In vitro and in silico multidimensional modeling of oncolytic tumor virotherapy dynamics.

Tumor therapy with replication competent viruses is an exciting approach to cancer eradication where viruses are engineered to specifically infect, replicate, spread and kill tumor cells. The outcome of tumor virotherapy is complex due to the variable interactions between the cancer cell and virus populations as well as the immune response. Oncolytic viruses are highly efficient in killing tumor cells in vitro, especially in a 2D monolayer of tumor cells, their efficiency is significantly lower in a 3D environment, both in vitro and in vivo. This indicates that the spatial dimension may have a major influence on the dynamics of virus spread. We study the dynamic behavior of a spatially explicit computational model of tumor and virus interactions using a combination of in vitro 2D and 3D experimental studies to inform the models. We determine the number of nearest neighbor tumor cells in 2D (median = 6) and 3D tumor spheroids (median = 16) and how this influences virus spread and the outcome of therapy. The parameter range leading to tumor eradication is small and even harder to achieve in 3D. The lower efficiency in 3D exists despite the presence of many more adjacent cells in the 3D environment that results in a shorter time to reach equilibrium. The mean field mathematical models generally used to describe tumor virotherapy appear to provide an overoptimistic view of the outcomes of therapy. Three dimensional space provides a significant barrier to efficient and complete virus spread within tumors and needs to be explicitly taken into account for virus optimization to achieve the desired outcome of therapy.

Activation of ß-noradrenergic receptors enhances rhythmic bursting in mouse olfactory bulb external tufted cells.

The main olfactory bulb (MOB) receives a rich noradrenergic innervation from the nucleus locus coeruleus. Despite the well-documented role of norepinephrine and ß-adrenergic receptors in neonatal odor preference learning, identified cellular physiological actions of ß-receptors in the MOB have remained elusive. ß-Receptors are expressed at relatively high levels in the MOB glomeruli, the location of external tufted (ET) cells that exert an excitatory drive on mitral and other cell types. The present study investigated the effects of ß-receptor activation on the excitability of ET cells with patch-clamp electrophysiology in mature mouse MOB slices. Isoproterenol and selective ß2-, but not ß1-, receptor agonists were found to enhance two key intrinsic currents involved in ET burst initiation: persistent sodium (INaP) and hyperpolarization-activated inward (Ih) currents. Together, the positive modulation of these currents increased the frequency and strength of ET cell rhythmic bursting. Rodent sniff frequency and locus coeruleus neuronal firing increase in response to novel stimuli or environments. The increase in ET excitability by ß-receptor activation may better enable ET cell rhythmic bursting, and hence glomerular network activity, to pace faster sniff rates during heightened norepinephrine release associated with arousal.

Characteristics of GABAergic and cholinergic neurons in perinuclear zone of mouse supraoptic nucleus.

The perinuclear zone (PNZ) of the supraoptic nucleus (SON) contains some GABAergic and cholinergic neurons thought to innervate the SON proper. In mice expressing enhanced green fluorescent protein (eGFP) in association with glutamate decarboxylase (GAD)65 we found an abundance of GAD65-eGFP neurons in the PNZ, whereas in mice expressing GAD67-eGFP, there were few labeled PNZ neurons. In mice expressing choline acetyltransferase (ChAT)-eGFP, large, brightly fluorescent and small, dimly fluorescent ChAT-eGFP neurons were present in the PNZ. The small ChAT-eGFP and GAD65-eGFP neurons exhibited a low-threshold depolarizing potential consistent with a low-threshold spike, with little transient outward rectification. Large ChAT-eGFP neurons exhibited strong transient outward rectification and a large hyperpolarizing spike afterpotential, very similar to that of magnocellular vasopressin and oxytocin neurons. Thus the large soma and transient outward rectification of large ChAT-eGFP neurons suggest that these neurons would be difficult to distinguish from magnocellular SON neurons in dissociated preparations by these criteria. Large, but not small, ChAT-eGFP neurons were immunostained with ChAT antibody (AB144p). Reconstructed neurons revealed a few processes encroaching near and passing through the SON from all types but no clear evidence of a terminal axon arbor. Large ChAT-eGFP neurons were usually oriented vertically and had four or five dendrites with multiple branches and an axon with many collaterals and local arborizations. Small ChAT-eGFP neurons had a more restricted dendritic tree compared with parvocellular GAD65 neurons, the latter of which had long thin processes oriented mediolaterally. Thus many of the characteristics found previously in unidentified, small PNZ neurons are also found in identified GABAergic neurons and in a population of smaller ChAT-eGFP neurons.

