Electrophysiology of Interneurons in the Glomerular Layer of the Rat Olfactory Bulb

A. Rory McQuiston and Lawrence C. Katz

Howard Hughes Medical Institute and Department of Neurobiology, Duke University School of Medicine, Durham, North Carolina 27710


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

McQuiston, A. Rory and Lawrence C. Katz. Electrophysiology of Interneurons in the Glomerular Layer of the Rat Olfactory Bulb. J. Neurophysiol. 86: 1899-1907, 2001. In the mammalian olfactory bulb, glomeruli are surrounded by a heterogeneous population of interneurons called juxtaglomerular neurons. As they receive direct input from olfactory receptor neurons and connect with mitral cells, they are involved in the initial stages of olfactory information processing, but little is known about their detailed physiological properties. Using whole cell patch-clamp techniques, we recorded from juxtaglomerular neurons in rat olfactory bulb slices. Based on their response to depolarizing pulses, juxtaglomerular neurons could be divided into two physiological classes: bursting and standard firing. When depolarized, the standard firing neurons exhibited a range of responses: accommodating, nonaccommodating, irregular firing, and delayed to firing patterns of action potentials. Although the firing pattern was not rigorously predictive of a particular neuronal morphology, most short axon cells fired accommodating trains of action potentials, while most delayed to firing cells were external tufted cells. In contrast to the standard firing neurons, bursting neurons produced a calcium-channel-dependent low-threshold spike when depolarized either by current injection or by spontaneous or evoked postsynaptic potentials. Bursting neurons also could oscillate spontaneously. Most bursting cells were either periglomerular cells or external tufted cells. Based on their mode of firing and placement in the bulb circuit, these bursting cells are well situated to drive synchronous oscillations in the olfactory bulb.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The structure and function of the olfactory system is conserved across many vertebrate and invertebrate species, thus providing insights into general principles of sensory processing (for reviews, see Hildebrand and Shepherd 1997; Mori 1987; Shipley and Ennis 1996). The first stage of olfactory information processing occurs in the olfactory bulb (OB), where olfactory sensory neurons make their first synapses in spherical structures called glomeruli. Individual glomeruli consist of a shell of small neurons and glial cells within which axons of sensory neurons synapse onto the dendrites of both principal cells (mitral/tufted cells) and interneurons surrounding the glomerulus (Pinching and Powell 1971a,b; White 1972, 1973), which have been termed juxtaglomerular cells (Shipley and Ennis 1996). Synaptic interactions between the dendrites of mitral/tufted cells (M/T) and juxtaglomerular cells also occur within glomeruli. Despite the obviously important position of juxtaglomerular (JG) cells at this first stage of olfactory processing, understanding of their functional repertoire is incomplete.

JG cells include several different types of interneurons such as periglomerular cells, short axon cells, and external tufted cells. In the original descriptions (Pinching and Powell 1971a-c), each class of cell was considered homogeneous. Periglomerular cells were proposed to inhibit the mitral/tufted cells via dendrodendritic synapses and lateral axonal collaterals, short axon cells to exclusively inhibit other short axon and periglomerular cells via axon collaterals, and external tufted cells to excite inhibitory cells of glomeruli and possibly granule cells of the granule cell layer.

More recent experiments have demonstrated that JG cells are far more heterogeneous (for review, see Kosaka et al. 1998). JG cells express a variety of different transmitters, including but not limited to GABA (Ribak et al. 1977), nitric oxide/NADPH (Scott et al. 1987), dopamine (Halasz et al. 1977), and neuropeptide Y (Scott et al. 1987). Furthermore the dendrites of different periglomular cells target specific functional domains of glomeruli. The dendrites of one subclass, for example, avoid regions innervated by sensory axons and only receive input from M/T cells, while other periglomerular cells receive both sensory neuron and M/T cell input (Kosaka et al. 1998).

JG neurons were initially described as bursting cells (Shepherd 1963), some of which were likely to be inhibitory (Duchamp-Viret et al. 1993; Getchell and Shepherd 1975). Others, however, have suggested that the JG neurons may all be excitatory (Freeman 1974a,b) and that GABAergic synaptic transmission in the glomeruli may be depolarizing (Siklos et al. 1995). Although others have observed the bursting behavior of JG neurons (specifically periglomerular neurons) (Wellis and Scott 1990), some experiments using whole cell patch clamping in olfactory bulb slices have failed to observe bursting neurons in the glomerular region. These studies have described JG neurons [including anatomically identified periglomerular (PG) neurons] as producing single action potentials or trains of a few action potentials in response to depolarizing current injections (Bardoni et al. 1995; Bufler et al. 1992; Puopolo and Belluzzi 1996, 1998). In contrast, PG neurons of very young rabbit (P4) olfactory bulb slices have been described as not spiking at all, where other JG neurons produce a train of spikes in response to depolarizing current injection (Bufler et al. 1992). Thus there are a number of discrepancies describing the physiological properties of JG neurons of the olfactory bulb that need to be addressed for the physiological nature of JG neurons to be fully understood.

