Reciprocal Dendrodendritic Synapses Research Articles

Local Postsynaptic Signaling on Slow Time Scales in Reciprocal Olfactory Bulb Granule Cell Spines Matches Asynchronous Release.

In the vertebrate olfactory bulb (OB), axonless granule cells (GC) mediate self- and lateral inhibitory interactions between mitral/tufted cells via reciprocal dendrodendritic synapses. Locally triggered release of GABA from the large reciprocal GC spines occurs on both fast and slow time scales, possibly enabling parallel processing during olfactory perception. Here we investigate local mechanisms for asynchronous spine output. To reveal the temporal and spatial characteristics of postsynaptic ion transients, we imaged spine and adjacent dendrite Ca2 +- and Na+-signals with minimal exogenous buffering by the respective fluorescent indicator dyes upon two-photon uncaging of DNI-glutamate in OB slices from juvenile rats. Both postsynaptic fluorescence signals decayed slowly, with average half durations in the spine head of t1/2_Δ[Ca2 +]i ∼500 ms and t1/2_Δ[Na+]i ∼1,000 ms. We also analyzed the kinetics of already existing data of postsynaptic spine Ca2 +-signals in response to glomerular stimulation in OB slices from adult mice, either WT or animals with partial GC glutamate receptor deletions (NMDAR: GluN1 subunit; AMPAR: GluA2 subunit). In a large subset of spines the fluorescence signal had a protracted rise time (average time to peak ∼400 ms, range 20 to >1,000 ms). This slow rise was independent of Ca2 + entry via NMDARs, since similarly slow signals occurred in ΔGluN1 GCs. Additional Ca2 + entry in ΔGluA2 GCs (with AMPARs rendered Ca2 +-permeable), however, resulted in larger ΔF/Fs that rose yet more slowly. Thus GC spines appear to dispose of several local mechanisms to promote asynchronous GABA release, which are reflected in the time course of mitral/tufted cell recurrent inhibition.

α2-Adrenergic receptor activation promotes long-term potentiation at excitatory synapses in the mouse accessory olfactory bulb.

The formation of mate recognition memory in mice is associated with neural changes at the reciprocal dendrodendritic synapses between glutamatergic mitral cell (MC) projection neurons and GABAergic granule cell (GC) interneurons in the accessory olfactory bulb (AOB). Although noradrenaline (NA) plays a critical role in the formation of the memory, the mechanism by which it exerts this effect remains unclear. Here we used extracellular field potential and whole-cell patch-clamp recordings to assess the actions of bath-applied NA (10 µM) on the glutamatergic transmission and its plasticity at the MC-to-GC synapse in the AOB. Stimulation (400 stimuli) of MC axons at 10 Hz but not at 100 Hz effectively induced N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP), which exhibited reversibility. NA paired with subthreshold 10-Hz stimulation (200 stimuli) facilitated the induction of NMDA receptor-dependent LTP via the activation of α2-adrenergic receptors (ARs). We next examined how NA, acting at α2-ARs, facilitates LTP induction. In terms of acute actions, NA suppressed GC excitatory postsynaptic current (EPSC) responses to single pulse stimulation of MC axons by reducing glutamate release from MCs via G-protein coupled inhibition of calcium channels. Consequently, NA reduced recurrent inhibition of MCs, resulting in the enhancement of evoked EPSCs and spike fidelity in GCs during the 10-Hz stimulation used to induce LTP. These results suggest that NA, acting at α2-ARs, facilitates the induction of NMDA receptor-dependent LTP at the MC-to-GC synapse by shifting its threshold through disinhibition of MCs.

Direct Recording of Dendrodendritic Excitation in the Olfactory Bulb: Divergent Properties of Local and External Glutamatergic Inputs Govern Synaptic Integration in Granule Cells.