Activation of group I metabotropic glutamate receptors enhances persistent sodium current and rhythmic bursting in main olfactory bulb external tufted cells.

Rhythmically bursting olfactory bulb external tufted (ET) cells are thought to play a key role in synchronizing glomerular network activity to respiratory-driven sensory input. Whereas spontaneous bursting in these cells is intrinsically generated by interplay of several voltage-dependent currents, bursting strength and frequency can be modified by local intrinsic and centrifugal synaptic input. Activation of metabotropic glutamate receptors (mGluRs) engages a calcium-dependent cation current (I(CAN)) that increases rhythmic bursting, but mGluRs may also modulate intrinsic mechanisms involved in bursting. Here, we used patch-clamp electrophysiology in rat olfactory bulb slices to investigate whether mGluRs modulate two key intrinsic currents involved in ET cell burst initiation: persistent sodium (I(NaP)) and hyperpolarization-activated cation (Ih) currents. Using a BAPTA-based internal solution to block I(CAN), we found that the mGluR1/5 agonist DHPG enhanced I(NaP) but did not alter Ih. I(NaP) enhancement consisted of increased current at membrane potentials between -60 and -50 mV and a hyperpolarizing shift in activation threshold. Both effects would be predicted to shorten the interburst interval. In agreement, DHPG modestly depolarized (∼3.5 mV) ET cells and increased burst frequency without effect on other major burst parameters. This increase was inversely proportional to the basal burst rate such that slower ET cells exhibited the largest increases. This may enable ET cells with slow intrinsic burst rates to pace with faster sniff rates. Taken with other findings, these results indicate that multiple neurotransmitter mechanisms are engaged to fine-tune rhythmic ET cell bursting to context- and state-dependent changes in sniffing frequency.

Nonlinear effects of noradrenergic modulation of olfactory bulb function in adult rodents.

The mammalian main olfactory bulb receives a significant noradrenergic input from the locus coeruleus. Norepinephrine (NE) is involved in acquisition of conditioned odor preferences in neonatal animals, in some species-specific odor-dependent behaviors, and in adult odor perception. We provide a detailed review of the functional role of NE in adult rodent main olfactory bulb function. We include cellular, synaptic, network, and behavioral data and use computational simulations to tie these different types of data together.

Mutations in the stalk region of the measles virus hemagglutinin inhibit syncytium formation but not virus entry.

Measles virus (MV) entry requires at least 2 viral proteins, the hemagglutinin (H) and fusion (F) proteins. We describe the rescue and characterization of a measles virus with a specific mutation in the stalk region of H (I98A) that is able to bind normally to cells but infects at a lower rate than the wild type due to a reduction in fusion triggering. The mutant H protein binds to F more avidly than the parent H protein does, and the corresponding virus is more sensitive to inhibition by fusion-inhibitory peptide. We show that after binding of MV to its receptor, H-F dissociation is required for productive infection.

The Interdependency and Co-Regulation of the Vitamin D and Cholesterol Metabolism.