Given their diversity, their importance at the first stage of sensory processing, and the paucity of coherent information regarding their physiological properties, we investigated the physiological properties of JG neurons in the rat olfactory bulb. In addition to this chemical and connectivity diversity, we found considerable heterogeneity of spiking behavior. Of particular interest was the substantial population of these interneurons that exhibited spontaneous and evoked bursting behavior. Synchronous firing of neurons in the bulb has been implicated in olfactory coding; these neurons may be important components of the intrinsic bulbar networks that generate rhythmic firing.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Male Sprague Dawley rats (13-21 days old) were anesthetized with isoflurane and killed by decapitation. The olfactory bulbs (OB) were removed and placed in cold, oxygenated saline [which contained (in mM) 119 NaCl, 2.5 KCl, 1.0 CaCl2, 4 MgCl2, 1 NaHPO4, 26.2 NaHCO3, 11 glucose, and 1 kynurenic acid, pH 7.4 when bubbled with 95% O2-5% CO2]. The OBs were sectioned coronally or horizontally (300-350 µm thick) on a Vibratome (Lancer, Technical Products, St. Louis, MO), maintained submerged in saline (as in the preceding text but without kynurenic acid) supported on a nylon mesh. Slices were incubated at 30°C for the first 30 min and then at room temperature (~23°C); under these conditions slices remained healthy for 2-4 h. Although slices were usable for longer periods of time, clear deterioration of slice health (apoptotic cells) were obvious after several hours in the incubation chamber. Recordings lasted on average 30 min. Prolonged recordings and continued activation of the low-threshold spike (LTS) caused significant run down of the LTS making interpretation of the results difficult.

For whole cell patch-clamp recording, the tissue slice was submerged in a recording chamber mounted on a custom made stage under a Zeiss Axioscope FS microscope (Carl Zeiss, Thornwood, NY). The slice was supported by a cover slip, which formed the bottom of the recording chamber and was superfused with saline (mostly at room temperature, some at 34°C) consisting of (in mM) 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1 NaHPO4, 26.2 NaHCO3, and 11 glucose, pH 7.4). Slices were visualized using a ×63 water-immersion objective trans-illuminated with infrared light. The image was collected by a Hamamatsu C2400 CCD camera (Hamamatsu, Bridgewater, NJ) with contrast enhancement and displayed on a video monitor so that glass patch pipettes could be visually advanced through the slice to the surface of a cell (Dodt and Zieglgansberger 1990; MacVicar 1984). Whole cell patch-clamp recordings were made from visually identified JG cells in the glomerular layer of the OB.

Patch pipettes were fabricated from borosilicate glass (KG33; 1.5 mm OD, 1.0 mm ID; Garner Glass, Claremont, CA) pulled on a two-stage vertical electrode puller (Narishige PP 83, East Meadow, NY). The intracellular solution consisted of (in mM)120 K methylsulfate, 8 NaCl, 10 HEPES, 2 alpha -methyl-alpha -phenylsuccinimide, paraformaldehyde (MgATP), 0.3 Na3GTP, and 0.1 BAPTAK4, biocytin, 0.2-0.5%, pH 7.25. Membrane potentials were monitored with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA), and data were collected onto a Pentium II personal computer using software written in LabView (National Instruments; by Eric Schaible and Paul Pavlidis, Stanford University). Data were analyzed using LabView and Axum (Mathsoft, Cambridge, MA) software packages. Statistical significance was determined by a two-tailed unpaired Student's t-test for data of unequal variance. Values are reported as means ± SE. Drugs were applied by bath superfusion or by flash photolysis from a mercury arc lamp (Oriel, Stratford, CT) focused into a 60-µm fiber optic (Ceram) placed above the slice and cell of interest (Kandler et al. 1998). Synaptic responses were evoked by bipolar tungsten electrodes placed in the sensory nerve layer (FHC, Bowdoinham, Maine).

Following physiological experiments, cells were processed for morphological analysis as biocytin had been included in the patch pipettes. Slices were fixed overnight in buffered formalin (0.1 M phosphate buffer, 4% paraformaldehyde), and processed without further sectioning. Slices were processed one of two ways---by using either a peroxidase conjugate to produce a dark reaction or a fluorescent streptavidin conjugate to produce a fluorescent cell (Alexa Fluor-488, Molecular Probes). To produce a dark reaction product, slices were permeabilized (0.5% Triton X-100), treated with 0.3% H2O2 to reduce background, and incubated 1-3 days in avidin-biotin-peroxidase complex (Elite Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Sections were then reacted with diaminobenzidine and intensified with nickel, mounted on slides, cleared, and coverslipped. Cells were subsequently reconstructed using a camera-lucida. For fluorescence, slices were permeabilized with Triton X-100, incubated with goat serum to reduce background, and incubated with streptavidin Alexa Fluor 488 for 1-2 days. Slices were then washed and mounted wet in Vectashield mounting medium (Vector Laboratories). Fluorescent cells were reconstructed with a BioRad MRC 600 confocal microscope in 0.5- to 1-µm sections.