The olfactory bulb contains excitatory principal cells (mitral and tufted cells) that project to cortical targets as well as inhibitory interneurons. How the local circuitry in this region facilitates odor-specific output is not known, but previous work suggests that GABAergic granule cells plays an important role, especially during fine odor discrimination. Principal cells interact with granule cells through reciprocal dendrodendritic connections that are poorly understood. While many studies examined the GABAergic output side of these reciprocal connections, little is known about how granule cells are excited. Only two previous studies reported monosynaptically coupled mitral/granule cell connections and neither attempted to determine the fundamental properties of these synapses. Using dual intracellular recordings and a custom-built loose-patch amplifier, we have recorded unitary granule cell EPSPs evoked in response to mitral cell action potentials in rat (both sexes) brain slices. We find that the unitary dendrodendritic input is relatively weak with highly variable release probability and short-term depression. In contrast with the weak dendrodendritic input, the facilitating cortical input to granule cells is more powerful and less variable. Our computational simulations suggest that dendrodendritic synaptic properties prevent individual principal cells from strongly depolarizing granule cells, which likely discharge in response to either concerted activity among a large proportion of inputs or coactivation of a smaller subset of local dendrodendritic inputs with coincidence excitation from olfactory cortex. This dual-pathway requirement likely enables the sparse mitral/granule cell interconnections to develop highly odor-specific responses that facilitate fine olfactory discrimination.SIGNIFICANCE STATEMENT The olfactory bulb plays a central role in converting broad, highly overlapping, sensory input patterns into odor-selective population responses. How this occurs is not known, but experimental and theoretical studies suggest that local inhibition often plays a central role. Very little is known about how the most common local interneuron subtype, the granule cell, is excited during odor processing beyond the unusual anatomical arraignment of the interconnections (reciprocal dendrodendritic synapses). Using paired recordings and two-photon imaging, we determined the properties of the primary input to granule cells for the first time and show that these connections bias interneurons to fire in response to spiking in large populations of principal cells rather than a small group of highly active cells.

Sniff-Like Patterned Input Results in Long-Term Plasticity at the Rat Olfactory Bulb Mitral and Tufted Cell to Granule Cell Synapse

During odor sensing the activity of principal neurons of the mammalian olfactory bulb, the mitral and tufted cells (MTCs), occurs in repetitive bursts that are synchronized to respiration, reminiscent of hippocampal theta-gamma coupling. Axonless granule cells (GCs) mediate self- and lateral inhibitory interactions between the excitatory MTCs via reciprocal dendrodendritic synapses. We have explored long-term plasticity at this synapse by using a theta burst stimulation (TBS) protocol and variations thereof. GCs were excited via glomerular stimulation in acute brain slices. We find that TBS induces exclusively long-term depression in the majority of experiments, whereas single bursts (“single-sniff paradigm”) can elicit both long-term potentiation and depression. Statistical analysis predicts that the mechanism underlying this bidirectional plasticity involves the proportional addition or removal of presynaptic release sites. Gamma stimulation with the same number of APs as in TBS was less efficient in inducing plasticity. Both TBS- and “single-sniff paradigm”-induced plasticity depend on NMDA receptor activation. Since the onset of plasticity is very rapid and requires little extra activity, we propose that these forms of plasticity might play a role already during an ongoing search for odor sources. Our results imply that components of both short-term and long-term olfactory memory may be encoded at this synapse.

Three-dimensional reconstruction of electron micrographs reveals intrabulbar circuit differences between accessory and main olfactory bulbs

Three-dimensional (3D) reconstruction of synaptic arrangement on a particular dendrite provides essential information regarding neuronal properties and neural microcircuits. Unconventional synapses are particularly good candidates for such steric attribution. In main and accessory olfactory bulbs (MOBs and AOBs), there are dendrodendritic reciprocal synapses (RSs) between excitatory projection neurons and inhibitory interneurons. Although the fine structure and configuration of these synapses have been investigated in MOB, their characteristics in AOB were unknown. In this study, we performed 3D AOB reconstruction using serial section transmission electron microscopy. We found numerous RSs on primary dendrites from glomeruli to mitral/tufted (MT) cell somas. These synapses formed between dendritic shafts of MT cells and large dendritic spines, or so-called gemmules, of granule (Gr) cells. This indicates that chemical signals received by a glomerulus are regulated in the primary dendrite of an MT cell before reaching its soma. In MOB, RSs are located on secondary dendrites and act as lateral and self-inhibiting following mitral cell depolarization. Our results indicate that AOB intrabulbar microcircuitry is quite different from that in the MOB.