Vitamin D and cholesterol metabolism overlap significantly in the pathways that contribute to their biosynthesis. However, our understanding of their independent and co-regulation is limited. Cardiovascular disease is the leading cause of death globally and atherosclerosis, the pathology associated with elevated cholesterol, is the leading cause of cardiovascular disease. It is therefore important to understand vitamin D metabolism as a contributory factor. From the literature, we compile evidence of how these systems interact, relating the understanding of the molecular mechanisms involved to the results from observational studies. We also present the first systems biology pathway map of the joint cholesterol and vitamin D metabolisms made available using the Systems Biology Graphical Notation (SBGN) Markup Language (SBGNML). It is shown that the relationship between vitamin D supplementation, total cholesterol, and LDL-C status, and between latitude, vitamin D, and cholesterol status are consistent with our knowledge of molecular mechanisms. We also highlight the results that cannot be explained with our current knowledge of molecular mechanisms: (i) vitamin D supplementation mitigates the side-effects of statin therapy; (ii) statin therapy does not impact upon vitamin D status; and critically (iii) vitamin D supplementation does not improve cardiovascular outcomes, despite improving cardiovascular risk factors. For (iii), we present a hypothesis, based on observations in the literature, that describes how vitamin D regulates the balance between cellular and plasma cholesterol. Answering these questions will create significant opportunities for advancement in our understanding of cardiovascular health.

Group I mGluR activation enhances Ca(2+)-dependent nonselective cation currents and rhythmic bursting in main olfactory bulb external tufted cells.

In the main olfactory bulb, activation of group I metabotropic glutamate receptors (mGluRs) by olfactory nerve stimulation generates slow (2 Hz) oscillations near the basal respiratory frequency. These oscillations arise in the glomerular layer and may be generated, in part, by the intrinsic neurons, the juxtaglomerular neurons. We investigated the physiological effects of group I mGluR agonists on one population of juxtaglomerular neurons, external tufted (ET) cells, which rhythmically burst at respiratory frequencies and synchronize the intraglomerular network. Electrophysiological studies in rat main olfactory bulb slices demonstrated that the mGluR agonist 3,4-dihydroxyphenylglycine (DHPG) amplified the strength of ET cell spike bursts, principally by increasing the number of spikes per burst. Voltage-clamp and Ca(2+)-imaging studies showed that DHPG elicits a Ca(2+)-dependent nonselective cation current (I(CAN)) in the dendrites of ET cells triggered by Ca(2+) release from internal stores. The DHPG effects on bursting and membrane current were attenuated by flufenamic acid and SKF96365, agents known to antagonize I(CAN) in a variety of neurons. DHPG also elicited slow membrane current oscillations and spikelets in ET cells when synaptic transmission and intrinsic membrane channels were inoperative. These findings indicate that DHPG may passively (by increasing burst strength) or actively (by increasing conductance of gap junctions) enhance the strength of electrical synapses between ET cells. Together, these findings indicate that activation of group I mGluRs on the dendrites of ET cells play a key role in the generation of slow rhythmic oscillation in the glomerular network, which is in turn tuned to sniffing of the animal in vivo.

Noradrenergic modulation of behavioral odor detection and discrimination thresholds in the olfactory bulb.

The mammalian main olfactory bulb (MOB) receives a significant noradrenergic input from the locus coeruleus. Norepinephrine (NE) is involved in the acquisition of conditioned odor preferences in neonatal animals and in some species-specific odor-dependent behaviors. Thus far, the role of NE in odor processing in adult rats remains less studied. We investigated the role of noradrenergic modulation in the MOB on odor detection and discrimination thresholds using behavioral and computational modeling approaches. Adult rats received bilateral MOB injections of vehicle, NE (0.1-1000 microM), noradrenergic receptor antagonists and NE + receptor antagonists combined. NE infusion improved odor detection and discrimination as a function of NE and odor concentration. The effect of NE on detection and discrimination magnitude at any given odor concentration varied in a non-linear function with respect to NE concentration. Receptor antagonist infusion demonstrated that alpha1 receptor activation is necessary for the modulatory effect of NE. Computational modeling showed that increases in the strength of alpha1 receptor activation leads to improved odor signal-to-noise ratio and spike synchronization in mitral cells that may underlie the behaviorally observed decrease of detection and discrimination thresholds. Our results are the first to show that direct infusion of NE or noradrenergic receptor antagonists into a primary sensory network modulates sensory detection and discrimination thresholds at very low stimulus concentrations.