All chemicals were purchased from Fluka (Milwaukee, WI), except for the following: tetrodotoxin (TTX; Calbiochem, La Jolla, CA); MgATP (Sigma, St. Louis, MO); and potassium methylsulfate (ICN, Costa Mesa, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Whole cell patch-clamp recordings were made from visually identified neurons in the periglomerular region of rat olfactory bulb slices. Under infrared illumination glomeruli were easily recognizable as lucent spherical objects surrounded by small cell bodies (5-15 µm diameter). We recorded from a total of 179 cells with intermediate soma diameters (8-12 µm), a range that includes neurons from the three broad classes of JG neurons in this region: periglomerular, short axon and external tufted cells (Pinching and Powell 1971a). The membrane potential (Vm) of the JG cells was maintained near their resting potential, 60-70 mV. The internal pipette solution contained biocytin (0.2-0.5%) to allow subsequent morphological analysis of a subset of recorded cells.

JG cells show diverse electrophysiological properties

JG cells exhibit a diverse array of firing properties in response to an artificial depolarization, which could be divided into two large groups (Figs. 1 and 2). One group (102 cells) fired standard trains of action potentials; the second group fired in bursts. The first group, which exhibited "standard" firing properties, consisted of cells that displayed one of four different firing patterns (Fig. 1). The second group (77 cells), which fired bursts of action potentials, was distinguished by the presence of a low-threshold calcium-dependent spike (Fig. 2).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1. Juxtaglomerular cells display a range of electrophysiological properties. Ai: the membrane potential (Vm) response of a juxtaglomerular cell (JG) to rectangular depolarizing current injection (600 ms, 100 pA). Aii: The Vm response of the same cell (Ai) to hyperpolarizing current injection (600 ms, 80 pA). Note the nonaccommodating action potentials during the depolarization (Ai) and the sag in the Vm toward resting Vm during the hyperpolarization (depolarizing short axon) (Aii). Following the hyperpolarization, the Vm overshoots rest and produced an action potential (anode break response, Aii). Bi: another JG cell produced accommodating action potentials in response to depolarizing current injection (600 ms, 120 pA). Bii: the same neuron (Bi) showed a small depolarizing sag during hyperpolarizing current injection (600 ms, 120 pA). Ci: a JG cell produced only an individual action potential in response to depolarizing current injection (600 ms, 100 pA). Cii: the same cell (Ci) produced a small depolarizing sag during hyperpolarizing current injection (600 ms, 40 pA). Di: a JG cell that displayed irregular firing in response to depolarizing current injection (600 ms, 100 pA). Dii: the same cell (Di) produced a depolarizing sag during hyperpolarizing current injection (600 ms, 120 pA), followed by an anode break response producing a burst of action potentials. Ei: a JG cell produced a delayed firing in response to depolarizing current injection (600 ms, 150 pA). The Vm showed a ramp-like delayed depolarization with subthreshold oscillations. Eii: the same cell (Ei) did not show any depolarizing sag during hyperpolarizing current injection (600 ms, 100 pA). All scale bars: vertical, 10 mV; horizontal, 200 ms.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2. A subclass of JG cells produce low-threshold spikes. Ai: depolarizing current injection into a JG cell produced a low-threshold spike (LTS; 600 ms, 80 pA). Aii: in the same JG cell (Ai), a hyperpolarizing current injection produced a depolarizing sag during the hyperpolarization, and an anode break response leading to an LTS following the hyperpolarization (top). Bottom: the LTSs following the hyperpolarization shown on an expanded time scale. Note the single action potential riding on the LTSs. Bi: another JG cell produced an LTS when depolarized (600 ms, 100 pA). Bii: this same cell (Bi) produced an anode break LTS following a hyperpolarization. The expanded time scale of the LTS (bottom) displays the 5 action potentials riding on top of the LTS. These action potentials showed decrementing amplitudes with time. Ci: a smaller population of JG cells produced plateau potentials following a depolarization (600 ms, 100 pA). Cii: the plateau potential could also be produced by an anode break response following a hyperpolarizing current injection (600 ms, 100 pA). Scale bars: horizontal traces, 10 mV; vertical, 200 ms for A and B (top), 35 ms for Aii and Bii (bottom), 2 s for C.

In the simplest case, the standard group responded to brief current injection through the patch pipette with a regularly spaced train of action potentials (Fig. 1Ai, n = 31). Because the intervals between action potentials in the train remained similar throughout current injections, we termed this type of cell nonaccommodating. On hyperpolarization, 71% (22/31 cells, Fig. 1Aii) of nonaccommodating neurons displayed a small depolarizing sag in the membrane potential (Vm) during the hyperpolarizing pulse. In the remaining 29% (9/31 cells), the sag was absent.

Another subset of cells in the standard group fired trains of action potentials that decreased in frequency during depolarization (Fig. 1Bi, n = 14) and were therefore termed accommodating cells. Most cells of this type produced a depolarizing sag during a hyperpolarizing current pulse (Fig. 1Bii, 11/14). A related group of cells, also termed accommodating, produced only one action potential during the depolarization regardless of the magnitude of the depolarizing current pulse (Fig. 1Ci, n = 21). The majority of the cells, (67%, 14/21), that fired single action potentials produced a depolarizing sag during hyperpolarizing steps (Fig. 1Cii).

Infrequently we encountered neurons that fired sporadically with no clear pattern when depolarized. Most of these irregular firing cells also produced depolarizing sags during hyperpolarizing steps (Fig. 1Dii, 4/5).