Noradrenaline‐induced enhancement of oscillatory local field potentials in the mouse accessory olfactory bulb does not depend on disinhibition of mitral cells

The olfactory bulb differs from other brain regions by its use of bidirectional synaptic transmission at dendrodendritic reciprocal synapses. These reciprocal synapses provide tight coupling of inhibitory feedback from granule cell interneurons to mitral cell projection neurons in the accessory olfactory bulb (AOB), at the first stage of vomeronasal processing. It has been proposed that both the mGluR2 agonist DCG-IV and noradrenaline promote mate recognition memory formation by reducing GABAergic feedback on mitral cells. The resultant mitral cell disinhibition is thought to induce a long-lasting enhancement in the gain of inhibitory feedback from granule to mitral cells, which selectively gates the transmission of the learned chemosensory information. However, we found that local infusions of both noradrenaline and DCG-IV failed to disinhibit AOB neural activity in urethane-anaesthetised mice. DCG-IV infusion had similar effects to the GABA(A) agonist isoguvacine, suggesting that it increased GABAergic inhibition in the AOB rather than reducing it. Noradrenaline infusion into the AOB also failed to disinhibit mitral cells in awake mice despite inducing long-term increases in power of AOB local field potentials, similar to those observed following memory formation. These results suggest that mitral cell disinhibition is not essential for the neural changes in the AOB that underlie mate recognition memory formation in mice.

Sparse Incomplete Representations: A Potential Role of Olfactory Granule Cells

Mitral/tufted cells of the olfactory bulb receive odorant information from receptor neurons and transmit this information to the cortex. Studies in awake behaving animals have found that sustained responses of mitral cells to odorants are rare, suggesting sparse combinatorial representation of the odorants. Careful alignment of mitral cell firing with the phase of the respiration cycle revealed brief transient activity in the larger population of mitral cells, which respond to odorants during a small fraction of the respiration cycle. Responses of these cells are therefore temporally sparse. Here, we propose a mathematical model for the olfactory bulb network that can reproduce both combinatorially and temporally sparse mitral cell codes. We argue that sparse codes emerge as a result of the balance between mitral cells' excitatory inputs and inhibition provided by the granule cells. Our model suggests functional significance for the dendrodendritic synapses mediating interactions between mitral and granule cells.

Interplay of periglomerular and granule cell inhibitory synapses on mitral cell spiking

Mitral cells in the olfactory bulb (OB) receive dendrodendritic inhibitory synaptic inputs at two different levels of OB: at the glomerular level, inhibitory input is provided by periglomerular (PG) cells and, at a deeper level (the external plexiform layer), inhibitory input is provided by granule (Gr) cells [1]. It is thought that both kinds of inhibition control mitral cell spiking in specific ways: PG early inhibition would regulate spike initiation while Gr inhibition would coordinate spike synchronization among mitral cells [1,2]. However, recent computational modeling studies have suggested that, although acting at separate locations, PG and Gr inhibitory synapses may have integrated effects on mitral cell spiking which may be beneficial for odor representation and processing [2]. In this work we constructed in NEURON [3] a model of a triad composed of reduced compartmental models of a mitral cell, a PG cell and a Gr cell to study the combined effect of the inhibitory dendrodendritic synapses of the PG and Gr cells on the firing frequency of the mitral cell. The mitral and Gr cell models were taken from [4] with all parameters and characteristics kept as in the original. The PG cell model was based on the five-compartment model used in [5]. The cells were coupled by models of their reciprocal dendrodendritic synapses. The main parameters investigated in our simulations were the conductances of the dendrodendritic synapses, the amplitudes, durations and initiation times of the excitatory inputs applied to the mitral and PG cells representing stimuli coming from olfactory sensory neurons, and the amplitude, duration and initiation time of the input applied to the Gr cell representing afferents from other cells in the olfactory bulb. We performed simulations in which these parameters were varied and studied their effects on the mitral cell’s spiking pattern. Our results show that the response pattern of the mitral cell is strongly dependent of the two inhibitory synapses, and that their interaction can regulate both the spike frequency and timing of the mitral cell. Figure ​Figure11 illustrates one of these results. Figure 1 Firing rate of mitral cell as a function of maximal conductances of inhibitory synapses made by PG (horizontal axis) and Gr (vertical axis) cells. The color-code bar on the right indicates frequency in Hz.