The guanine nucleotide exchange factors trio, Ect2, and Vav3 mediate the invasive behavior of glioblastoma.

Malignant gliomas are characterized by their ability to invade normal brain tissue. We have previously shown that the small GTPase Rac1 plays a role in both migration and invasion in gliomas. Here, we aim to identify Rac-activating guanine nucleotide exchange factors (GEFs) that mediate glioblastoma invasiveness. Using a brain tumor expression database, we identified three GEFs, Trio, Ect2, and Vav3, that are expressed at higher levels in glioblastoma versus low-grade glioma. The expression of these GEFs is also associated with poor patient survival. Quantitative real-time polymerase chain reaction and immunohistochemical analyses on an independent set of tumors confirmed that these GEFs are overexpressed in glioblastoma as compared with either nonneoplastic brain or low-grade gliomas. In addition, depletion of Trio, Ect2, and Vav3 by siRNA oligonucleotides suppresses glioblastoma cell migration and invasion. Depletion of either Ect2 or Trio also reduces the rate of cell proliferation. These results suggest that targeting GEFs may present novel strategies for anti-invasive therapy for malignant gliomas.

In Vivo Imaging of Oncolytic Measles Virus Propagation with Single-Cell Resolution.

Recombinant measles viruses (MVs) have oncolytic activity against a variety of human cancers. However, their kinetics of spread within tumors has been unexplored. We established an intravital imaging system using the dorsal skin fold chamber, which allows for serial, non-invasive imaging of tumor cells and replication of a fusogenic and a hypofusogenic MV. Hypofusogenic virus-infected cells were detected at the earliest 3 days post-infection (dpi), with peak infection around 6 dpi. In contrast, the fusogenic virus replicated faster: infected cells were detectable 1 dpi and cells were killed quickly. Infection foci were significantly larger with the fusogenic virus. Both viruses formed syncytia. The spatial relationships between cells have a major influence on the outcome of therapy with oncolytic viruses.

Activation of group I metabotropic glutamate receptors on main olfactory bulb granule cells and periglomerular cells enhances synaptic inhibition of mitral cells.

Granule and periglomerular cells in the main olfactory bulb express group I metabotropic glutamate receptors (mGluRs). The group I mGluR agonist 3,4-dihydroxyphenylglycine (DHPG) increases GABAergic spontaneous IPSCs (sIPSCs) in mitral cells, yet the presynaptic mechanism(s) involved and source(s) of the IPSCs are unknown. We investigated the actions of DHPG on sIPSCs and TTX-insensitive miniature IPSCs (mIPSCs) recorded in mitral and external tufted cells in rat olfactory bulb slices. DHPG, acting at mGluR1 and mGluR5, increased the rate but not amplitude of sIPSCs and mIPSCs in both cell types. The increase in mIPSCs depended on voltage-gated Ca2+ channels but persisted when ionotropic glutamate receptors and sodium spikes were blocked. Focal DHPG puffs onto granule cells or bath application after glomerular layer (GL) excision failed to increase mIPSCs in mitral cells. Additionally, GL excision reduced sIPSCs in mitral cells by 50%, suggesting that periglomerular cells exert strong tonic GABAergic inhibition of mitral cells. In contrast, GL DHPG puffs readily increased mIPSCs. These findings indicate that DHPG-evoked GABA release from granule cells requires spikes, whereas in the GL, DHPG facilitates periglomerular cell GABA release via both spike-dependent and spike-independent presynaptic mechanisms. We speculate that mGluRs amplify spike-driven lateral inhibition through the mitral-to-granule cell circuit, whereas GL mGluRs may play a more important role in amplifying intraglomerular inhibition after subthreshold input.