The last group of cells in the standard category fired action potentials with a variable delay, during which the Vm slowly ramped toward the action potential firing threshold during depolarization. These cells also produced subthreshold oscillations during the ramp period before the initial action potential (Fig. 1Ei, n = 10). In contrast to the other standard cells, 50% of these delay cells did not produce a depolarizing sag during a hyperpolarization (Fig. 1Eii, 5 of 10). The properties of this cell type resembled those observed in mitral cells (Chen and Shepherd 1997).

Based on their electrophysiological properties, we classified these four types of neurons as one group of JG cells. All had very large input resistances (1,027 ± 123 MOmega , n = 46).

JG cells with LTSs

The second large group of JG cells produced a LTS evoked by depolarization from resting Vm or from an anode break response following a hyperpolarizing current pulse (Fig. 2, A and B). All cells with an LTS also had a depolarizing sag during a hyperpolarization (Fig. 2, Aii and Bii). The presence of a depolarizing sag in all cells that produce an LTS is significant since other neurons---such as thalamocortical cells---that produce behaviorally important slow oscillations (during sleep), critically depend on the interplay between the LTS and a depolarizing sag to produce slow (~4 Hz) oscillations. Termination of the hyperpolarizing pulse produced an overshoot of the resting Vm and subsequently induced an LTS. Generally, fast action potentials accompanied the LTS (Fig. 2, Aii and Bii, bottom). A variable number of fast action potentials occurred on the crest of the LTS (0-10; mean 2.4 ± 0.2, n = 77; Fig. 2, Aii and Bii). The frequency of these fast action potentials was in the gamma range (67 ± 4 Hz, n = 38). The duration of the LTS from the point of inflection to the corresponding Vm on the falling phase varied between 75 and 960 ms (mean 298 ± 2 ms, n = 73).

Most experiments were performed at room temperature; however the presence of the LTS was sensitive to prolonged recording and particularly to elevated temperatures. At physiological temperatures (35-37°C), the LTS duration was significantly decreased by roughly half (141 ± 28 ms, n = 5, P < 0.002, range 70-215 ms), but physiological temperature did not significantly affect the number of action potentials (3.6 ± 0.9, n = 5, P > 0.25). Although the frequency of fast action potentials appeared to increase at physiological temperature, the difference did not reach significance, probably because of the low number of cells recorded (163 ± 34 Hz, n = 4, P > 0.05).

At room temperature a few cells (n = 3) produced plateau potentials (Fig. 2C, i and ii). Similar to cells with LTSs, plateau potentials could be produced by depolarizing the cell from resting Vm (Fig. 2Ci) or from an anode break response following a hyperpolarizing current injection (Fig. 2Cii). Plateau potentials produced fast action potentials (1-7 ms; mean, 3 ± 2) and plateau durations lasted longer than a second (1,450-7,000 ms; mean, 4,817 ± 1,708). However, given their infrequency, they were not investigated further.

We collectively termed the cells having an LTS and those producing plateau potentials as bursting cells. Both the "standard" firing cells and the bursting cells had indistinguishable, very high-input resistances [1,027 ± 123, n = 46 (bursting) vs. 1,005 ± 30 MOmega , n = 61 (standard), P > 0.88].

Ionic basis of LTSs

We next investigated the ionic basis of the LTS. To determine whether Na+ influx was required, we first replaced extracellular Na+ with choline (Fig. 3A). Choline eliminated fast action potentials, but the LTS persisted (Fig. 3A, middle, n = 3). In a second manipulation, we included QX314 (5 mM), an inhibitor of voltage-dependent sodium channels, in the intracellular solution (Fig. 3B). QX314 inhibited the generation of fast action potentials but did not prevent the activation of an LTS (n = 6). Therefore sodium influx does not appear to be required for the production of an LTS in JG neurons.



View larger version (7K):
[in this window]
[in a new window]
 
Fig. 3. LTSs do not depend on sodium influx. A: a JG cell produced an LTS when depolarized (top) or an anode break response following a hyperpolarization (bottom; control). When the extracellular sodium chloride was replaced with choline chloride (choline), LTSs still occurred when the cell was depolarized (top) or following hyperpolarizing current injection (bottom). However, the fast action potentials that occurred on top of the LTS in control were blocked by the removal of extracellular sodium. B: inclusion of QX314 (5 mM) in the recording pipette prevented activation of fast action potentials but did not prevent the activation of LTSs by depolarization (top) or an anode break response following hyperpolarization (bottom). Scale bars: 10 mV (vertical), 200 ms (horizontal).

LTSs in neurons and nonneuronal cells often result from activation of low-voltage-activated calcium currents (LVA) (Huguenard 1996). We tested this possibility by examining the effect of antagonists to these channels on the LTSs. High concentrations of nickel (1 mM) completely and reversibly inhibited the LTSs in JG neurons (Fig. 4A, n = 13). Lower concentrations of nickel did not completely inhibit the LTSs. Similarly, high concentrations (5 mM) of alpha-methyl-alpha-phenylsuccinimide (MPS), which are known to inhibit LVA calcium currents (Huguenard 1996), completely blocked the LTSs; lower concentrations (1 mM) of MPS suppressed but did not completely inhibit the LTSs (not shown) (Fig. 4B, n = 5). Therefore the LTSs in JG neurons appears to arise from the activation of LVA calcium channels.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. LTSs are inhibited by low-voltage-activated calcium channel antagonists. A: LTS produced by depolarization (Control, top) or hyperpolarization (control, bottom) is completely blocked by 1 mM nickel (middle). On removal of the nickel, the LTS recovered (right). B: LTS activated by depolarization (top left) or hyperpolarization (bottom left) is completely blocked by alpha -methyl-alpha -phenylsuccinimide (MPS, 5 mM; middle, top and bottom). MPS blocked the fast action potentials as well. The LTS recovered (right, top and bottom) on removal of MPS from the bath. Scale bars, 10 mV (vertical), 200 ms (horizontal).