Somatostatin interneurons delineate the inner part of the external plexiform layer in the mouse main olfactory bulb

Neuropeptides play a major role in the modulation of information processing in neural networks. Somatostatin, one of the most concentrated neuropeptides in the brain, is found in many sensory systems including the olfactory pathway. However, its cellular distribution in the mouse main olfactory bulb (MOB) is yet to be characterized. Here we show that approximately 95% of mouse bulbar somatostatin-immunoreactive (SRIF-ir) cells describe a homogeneous population of interneurons. These are restricted to the inner lamina of the external plexiform layer (iEPL) with dendritic field strictly confined to the region. iEPL SRIF-ir neurons share some morphological features of Van Gehuchten short-axon cells, and always express glutamic acid decarboxylase, calretinin, and vasoactive intestinal peptide. One-half of SRIF-ir neurons are parvalbumin-ir, revealing an atypical neurochemical profile when compared to SRIF-ir interneurons of other forebrain regions such as cortex or hippocampus. Somatostatin is also present in fibers and in a few sparse presumptive deep short-axon cells in the granule cell layer (GCL), which were previously reported in other mammalian species. The spatial distribution of somatostatin interneurons in the MOB iEPL clearly outlines the region where lateral dendrites of mitral cells interact with GCL inhibitory interneurons through dendrodendritic reciprocal synapses. Symmetrical and asymmetrical synaptic contacts occur between SRIF-ir dendrites and mitral cell dendrites. Such restricted localization of somatostatin interneurons and connectivity in the bulbar synaptic network strongly suggest that the peptide plays a functional role in the modulation of olfactory processing.

Calcium buffering in rodent olfactory bulb granule cells and mitral cells

In the mammalian olfactory bulb, axonless granule cells (GCs) mediate self- and lateral inhibitory interactions between mitral cells (MCs) via reciprocal dendrodendritic synapses. Calcium signals in the GC dendrites and reciprocal spines appear to decay unusually slowly, hence GC calcium handling might contribute to the known asynchronous release at this synapse. By recording fluorescence transients of different Ca(2+)-sensitive dyes at variable concentrations evoked by backpropagating action potentials (APs) and saturating AP trains we extrapolated Ca(2+) dynamics to conditions of zero added buffer for juvenile rat GC apical dendrites and spines and MC lateral dendrites. Resting [Ca(2+)] was at approximately 50 nM in both GC dendrites and spines. The average endogenous GC buffer capacities (kappa(E)) were within a range of 80-90 in the dendrites and 110-140 in the spines. The extrusion rate (gamma) was estimated as 570 s(-1) for dendrites and 870 s(-1) for spines and the decay time constant as approximately 200 ms for both. Single-current-evoked APs resulted in a [Ca(2+)] elevation of approximately 250 nM. Calcium handling in juvenile and adult mouse GCs appeared mostly similar. In MC lateral dendrites, we found AP-mediated [Ca(2+)] elevations of approximately 130 nM with a similar decay to that in GC dendrites, while kappa(E) and gamma were roughly 4-fold higher. In conclusion, the slow GC Ca(2+) dynamics are due mostly to sluggish Ca(2+) extrusion. Under physiological conditions this slow removal may well contribute to delayed release and also feed into other Ca(2+)-dependent mechanisms that foster asynchronous output from the reciprocal spine.