Noradrenergic modulation in the olfactory bulb influences spontaneous and reward-motivated discrimination, but not the formation of habituation memory.

The mammalian main olfactory bulb receives a significant noradrenergic input from the locus coeruleus. Norepinephrine is involved in acquisition of conditioned odor preferences in neonatal animals and in some species-specific odor dependent behaviors. Thus far, the role of norepinephrine in odor processing in adult rats remains less studied. We tested the role of noradrenergic modulation in the olfactory bulb of cannulated rats by bilateral injections of vehicle (6 microL saline), the alpha noradrenergic receptor antagonist phentolamine (3.15 or 10 mM), the beta noradrenergic receptor antagonist alprenolol (12 or 120 mM), the alpha1 noradrenergic receptor antagonist prazosin (1 or 10(-2) mM) and the alpha2 noradrenergic receptor antagonist yohimbine (2 or 0.02 mM) 20 min before two different behavioral tasks. We found that local blockade of noradrenergic receptors in the olfactory bulb did not affect the formation of habituation memory to an odorant over sequential presentations separated by 5-min intertrial intervals. However, spontaneous discrimination between chemically related odorants was impaired when noradrenergic receptors, and in particular alpha1 receptors, were blocked by local antagonist infusion into the olfactory bulb. By contrast, discrimination was improved when beta receptors were blocked. These results show that although the formation of a habituation memory to odorants is not affected by noradrenergic modulation, the specificity of this memory is affected. In contrast, reward-motivated discrimination learning was not impaired, but slowed down in rats in which both alpha and beta receptors had been blocked.

In Vivo Estimation of Oncolytic Virus Populations within Tumors.

The use of replication-competent viruses as oncolytic agents is rapidly expanding, with several oncolytic viruses approved for cancer therapy. As responses to therapy are highly variable, understanding the dynamics of therapy is critical for optimal application of virotherapy in practice. Although mathematical models have been developed to understand the dynamics of tumor virotherapy, a scarcity of in vivo data has made difficult parametrization of these models. To tackle this problem, we studied the in vitro and in vivo spread of two oncolytic measles viruses that induce expression of the sodium iodide symporter (NIS) in cells. NIS expression enabled infected cells to concentrate radioactive isotopes that could be reproducibly and quantitatively imaged using SPECT/CT. We observed a strong linear relationship in vitro between infectious virus particles, viral N and NIS gene expression, and radioactive isotope uptake. In vivo radioisotope uptake was highly correlated with viral N and NIS gene expression. Similar expression patterns between viral N and NIS gene expression in vitro and in vivo implied that the oncolytic virus behaved similarly in both scenarios. Significant titers of viable virus were consistently isolated from tumors explanted from mice that had been injected with oncolytic measle viruses. We observed a weaker but positive in vivo relationship between radioisotope uptake and the viable virus titer recovered from tumors; this was likely due to anisotropies in the viral distribution in vivo These data suggest that methods that enable quantitation of in vivo anisotropies are required for continuing development of oncolytic virotherapy.Significance: These findings address a fundamental gap in our knowledge of oncolytic virotherapy by presenting technology that gives insight into the behavior of oncolytic viruses in vivo Cancer Res; 78(20); 5992-6000. ©2018 AACR.

Inactivation of the periaqueductal gray attenuates antinociception elicited by stimulation of the rat medial preoptic area.

The medial preoptic area (MPOA) is a sexually dimorphic structure that plays key roles in gonado-steroidal regulation and thermoregulation. The MPOA may be involved in sex-based differences in nociceptive processing and steroid hormones effect on pain thresholds. Consistent with this, there is evidence that MPOA can produce antinociception or hyperalgesia. MPOA stimulation inhibits spinal cord or trigeminal neuronal responses to noxious stimuli or produces analgesia, yet most of these studies utilized electrical stimulation which antidromically activates periaqueductal gray (PAG) and rostroventromedial medulla (RVM) neurons involved in descending modulation of nociception. Effects of selective activation of MPOA neurons on behavioral indices of antinociception and the site-specificity of such responses are unknown. To address these questions, we examined the influence of MPOA microinjections of d,l homocysteate (DLH) on hindlimb and tail nocifensive reflexes in lightly anesthetized rats. DLH, but not saline, microinjections into several MPOA subregions markedly increased withdrawal response latencies to noxious thermal stimuli. Antinociceptive effects of MPOA activation were abolished by microinjection of lidocaine into PAG. These results suggest that activation of MPOA neurons produces antinociception that is at least partly mediated by projections to PAG.