Physiological stimuli activate LTSs in JG neurons

Although an LTS could be evoked by nonphysiological stimuli such as intracellular current injection, if they are involved in bulbar information processing, more physiologically relevant stimuli should activate them as well. Excitatory synaptic inputs to JG cells arise from the sensory neuron axons and mitral/tufted cell dendrites. Both use glutamate as the excitatory transmitter (Bardoni et al. 1996; Berkowicz et al. 1994; Chen and Shepherd 1997; Ennis et al. 1996; Trombley and Shepherd 1992). Given that an LTS could be evoked by depolarization from the resting Vm (Fig. 2, Ai and Bi), we tested whether an LTS could be evoked by excitatory synaptic input to the JG cells or by photolysis of caged glutamate onto the dendritic region of the cell (Kandler et al. 1998).

With a stimulating electrode in the sensory nerve layer, brief stimuli evoked excitatory synaptic input onto JG cells, which in turn elicited an LTS (Fig. 5A, n = 25). Paired synaptic stimulation with intervals varying between 100 ms and 1 s produced paired-pulse inhibition sufficient to prevent the occurrence of a second LTS (Fig. 5A). An LTS could also be evoked in JG neurons by flash photolysis of caged glutamate onto JG cell dendrites (Fig. 5B, up-arrow , n = 7). Interestingly, many JG cells displayed considerable spontaneous synaptic activity (Fig. 5D). In many JG cells maintained at their resting membrane potential, these spontaneous excitatory postsynaptic potentials (EPSPs) were sufficient to produce an individual spontaneous LTSs or a series of spontaneous LTSs (Fig. 5D, n = 25 cells). These results demonstrate that an LTS can be evoked in JG cells by excitatory synaptic input and activation of glutamate receptors.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5. LTSs are evoked by physiological stimuli. A: electrical stimulation of the sensory nerve layer via bipolar tungsten electrodes (100-500 µA, 100 µs) produced excitatory postsynaptic potentials (EPSPs) displaying paired-pulse depression (interval, 1 s). The 1st EPSP produced an LTS, but the paired-pulse depression of the 2nd pulse prevented the production of an LTS. B: photolysis of caged glutamate (up-arrow , 5-ms flash from a 60-µm fiber optic placed over the glomerulus of interest, with mercury arc lamp light source) produced a depolarization of the JG cell and LTS. C: an inhibitory postsynaptic potential, evoked by bipolar stimulating electrodes in the glomerular layer [in the presence of the glutamate antagonists NBQX (30 µM) and APV (50 µM)] produced a rebound LTS and plateau. D, top: spontaneous EPSPs (small fast depolarizations of the membrane potential) periodically produced spontaneous LTSs. Bottom: 2 spontaneous LTSs are displayed on an expanded time base showing the spontaneous EPSPs that contributed to the production of spontaneous LTSs. E: a hyperpolarizing pulse produced rebound LTS and an oscillation of the membrane potential leading to rhythmic periodic bursts of action potentials. Scale bars: vertical, 10 mV; horizontal, 200 ms (A-C), 1 s (D, top), 100 ms (D, bottom), 500 ms (E).

Given that the LTSs could be produced following a hyperpolarization, we tested whether an LTS in JG cells could also be produced by an inhibitory PSP (IPSP). By placing a stimulating electrode in the glomerular region, we could periodically evoke IPSPs. On occasion this IPSP could produce an LTS (Fig. 5C, n = 3). Therefore as in other regions of the CNS (McCormick and Bal 1997), an IPSP in JG cells can elicit an LTS following the return of the membrane potential to rest. In addition to these various methods of evoking an LTS, we found that JG cells could also exhibit spontaneous rhythmic bursting (Fig. 5E, n = 11). The oscillations in JG cells varied in frequency but fell in the delta range (1.7 ± 0.2 Hz) as seen in the thalamus (McCormick and Bal 1997).

Therefore an LTS can be activated by a range of synaptic mechanisms, including spontaneous and evoked EPSPs and IPSPS; these LTSs also endow JG cells with the ability to oscillate spontaneously.

Morphology of JG neurons exhibiting LTSs

Based on morphological criteria, neurons in the JG region comprise several different classes (Shipley and Ennis 1996), and each class is heterogeneous (Kosaka et al. 1998). Given that not all neurons in the glomerular region produced an LTS (Fig. 1), we attempted to correlate the morphological classes of the JG neurons with their observed firing patterns.