Role of Cortical Feedback in Regulating Inhibitory Microcircuits

The olfactory bulb contains an impressive array of specialized inhibitory local circuits. The most frequent inhibitory microcircuit in this brain region is the reciprocal dendrodendritic synapse formed between the lateral dendrites of mitral cells and distal dendritic spines of GABAergic granule cells. Recent work discussed in this review suggests that release of GABA from granule cell spines may reflect near-coincident activation of both mitral cell-to-granule cell synapses and proximal excitatory synapses on granule cells that originate from pyramidal cells in piriform cortex. Recent work using two-photon guided microstimulation demonstrated that proximal and distal excitatory synapses onto granule cells exhibit different forms of short-term plasticity, with feedback inputs from piriform cortex facilitating when tested with brief ( approximately 50 ms) interstimulus intervals. One consequence of this synaptic plasticity is that short duration, gamma-frequency, oscillatory discharges in piriform cortical cells evoke summating excitatory postsynaptic potentials (EPSPs) in granule cells that effectively trigger action potentials. Piriform cortex stimulation can gate dendrodendritic inhibition onto mitral cells, presumably through the ability of EPSP-driven action potentials in granule cells to temporarily relieve the tonic blockade of NMDA receptors by extracellular Mg(2+) ions. Feedback projections in other CNS systems also may target inhibitory neurons, such as the backprojection from CA3 pyramidal neurons to GABAergic hilar interneurons. The ability of downstream processing areas to rapidly and selectively activate inhibitory interneurons in other brain regions may provide an important mechanism to dynamically modulate biological information processing.

Presynaptic Muscarinic Receptors Enhance Glutamate Release at the Mitral/Tufted to Granule Cell Dendrodendritic Synapse in the Rat Main Olfactory Bulb

The mammalian olfactory bulb receives multiple modulatory inputs, including a cholinergic input from the basal forebrain. Understanding the functional roles played by the cholinergic input requires an understanding of the cellular mechanisms it modulates. In an in vitro olfactory bulb slice preparation we demonstrate cholinergic muscarinic modulation of glutamate release onto granule cells that results in gamma-aminobutyric acid (GABA) release onto mitral/tufted cells. We demonstrate that the broad-spectrum cholinergic agonist carbachol triggers glutamate release from mitral/tufted cells that activates both AMPA and NMDA receptors on granule cells. Activation of the granule cell glutamate receptors leads to calcium influx through voltage-gated calcium channels, resulting in spike-independent, asynchronous GABA release at reciprocal dendrodendritic synapses that granule cells form with mitral/tufted cells. This cholinergic modulation of glutamate release persists through much of postnatal bulbar development, suggesting a functional role for cholinergic inputs from the basal forebrain in bulbar processing of olfactory inputs and possibly in postnatal development of the olfactory bulb.

Vomeronasal neurons promote synaptic formation on dendritic spines but not dendritic shafts in primary culture of accessory olfactory bulb neurons

To investigate the morphological changes of accessory olfactory bulb (AOB) neurons arising from pheromonal signals, a coculture system of AOB neurons and vomeronasal (VN) neurons had been established. Our previous study indicates that under coculture condition, the density of dendritic spines of an AOB neuron is less and the individual spine-head volume is larger than those under monoculture condition. In this study, to determine whether these differences in the dendrites of AOB neurons reflect the differences in synapse formation and synaptic properties, we observed these cultured cells by electron microscopy. Various synapses were observed under each culture condition. Synapses were classified on the basis of their postsynaptic structure and the size of postsynaptic density (PSD) was measured. Under the coculture condition with VN neurons, synapses on dendritic spines, which formed between AOB neurons, were observed frequently. In contrast, many synapses were formed on dendritic shafts under monoculture condition. The PSD of asymmetrical synapses on the spines under coculture condition was larger than that under monoculture condition. Moreover, some dendrodendritic reciprocal synapses were found only in coculture. We confirmed synapse formation between VN axons and AOB dendrites by immunohistochemical electron microscopy; thus, the characteristics of synapses between AOB neurons are considered to be modified by the synaptic contacts with VN axons.