Activation of Group II Metabotropic Glutamate Receptors Suppresses Excitability of Mouse Main Olfactory Bulb External Tufted and Mitral Cells.

Metabotropic glutamate receptors (mGluRs) are abundantly expressed in the rodent main olfactory bulb. The function of Group I mGluRs has been investigated in a number of studies, while the actions of Group II mGluRs, which include the mGluR2 and mGluR3 subtypes, have been less well explored. Here, we used electrophysiological approaches in mouse olfactory bulb slices to investigate how Group II mGluR activation and inactivation modifies the activity of external tufted (ET) and mitral cells. The Group II mGluR agonist DCG-IV was found to directly and uniformly reduce the spontaneous discharge of ET and mitral cells. The inhibitory effect of DCG-IV was absent in mitral cells with truncated apical dendrites, indicating a glomerular site of action. DCG-IV did not influence olfactory nerve-evoked monosynaptic responses in ET or mitral cells, indicating that Group II mGluRs do not presynaptically modulate glutamate release from olfactory nerve terminals. In contrast, DCG-IV suppressed polysynaptic responses in periglomerular cells evoked by olfactory nerve stimulation. DCG-IV also inhibited glutamate release from ET cells, and suppressed the spontaneous and olfactory nerve-evoked long-lasting depolarization in mitral cells. Applied alone, Group II receptor antagonists were without effect, suggesting that basal activation of these receptors is nil. These findings suggest that Group II mGluRs inhibit ET and mitral cell activity and further dampen intraglomerular excitatory circuits by suppressing glutamate release.

Olfactory bulb external tufted cells are synchronized by multiple intraglomerular mechanisms.

In rat olfactory bulb slices, external tufted (ET) cells spontaneously generate spike bursts. Only ET cells affiliated with the same glomerulus exhibit significant synchronous activity, suggesting that synchrony results mainly from intraglomerular interactions. The intraglomerular mechanisms underlying their synchrony are unknown. Using dual extracellular and patch-clamp recordings from ET cell pairs of the same glomerulus, we found that the bursting of ET cells is synchronized by several mechanisms. First, ET cell pairs of the same glomerulus receive spontaneous synchronous fast excitatory synaptic input that can also be evoked by olfactory nerve stimulation. Second, they exhibit correlated spontaneous slow excitatory synaptic currents that can also be evoked by stimulation of the external plexiform layer. These slow currents may reflect the repetitive release of glutamate via spillover from the dendritic tufts of other ET or mitral/tufted cells affiliated with the same glomerulus. Third, ET cells exhibit correlated bursts of inhibitory synaptic activity immediately after the synchronous fast excitatory input. These bursts of IPSCs were eliminated by CNQX and may therefore reflect correlated feedback inhibition from periglomerular cells that are driven by ET cell spike bursts. Fourth, in the presence of fast synaptic blockers, ET cell pairs exhibit synchronous slow membrane current oscillations associated with rhythmic spikelets, which were sensitive to the gap junction blocker carbenoxolone. These findings suggest that coordinated synaptic transmission and gap junction coupling synchronize the spontaneous bursting of ET cells of the same glomerulus.

Increased olfactory bulb acetylcholine bi-directionally modulates glomerular odor sensitivity.