JG neurons that produced an LTS fell into two morphological categories (Fig. 6). One group had morphological characteristics typical of external tufted (ET) cells. For the most part, the dendrites were devoid of spine-like appendages (Fig. 6C, i and ii), although some dendrites showed sparse spines (Fig. 6Aii, arrow). These cells often had axonal projections emerging from the glomerular region and penetrating into the internal plexiform layer (IPL) and/or granule cell layer (Fig. 6C). All these characteristics are well-defined features of ET cells (Pinching and Powell 1971a).



View larger version (50K):
[in this window]
[in a new window]
 
Fig. 6. Morphology of JG cells with LTSs. Ai: a confocal reconstruction of a biocytin-filled JG cell that produced LTSs (left). Notice the axon leaving the cell body (arrowhead). The axon sent collaterals into the granule cell layer and laterally in the glomerular layer (not shown). Aii: an enlargement of the area in the rectangle on the left of Ai. Arrows point to some small spine-like appendages. Bi: a confocal reconstruction of another biocytin-filled JG cell that produced LTSs. Bii: an enlargement of the rectangle in Bi. Notice the many large complex spine-like appendages (gemmules), some examples are indicated by arrows. The axon left the cell body (arrow head) and sent collaterals in the glomerular layer, 2 glomeruli distant. C, i and ii: reconstructions of biocytin-filled JG cells that produced LTSs. Both lacked spines and had axons that ran toward or into the inner plexiform layer. The dendrites of the cell in Ci arborized in 2 glomeruli, whereas dendrites of the cell in Cii were confined to 1 glomerulus. GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, inner plexiform layer. Scale bars: 30 µm (Ai), 5 µm (Aii and Bii), 25 µm (Bi), 50 µm (C).

However, not all JG neurons that produced an LTS displayed morphological features of external tufted cells. The dendrites of some cells did not completely fill the glomerulus, their dendrites had many large spine-like appendages (gemmules), and their axons were confined to the glomerular region (Fig. 6B, i and ii). Based on previous morphological studies, these cells have the characteristics of inhibitory periglomerular cells (Pinching and Powell 1971a). Therefore at least two morphological classes of JG cells can produce an LTS.

JG cells that did not produce an LTS had morphological features that overlapped with cells that did produce an LTS (Fig. 7). However, short axon cells rarely produced an LTS (Fig. 7A). Short axon cells had sparse dendrites and axons whose arbors avoided glomeruli and were confined to the periglomerular region (Fig. 7A). Some external tufted cells, characterized by their lack of spines, beaded dendrites (Fig. 7B), and/or presence of secondary dendrites along the bottom of the glomerular layer (Fig. 7C) lacked an LTS (Fig. 7, B and C). Some periglomerular neurons also did not produce an LTS (Fig. 7D). These cells were usually smaller than those that did exhibit an LTS but otherwise had dendritic gemmules and axons confined to the glomerular layer characteristic of cells of this class.



View larger version (86K):
[in this window]
[in a new window]
 
Fig. 7. Morphology of JG cells lacking LTSs. A: confocal reconstruction of a biocytin-filled JG cell. Notice the sparse dendrites with little branching. The dendrites clasped a glomerulus, remained in the periglomerular region, and did not enter the glomerulus. B: confocal reconstruction of a biocytin-filled JG cell. Notice the primary dendrite with straight aspiny secondary branches. Subsequent dendrites were very beaded and filled the glomerulus. Notice the axon leaving the cell body (arrow head). C, top: photograph of a biocytin-filled cell with a tuft of straight aspiny dendrites in 1 glomerulus and a single large horizontal dendrite projecting along the bottom of the glomerular layer. Bottom: reconstruction of the same cell showing its position relative to the layers of the olfactory bulb. D, top: photograph of a JG cell with multiple primary dendrites (spines, although present, cannot be seen in photograph) confined to a single glomerulus. The axon leaves the cell body and projects a short distance toward the sensory nerve layer. Bottom: a drawing of the cell showing its orientation relative to the layers of the olfactory bulb. Scale bars: 30 µm (A and B), 50 µm (C and D).

While considerable heterogeneity in both morphology and physiology was observed, some trends were clear (Tables 1 and 2). Roughly two-thirds of external tufted and periglomerular cells produced an LTS, while two-thirds of short axon cells did not. Table 2 summarizes the firing properties of 25 morphological neurons that did not produce an LTS. There was no clear single type of firing property for periglomerular or external tufted cells; however, most short axon neurons produced accommodating trains of action potentials. Two of these neurons that fired with a delay were external tufted cells, which is not surprising given that the delay to firing pattern is similar to that seen in the other excitatory neurons in the olfactory bulb, the mitral cells (Chen and Shepherd 1997).


                              
View this table:
[in this window]
[in a new window]
 
Table 1. Representation of bursting and nonbursting cells in different morphological classes of olfactory bulb interneurons


                              
View this table:
[in this window]
[in a new window]
 
Table 2. Spiking properties of olfactory bulb interneurons neurons lacking LTSs


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Based on modes of firing, we have found that JG cells fall into two different physiological classes: standard nonbursting cells and bursting neurons that exhibit a low-threshold calcium-dependent spike. Nonbursting neurons exhibited a range of firing properties, from regular trains of action potentials to highly irregular patterns of spikes. Firing patterns did not strongly correlate with the morphological class of JG cell from which we recorded. A similar lack of correspondence between interneuron morphology and physiology has been observed in other cortical structures such as the hippocampus, where interneurons of similar morphology have striking differences in their passive electrical properties and action potential firing patterns (Cauli et al. 1997; Mott et al. 1997; Parra et al. 1998). This heterogeneity could be explained by numerous factors, including the distribution of ion channels; indeed there is evidence for considerable heterogeneity in the distribution of potassium channels in JG cells, which would be expected to alter firing patterns (Puopolo and Belluzzi 1998).