Synaptic sodium spikes trigger long‐lasting depolarizations and slow calcium entry in rat olfactory bulb granule cells

In the mammalian olfactory bulb, axonless granule cells mediate self- and lateral inhibitory interactions between mitral/tufted cells via reciprocal dendrodendritic synapses. Synaptic output from granule cells occurs on both fast and slow timescales, allowing for multiple granule cell functions during olfactory processing. We find that granule cell sodium action potentials evoked by synaptic activation of the sensory input via mitral/tufted cells are followed by a long-lasting depolarization that is not observed after current-evoked action potentials or large excitatory postsynaptic potentials in the same cell. Using two-photon imaging in acute rat brain slices, we demonstrate that this prolonged electrical response is paralleled by an unusual, long-lasting postsynaptic calcium signal. We find that this slow synaptic Ca(2+) signal requires sequential activation of NMDA receptors, a nonselective cation conductance I(CAN) and T-type voltage-dependent Ca(2+) channels. Remarkably, T-type Ca(2+) channels are of critical importance for the 'globalization' of Ca(2+) transients. In individual active spines, the local synaptic Ca(2+) signal summates at least linearly with the global spike-mediated Ca(2+) signal. We suggest that this robust slow synaptic Ca(2+) signal triggers dendritic transmitter release and thus contributes to slow synaptic output of the granule cell. Therefore, the synaptic sodium spike signal could represent a special adaptation of granule cells to the wide range of temporal requirements for their dendritic output. Our findings demonstrate with respect to neuronal communication in general that action potentials evoked by somatic current injection may lack some of the information content of 'true' synaptically evoked spikes.

Dendritic transmitter release and analog computation in mitral cells

Mitral cells of the accessory olfactory bulb contain between 2–10 principal dendrites, each of which terminates in a densely branched tuft contained in the glomerular layer. This multi-tufted morphology has led to the proposal that tufts may serve as local subthreshold processing stations that function independently of one another and in relative isolation from the soma. Consistent with this, we have found that focal synaptic stimulation of individual tufts results in regenerative calcium spikes restricted to the tuft (n = 7). In addition, tufts are closely associated with local interneurons called periglomerular (PG) cells, with which they from reciprocal dendrodendritic synapses. In experiments in which mitral cells were patched and filled with the calcium indicator Calcium Orange, we found a strong covariation between tuft calcium influx and inhibition received by mitral cells (n = 8), suggesting that tufts may self regulate their excitability via subthreshold synaptic mechanisms. To test this more directly, we first patched mitral cells with normal internal and evoked inhibition by activation of tuft spikes via afferent inputs. We then repatched with internal containing 1 mM BAPTA to buffer intracellular calcium concentration, which should reduce release from the mitral cell (n = 5). The magnitude of inhibition received by the mitral cell decreased by 75% ± 27% within 2 minutes of BAPTA repatch. In a final set of experiments, we performed paired recordings of mitral and PG cells and found that synaptically evoked tuft calcium transients were highly correlated with the amplitude of EPSPs recorded in PG cells. Moreover, the synaptic input to PG cells was reduced by hyperpolarizing the mitral cell. The above data motivate a model of synaptic integration in AOB mitral cells in which each tuft acts as an independent processing station capable of providing synaptic output to local interneurons. We are currently exploring the computational consequences of this phenomenon by modeling AOB mitral cells as binary classifiers and exploring how dendritic transmitter release contributes to their ability to discriminate among analog patterns.

Long-term Potentiation in the Accessory Olfactory Bulb: A Mechanism for Olfactory Learning