The glomerular layer of the olfactory bulb (OB) receives heavy cholinergic input from the horizontal limb of the diagonal band of Broca (HDB) and expresses both muscarinic and nicotinic acetylcholine (ACh) receptors. However, the effects of ACh on OB glomerular odor responses remain unknown. Using calcium imaging in transgenic mice expressing the calcium indicator GCaMP2 in the mitral/tufted cells, we investigated the effect of ACh on the glomerular responses to increasing odor concentrations. Using HDB electrical stimulation and in vivo pharmacology, we find that increased OB ACh leads to dynamic, activity-dependent bi-directional modulation of glomerular odor response due to the combinatorial effects of both muscarinic and nicotinic activation. Using pharmacological manipulation to reveal the individual receptor type contributions, we find that m2 muscarinic receptor activation increases glomerular sensitivity to weak odor input whereas nicotinic receptor activation decreases sensitivity to strong input. Overall, we found that ACh in the OB increases glomerular sensitivity to odors and decreases activation thresholds. This effect, along with the decreased responses to strong odor input, reduces the response intensity range of individual glomeruli to increasing concentration making them more similar across the entire concentration range. As a result, odor representations are more similar as concentration increases.

External tufted cells: a major excitatory element that coordinates glomerular activity.

The glomeruli of the olfactory bulb are the first site of synaptic processing in the olfactory system. The glomeruli contain three types of neurons that are referred to collectively as juxtaglomerular (JG) cells: external tufted (ET), periglomerular (PG), and short axon (SA) cells. JG cells are thought to interact synaptically, but little is known about the circuitry linking these neurons or their functional roles in olfactory processing. Single and paired whole-cell recordings were performed to investigate these questions. ET cells spontaneously fired rhythmic spike bursts in the theta frequency range and received monosynaptic olfactory nerve (ON) input. In contrast, all SA and most PG cells lacked monosynaptic ON input. PG and SA cells exhibited spontaneous, intermittent bursts of EPSCs that were highly correlated with spike bursts of ET cells in the same but not in different glomeruli. Paired recording experiments demonstrated that ET cells provide monosynaptic excitatory input to PG/SA cells; the ET to PG/SA cell synapse is mediated by glutamate. ET cells thus are a major excitatory linkage between ON input and other JG cells. Spontaneous bursting is highly correlated among ET cells of the same glomerulus, and ET cell activity remains correlated when all fast synaptic activity is blocked. The findings suggest that multiple, synchronously active ET cells synaptically converge onto single PG/SA cells. Synchronous ET cell bursting may function to amplify transient sensory input and coordinate glomerular output.

Olfactory bulb glomeruli: external tufted cells intrinsically burst at theta frequency and are entrained by patterned olfactory input.

Glomeruli, the initial sites of synaptic processing in the olfactory system, contain at least three types of neurons collectively referred to as juxtaglomerular (JG) neurons. The role of JG neurons in odor processing is poorly understood. We investigated the morphology, spontaneous, and sensory-evoked activity of one class of JG neurons, external tufted (ET) cells, using whole-cell patch-clamp and extracellular recordings in rat olfactory bulb slices. ET cells have extensive dendrites that ramify within a single glomerulus or, rarely, in two adjacent glomeruli. All ET neurons exhibit spontaneous rhythmic bursts of action potentials (approximately 1-8 bursts/sec). Bursting is intrinsically generated; bursting persisted and became more regular in the presence of ionotropic glutamate and GABA receptor antagonists. Burst frequency is voltage dependent; frequency increased at membrane potentials depolarized relative to rest and decreased during membrane potential hyperpolarization. Spontaneous bursting persisted in blockers of calcium channels that eliminated low-threshold calcium spikes (LTS) in ET cells. ET cells have a persistent sodium current available at membrane potentials that generate spontaneous bursting. Internal perfusion with a fast sodium channel blocker eliminated spontaneous bursting but did not block the LTS. These results suggest that persistent sodium channels are essential for spontaneous burst generation in ET cells. ET cell bursts were entrained to ON stimuli delivered over the range of theta frequencies. Thus, ET cells appear to be tuned to the frequency of sniffing.