However, some consistencies were observed. The majority of short axon cells fired accommodating trains of action potentials, and two of three cells that showed delay to firing when depolarized were external tufted cells. This is consistent with previous results showing that the delay to firing pattern is characteristic of mitral and deep tufted cells (Chen and Shepherd 1997).

Our studies show additional similarities to previous investigations. Studies in vivo using both extracellular and intracellular techniques have demonstrated a class of neuron in the glomerular layer that fire bursts of action potentials similar to our bursting neurons (Freeman 1974a; Shepherd 1963; Wellis and Scott 1990). There are some differences in the bursting we observed and that of previous studies. Most obvious is that the duration of our bursts is 100-200 ms longer than those reported in vivo. There are a number of possible reasons for this discrepancy. First, most of our experiments were conducted at room temperature. Increasing the temperature of the slice did reduce the duration of our bursts, albeit not down to the durations observed in vivo. We produced and measured our bursts by directly changing the membrane potential of neuron. The in vivo studies used synaptic activation that would activate additional neurons that could inhibit and reduce the duration of the burst. The extracellular studies measured the duration of spiking (Freeman 1974a; Shepherd 1963), which is considerably shorter than the entire envelope of the burst. And finally, there could be a difference created by the very difficult experiments obtained with leaky intracellular recordings (Wellis and Scott 1990) and those obtained with tight whole-seal recordings.

In contrast, previous studies in vitro have not observed bursting cells in the glomerular layer. Whole cell recordings in olfactory bulb slices have claimed that all JG neurons fire single or trains of spikes similar to our standard firing cells (Bardoni et al. 1995; Puopolo and Belluzzi 1996, 1998). A different study showed that PG neurons do not fire spikes at all (Bufler et al. 1992). A possible reason previous studies did not observe the bursting behavior in vitro may be due to the whole cell patch-clamping technique; we observed that the bursting phenomenon was sensitive to prolonged recording and activation. Prolonged recording and excessive activation resulted in a hyperpolarization of the bursting JG cell resting potential, a reduction in the input resistance of the cell, and the ability to fire only one fast action potential. The previous observation that PG cells do not fire any spikes at all may differ from our observations because of species and developmental differences. Previous studies used P4 rabbits, while we used P13-21 rats.

The neurons generating bursts based on LTSs are of particular interest. The behavior of these cells---especially the circumstances that elicit LTSs---resembles that of the reticular nucleus of the thalamus and thalamocortical neurons (Andersen et al. 1968; Deschenes et al. 1984; Huguenard and Prince 1992; Roy et al. 1984). LTSs in both of these types of thalamic neurons results from activation of a LVA calcium current (Huguenard and Prince 1992; Jahnsen and Llinas 1984a,b). LVA calcium channels also appear to underlie the LTSs in JG neurons, as antagonists of LVA channels could completely inhibit the LTSs. The concentrations of antagonists required to inhibit the LTSs in JG cells were high but within the widely variable range of concentrations observed to inhibit LVA currents in different cell types (for review, see Huguenard 1996; Perez-Reyes 1998). Furthermore, inhibition of voltage-dependent sodium channels by the antagonist QX314, and inhibition of sodium influx by the removal of extracellular sodium, did not inhibit the LTSs. Consistent with our findings, a LVA calcium channel has been cloned from mouse (Klugbauer et al. 1999) and is expressed widely throughout the olfactory bulb. However, two potential alternative explanations for the mechanism producing the LTSs might be: 1) the release of glutamate from the recorded JG neuron acts on glutamatergic autoreceptors to produce the LTSs and 2) a network mechanism in which glutamate is released from the recorded JG neuron, resulting in excitatory feedback from other neurons to produce the LTSs. However, these alternatives are inconsistent with our data. First, LTSs could be produced in the presence of glutamatergic receptor antagonists that should inhibit both glutamatergic autoreceptors and network activity. Second, LTSs were observed in the presence of sodium channel blockers that should inhibit synaptic transmission. Finally, LTSs could be observed in JG neurons with morphological characteristics of GABAergic interneurons, suggesting that activation of some JG neurons should produce inhibition, not excitation. Taken together our data are most consistent with LTSs arising from the activation of LVA calcium channels.

Morphology of neurons with LTSs

The morphological studies of Pinching and Powell (1971a-c) classified JG neurons into different categories: PG, ET, and short axon neurons. The PG neurons, which are thought to be inhibitory (Duchamp-Viret et al. 1993; Pinching and Powell 1971a-c; Puopolo and Belluzzi 1998; Shepherd 1971; but see Freeman 1974a; Siklos et al. 1995), have small cell bodies, spine-like processes (gemmules), and dendrites that usually did not completely fill a glomerulus. Short axon cells, also thought to be inhibitory, have spineless dendrites with processes that avoided entering glomeruli. The excitatory ET cells exhibit dendrites with few spines. These three cell types are richly interconnected, with PG cells forming local dendrodendritic and lateral axodendritic connections with ET, mitral-tufted, and other PG cells. ET cells form dendrodendritic connections with PG cells and lateral axonal connections onto both short axon and PG cells. Importantly, axons of ET cells synapse on olfactory bulb neurons in other layers, including granule cells (for review, see Shipley and Ennis 1996). Short axon neurons appeared to be interneuron selective, only forming axonal connections with PG cells.