One context of olfactory learning that has been investigated in some detail concerns the memory, established at mating, and formed by the female mouse to the odours (pheromones) of the mating male. This olfactory memory is vital for mitigating pregnancy block that might otherwise be induced by his pheromones (Keverne and Rosser, 1986). Pheromones from an unfamiliar male, for which no memory has been formed, activate the vomeronasal system with their receptors in the vomeronasal organ, thereby initiating a neuroendocrine reflex that suppresses prolactin secretion from the pituitary (Keverne, 1983). The removal of luteotrophic support results in a fall in progesterone levels and a return to oestrus. Therefore, it has been hypothesized that the pheromonal memory functions as a gate to suppress or modulate the specific pheromonal signal (Brennan et al., 1990; Kaba and Nakanishi, 1995; Brennan and Keverne, 1997; Brennan, 2001). The pheromonal memory is acquired with one trial learning, depends upon mating and lasts for several weeks (Keverne and de la Riva, 1982; Kaba et al., 1988). The neural changes underlying memory formation occur in the accessory olfactory bulb (AOB), the first relay in the vomeronasal system, independently of the main olfactory system and the hippocampus (Brennan et al., 1990; Kaba and Nakanishi, 1995; Brennan and Keverne, 1997). Microcircuits in the AOB include the prominent reciprocal dendrodendritic synapse between mitral cells, a single class of projection neurons and granule cell interneurons. Glutamate released from mitral cell dendrites activates the dendrites of granule cells, which in turn mediate GABAergic dendrodendritic inhibition back onto mitral cell dendrites (Jia et al., 1999; Taniguchi and Kaba, 2001). This feedback inhibition at the reciprocal synapses regulates mitral cell activity (Jia et al., 1999; Taniguchi and Kaba, 2001). The formation of the pheromonal memory requires the association of the pheromonal and the mating signals in the AOB. The mating signal is conveyed by noradrenergic projections from the locus coeruleus. Artificial vaginocervical stimulation (Rosser and Keverne, 1985) or mating (Brennan et al., 1995) promotes the release of noradrenaline (NA) in the AOB. Blockade of α-adrenergic receptors in the AOB immediately after mating prevents the formation of the pheromonal memory (Kaba and Keverne, 1988), as does removal of noradrenergic innervation of the AOB prior to mating (Rosser and Keverne, 1985). Furthermore, memory formation is associated with neurochemical and morphological changes at the mitral–granule cell reciprocal synapses (Brennan et al., 1995; Matsuoka et al., 1997, 2004). Despite advances such as these, however, an important void in our knowledge of electrophysiological aspects has remained: a longlasting increase in synaptic strength, known as long-term potentiation (LTP), has been little investigated. Moreover, the cellular and synaptic mechanisms underlying noradrenergic modulation of pheromonal learning are also unknown. To address these questions, we have carried out a series of experiments in AOB slices. Induction of LTP at the mitral-to-granule cell synapse in the AOB

Pre‐ and postsynaptic GABAA receptors at reciprocal dendrodendritic synapses in the olfactory bulb

Presynaptic ionotropic receptors are important regulators of synaptic function; however, little is known about their organization in the presynaptic membrane. We show here a different spatial organization of presynaptic and postsynaptic GABA(A) receptors at reciprocal dendrodendritic synapses between mitral and granule cells in the rat olfactory bulb. Using postembedding electron microscopy, we have found that mitral cell dendrites express GABA(A) receptors at postsynaptic specializations of symmetric (GABAergic) synapses, as well as at presynaptic sites of asymmetric (glutamatergic) synapses. Analysis of the subsynaptic distribution of gold particles revealed that in symmetric synapses GABA(A) receptors are distributed along the entire postsynaptic membrane, whereas in asymmetric synapses they are concentrated at the edge of the presynaptic specialization. To assess the specificity of immunogold labelling, we analysed the olfactory bulbs of mutant mice lacking the alpha1 subunit of GABA(A) receptors. We found that in wild-type mice alpha1 subunit immunoreactivity was similar to that observed in rats, whereas in knockout mice the immunolabelling was abolished. These results indicate that in mitral cell dendrites GABA(A) receptors are distributed in a perisynaptic domain that surrounds the presynaptic specialization. Such presynaptic receptors may be activated by spillover of GABA from adjacent inhibitory synapses and modulate glutamate release, thereby providing a novel mechanism regulating dendrodendritic inhibition in the olfactory bulb.

