recurrent excitation, we applied APV (500 µM) locally to the GL to block NMDA receptors in the apical dendrites. Figure 3A shows that when NMDA receptors on the apical dendrites were blocked, as evidenced by the blockade of the ON-evoked NMDA-receptor-mediated EPSP, the prolonged depolarization evoked by intracellular pulses was only reduced (29.7 ± 3.7%, mean ± SE, n = 4); the residual depolarization was blocked by bath-applied APV. This experiment shows that a major portion of the postpulse depolarization is generated by the lateral dendrites. The reduction of the postpulse depolarization by application of APV to the GL could imply that part of this response is generated in the apical dendrites. However, it is possible that APV diffused into the EPL, thereby reducing the response of the lateral dendrites.



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Fig. 3. Both the lateral and the apical dendrites of mitral cells display recurrent excitation. A: recordings are in Mg2+-free medium and in the presence of BMCl (10 µM) and CNQX (10 µM). ON shocks and 1-nA, 30-ms depolarizing pulses were applied alternately to this cell. When NMDA receptors in the apical dendrites were blocked by local APV (500 µM) application to the border of the ONL/GL, the ON-evoked response was blocked, but the intracellular pulse-evoked depolarization was only reduced. Traces are averages of 5-10 sweeps. Action potentials have been truncated. B: laminar current source density (CSD) distribution in the MOB, evoked by selective stimulation of the LOT in Mg2+-free medium and CNQX (10 µM). In addition to the granule cell currents (see text and Aroniadou-Anderjaska et al. 1999), a sink is present in the glomerular layer (S1LOT). The location of the corresponding sources in the EPL indicates that S1LOT is produced in the glomerular dendritic tufts of M/T cells. Bath-applied APV (50 µM) blocked all synaptic currents.

To determine whether recurrent excitation occurs in the glomerular dendritic tufts of M/T cells, we used the CSD method to calculate the glomerular currents generated in response to antidromic activation of M/T cells. We recently reported that the glomerular dendritic arborizations of M/T cells produce a glutamatergic sink in response to stimulation in the MCL (Aroniadou-Anderjaska et al. 1999). This sink was very small in normal medium but was enhanced markedly in zero extracellular Mg2+. We suggested that glutamate released from the apical dendrites of M/T cells feeds back and excites the same and/or neighboring M/T cell dendrites. However, stimulation in the MCL, in addition to depolarizing directly M/T cells, also may activate centrifugal fibers a few of which reach the GL. Thus we could not exclude that centrifugal inputs played a role in the generation of the glomerular sink. For this reason, in the present study we prepared slices that would allow us to stimulate selectively the LOT (Fig. 1B). Figure 3B shows the laminar CSD distribution in the MOB evoked by single pulses applied to the LOT, in Mg2+-free medium and in the presence of CNQX (n = 5). In addition to the granule cell currents (S2LOT and S3LOT) (see Aroniadou-Anderjaska et al. 1999), a current sink (S1LOT) is present in the glomerular layer. The corresponding sources are in the EPL, which suggests that S1LOT is generated by M/T cells. APV blocked all synaptic currents (Fig. 3B). The effects of APV were reversible (not shown, see Aroniadou-Anderjaska et al. 1999). These results suggest that the apical dendrites of M/T cells also display recurrent and/or neighboring excitation.


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Physiological studies have demonstrated that glutamate released from the lateral and apical dendrites of M/T cells activates granule cells (Aroniadou-Anderjaska et al. 1999; Isaacson and Strowbridge 1998; Schoppa et al. 1998; Wellis and Kauer 1994) and glomerular interneurons (Bardoni et al. 1996), respectively. These interactions occur at anatomically defined, dendrodendritic synapses (Pinching and Powell 1971; Price and Powell 1970; Shepherd 1972). The present findings demonstrate that glutamate released from M/T cell dendrites also can excite the parent and neighboring M/T cells. However, there are thought to be no synapses between the dendrites of M/T cells (Toida et al. 1996; White 1972). Therefore the most plausible interpretation of the present findings is that dendritically released glutamate diffuses locally to activate NMDA receptors on neighboring dendrites and on the parent cell. It is also possible that glutamate activates local interneurons, which in turn excite M/T cells, but at present there is no evidence for such hypothetical excitatory interneurons. Recurrent excitation via axon collaterals of M/T cells is unlikely because these collaterals do not enter in the EPL (Orona et al. 1984) where the lateral dendrites of M/T cells extend. In addition, the observation that sodium spikes are not required to elicit the postpulse, NMDA-receptor-dependent depolarization in mitral cells further weakens the possibility for involvement of recurrent axon collaterals.

Recent studies suggest that glutamate may escape from the synaptic cleft and activate extrasynaptic NMDA receptors (Asztely et al. 1997; Rusakov and Kullmann 1998). Because of the lower affinity of kainate/AMPA receptors for glutamate (Patneau and Mayer 1990), their activation requires high glutamate concentrations that probably are achieved only within the synaptic cleft. This may explain the apparent absence of kainate/AMPA receptor contribution to the prolonged depolarizations evoked by intracellular pulses or antidromic stimulation of the LOT. However, we cannot exclude that a kainate/AMPA component is present but too small or remote to be detected at the soma.

The present experiments were conducted under conditions that facilitate activation of NMDA receptors. During normal functioning of the MOB, it is possible that these conditions are created in some M/T cells by the activity patterns within the MOB network or by modulatory inputs. For example, noradrenergic inputs that seem to disinhibit mitral cells (Ciombor et al. 1999; Jiang et al. 1996) could facilitate expression of recurrent excitation.

The apical dendrites of M/T cells generate a remarkably prolonged NMDA-receptor-dependent EPSP in response to ON stimulation even in normal medium (Aroniadou-Anderjaska et al. 1999). In some mitral cells, this EPSP is associated with prolonged spiking (Aroniadou-Anderjaska et al. 1997; Ennis et al. 1996). Whether or not the recurrent and neighboring excitation of M/T cell dendrites reported here contribute to these prolonged responses remains to be determined.

Dendritic neurotransmitter release is not unique to the olfactory bulb. It also has been demonstrated in the dopaminergic neurons of the substantia nigra (Cheramy et al. 1981; Jaffe et al. 1998), and recent evidence suggests that other neuron types also may release neurotransmitter from their somata or dendrites (Glitsch et al. 1996; Huang and Neher 1996; Lledo et al. 1998; Maletic-Savatic and Malinow 1998; Morishita et al. 1998). Thus local dendrodendritic interactions may be a more common theme in neuronal communication than previously recognized.


    ACKNOWLEDGMENTS

We thank Dr. Adam C. Puche for assistance and Drs. Asaf Keller, Nevin A. Lambert, and Greg C. Carlson for helpful discussions.

This work was supported by National Institutes of Health Grants DC-03195, DC-00347, DC-02588, and NS-36940.


    FOOTNOTES

Address for reprint requests: V. Aroniadou-Anderjaska, Dept. of Anatomy and Neurobiology, HSF, Univ. of Maryland, School of Medicine, 685 W. Baltimore St., Baltimore, MD, 21201.

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 13 January 1999; accepted in final form 15 March 1999.


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