Although our results show that examples of all three classes that could occasionally produce an LTS, almost all (86%) of JG cells producing LTSs were either external tufted or periglomerular cells. Thus both excitatory and inhibitory JG neurons appear to be capable of producing an LTS. Given that these JG neurons are richly interconnected to one another, and to neurons in other regions of the olfactory bulb, the LTSs in these cell types may have important physiological functions in olfactory processing within the olfactory bulb.

Physiological implications of the LTS in JG neurons

We found that the LTSs in JG cells could be activated by physiological phenomenon such as IPSPs and EPSPs; the LTSs also endowed JG cells with the ability to spontaneously oscillate. In vivo studies have previously demonstrated that superficial cells in the bulb produce bursts of spikes (Freeman 1974a; Schneider and Scott 1983; Shepherd 1963). Subsequent studies demonstrated that at least a subset of these neurons were PG neurons and that these fired in bursts or plateaus (Wellis and Scott 1990). Interestingly, these cells could be activated by stimulating anywhere in the sensory nerve layer, outside the area that would directly stimulate sensory input to the recorded cell, which implies extensive interconnections among these bursting superficial cells (Freeman 1974a,b; Wellis and Scott 1990). Superficial cells could also be rhythmically activated during an artificial sniff cycle, even in the absence of an odor (Onoda and Mori 1980). Interestingly, these superficial cells fired either in phase, or out of phase with inspiration (Onoda and Mori 1980). The out of phase cells were spontaneously active when the receptor neurons were not activated, whereas the in phase cells were silent. On inspiration and stimulation of the receptor neurons, the in-phase cells burst, and the out of phase became silent, possibly due to the inhibition of the out of phase cells by the in phase cells. This suggests that a network oscillation driven by the inspiration cycle may occur between neurons in the glomerular layer. Furthermore, the granule cell layer produced a synchronous population oscillation during the inspiration that was delayed relative to the activation of in phase JG cells. In contrast, the output mitral and tufted cells showed only a small amount of firing, which was rarely correlated with the inspiration cycle or was very complicated relative to the glomerular cells and granule cell rhythm.

There is a significant amount of evidence suggesting that oscillations occurring in the deeper layers arise from the synaptic interactions between mitral/tufted cells and granule cells (Desmaison et al. 1999; Freeman 1974a; Kashivadini et al. 1999; Mori and Takagi 1978). However, the experiments of Onoda and Mori (1980) show that mitral/tufted cells do not show coherent activity when oscillations occur in the granule cell layer. The question arises, is this minimal activity in mitral/tufted cells alone enough to generate the rhythmicity in granule cells? We suggest that because activation of JG neurons during inspiration precedes oscillations in the granule cell layer and that many bursting neurons in the glomerular layer have axons that project toward the granule cells, that the projection of bursting neurons to deeper layers of the olfactory bulb may generate oscillations in the granule cell layer.

This rhythmic synchronization of action potentials appears to play an important function in olfactory processing in the olfactory bulb. Synchronization from distributed principal cells in a variety of different organisms is associated with olfactory discrimination tasks (for reviews, see Laurent 1996, 1999; Mori et al. 1999). Slow oscillations, such as those that the LTS in JG cells can produce, are evident in the olfactory bulb of several species (Delaney and Hall 1996; Lam et al. 2000). In rats, spatially distributed principal cells synchronize at 30-60 Hz, much faster than the bursting of JG cells. However, these fast oscillations are modulated by the slower rhythms associated with the breathing rhythm or superimposed on top of the slower rhythm (Freeman 1978; Freeman and Viana Di Prisco 1986).

Interestingly, the properties of the LTS in JG cells, the phenomena that activates this LTS, the cell types that produce an LTS, and the rich synaptic interactions between cells that produce an LTS are similar to the thalamus. It is these properties of the thalamic neurons that allow them to oscillate and generate biologically important rhythms such as spindle waves and slow wave sleep (for review see McCormick and Bal 1997). Given the similarities of JG neurons to rhythmic thalamic neurons, that JG neurons may rhythmically burst in vivo, and that JG neurons with an LTS project extensively throughout the bulb, we propose that the LTSs in JG cells may be important for contributing to the generation of synchronous rhythms throughout the olfactory bulb during olfactory processing.


    ACKNOWLEDGMENTS

The authors thank M. Gray for assistance with the histology and Drs. Leo Belluscio and Tom Tucker for helpful discussions.


    FOOTNOTES

Address for reprint requests: L. C. Katz, Duke University Medical Center, Dept. of Neurobiology, Box 3209, Bryan Research Building, Research Dr., Durham, NC 27710 (E-mail: larry{at}neuro.duke.edu).

Received 8 January 2001; accepted in final form 26 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society