Mechanisms of Lateral Inhibition in the Olfactory Bulb: Efficiency and Modulation of Spike-Evoked Calcium Influx into Granule Cells

Granule cells are axonless local interneurons that mediate lateral inhibitory interactions between the principal neurons of the olfactory bulb via dendrodendritic reciprocal synapses. This unusual arrangement may give rise to functional properties different from conventional lateral inhibition. Although granule cells spike, little is known about the role of the action potential with respect to their synaptic output. To investigate the signals that underlie dendritic release in these cells, two-photon microscopy in rat brain slices was used to image calcium transients in granule cell dendrites and spines. Action potentials evoked calcium transients throughout the dendrites, with amplitudes increasing with distance from soma and attaining a plateau level within the external plexiform layer, the zone of granule cell synaptic output. Transient amplitudes were, on average, equal in size in spines and adjacent dendrites. Surprisingly, both spine and dendritic amplitudes were strongly dependent on membrane potential, decreasing with depolarization and increasing with hyperpolarization from rest. Both the current-voltage relationship and the time course of inactivation were consistent with the known properties of T-type calcium channels, and the voltage dependence was blocked by application of the T-type calcium channel antagonists Ni2+ and mibefradil. In addition, mibefradil reduced action potential-mediated synaptic transmission from granule to mitral cells. The implication of a transiently inactivating calcium channel in synaptic release from granule cells suggests novel mechanisms for the regulation of lateral inhibition in the olfactory bulb.

Organization of postsynaptic density proteins and glutamate receptors in axodendritic and dendrodendritic synapses of the rat olfactory bulb.

Glutamate neurotransmission in the olfactory bulb involves both axodendritic synapses and dendrodendritic reciprocal synapses and possibly also extrasynaptic receptors. By using a sensitive immunogold procedure, we have investigated the organization of two synaptic scaffolding molecules, PSD-95 and PSD-93, as well as N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors, at these heterogeneous glutamate signaling sites. Immunolabeling for PSD-95 and PSD-93 was present in all major types of putative glutamatergic synapse, suggesting that these proteins are essential components of the synaptic signaling apparatus. The linear density and the subsynaptic distribution of PSD-95/PSD-93 gold particles did not differ significantly between axodendritic and dendrodendritic synapses. Antibodies recognizing NMDA and AMPA receptor subunits also labeled asymmetric synapses throughout the olfactory bulb. Immunolabeling for the AMPA receptor subunits GluR2/3 was similar in all types of synapse. In contrast, immunogold signals for the NR1 subunit of NMDA receptors varied significantly among different synapse populations, with olfactory nerve synapses in the glomerular layer showing the lowest labeling intensity. Although the lateral dendrites of mitral and tufted cells have been reported to respond to glutamate, they did not display significant plasma membrane labeling for the NR1 subunit or for PSD-95, suggesting that the physiological effects of glutamate at these sites are mediated by NMDA autoreceptors that are not clustered and occur only at a low density on the dendritic surface. Our quantitative analysis of olfactory bulb synapses indicates that the density of NMDA receptors is not determined by the complement of PSD-95/PSD-93. The latter molecules appear to be expressed in an all-or-none fashion and may form a standard lattice common to different types of glutamatergic synapse.

Gamma-frequency excitatory input to granule cells facilitates dendrodendritic inhibition in the rat olfactory Bulb.

Recurrent and lateral inhibition play a prominent role in patterning the odor-evoked discharges in mitral cells, the output neurons of the olfactory bulb. Inhibitory responses in this brain region are mediated through reciprocal synaptic connections made between the dendrites of mitral cells and GABAergic interneurons. Previous studies have demonstrated that N-methyl-D-aspartate (NMDA) receptors on interneurons play a critical role in eliciting GABA release at reciprocal dendrodendritic synapses. In acute olfactory bulb slices, these receptors are tonically blocked by extracellular Mg2+, and recurrent inhibition is disabled. In the present study, we examined the mechanisms by which this tonic blockade could be reversed. We demonstrate that near-coincident activation of an excitatory pathway to the proximal dendrites of GABAergic interneurons relieves the Mg2+ blockade of NMDA receptors at reciprocal dendrodendritic synapses and greatly facilitates recurrent inhibition onto mitral cells. Gating of recurrent and lateral inhibition in the presence of extracellular Mg2+ requires gamma-frequency stimulation of glutamatergic axons in the granule cell layer. Long-range excitatory axon connections from mitral cells innervated by different subpopulations of olfactory receptor neurons may provide a gating input to granule cells, thereby facilitating the mitral cell lateral inhibition that contributes to odorant encoding.