Dendritic integration in olfactory bulb granule cells upon simultaneous multispine activation: Low thresholds for nonlocal spiking activity.

The inhibitory axonless olfactory bulb granule cells form reciprocal dendrodendritic synapses with mitral and tufted cells via large spines, mediating recurrent and lateral inhibition. As a case in point for dendritic transmitter release, rat granule cell dendrites are highly excitable, featuring local Na+ spine spikes and global Ca2+- and Na+-spikes. To investigate the transition from local to global signaling, we performed holographic, simultaneous 2-photon uncaging of glutamate at up to 12 granule cell spines, along with whole-cell recording and dendritic 2-photon Ca2+ imaging in acute juvenile rat brain slices. Coactivation of less than 10 reciprocal spines was sufficient to generate diverse regenerative signals that included regional dendritic Ca2+-spikes and dendritic Na+-spikes (D-spikes). Global Na+-spikes could be triggered in one third of granule cells. Individual spines and dendritic segments sensed the respective signal transitions as increments in Ca2+ entry. Dendritic integration as monitored by the somatic membrane potential was mostly linear until a threshold number of spines was activated, at which often D-spikes along with supralinear summation set in. As to the mechanisms supporting active integration, NMDA receptors (NMDARs) strongly contributed to all aspects of supralinearity, followed by dendritic voltage-gated Na+- and Ca2+-channels, whereas local Na+ spine spikes, as well as morphological variables, barely mattered. Because of the low numbers of coactive spines required to trigger dendritic Ca2+ signals and thus possibly lateral release of GABA onto mitral and tufted cells, we predict that thresholds for granule cell-mediated bulbar lateral inhibition are low. Moreover, D-spikes could provide a plausible substrate for granule cell-mediated gamma oscillations.

Olfactory bulb granule cells: specialized to link coactive glomerular columns for percept generation and discrimination of odors.

The role of granule cells in olfactory processing is surrounded by several enigmatic observations, such as the purpose of reciprocal spines and the mechanisms for GABA release, the apparently low firing activity and recurrent inhibitory drive of granule cells, the missing proof for functional reciprocal connectivity, and the apparently negligible contribution to lateral inhibition. Here, we summarize recent results with regard to both the mechanisms of GABA release and the behavioral relevance of granule cell activity during odor discrimination. We outline a novel hypothesis that has the potential to resolve most of these enigmas and allows further predictions on the function of granule cells in odor processing. Briefly, recent findings imply that GABA release from the reciprocal spine requires a local spine action potential and the cooperative action of NMDA receptors and high voltage-activated Ca2+ channels. Thus, lateral inhibition is conditional on activity in the principal neurons connected to a granule cell and tightly intertwined with recurrent inhibition. This notion allows us to infer that lateral inhibition between principal neurons occurs "on demand," i.e., selectively on coactive mitral and tufted cells, and thus can provide directed, dynamically switched lateral inhibition in a sensory system with 1000 input channels organized in glomerular columns. The mechanistic underpinnings of this hypothesis concur with findings from odor discrimination behavior in mice with synaptic proteins deleted in granule cells. In summary, our hypothesis explains the unusual microcircuit of the granule cell reciprocal spine as a means of olfactory combinatorial coding.

Coincidence Detection within the Excitable Rat Olfactory Bulb Granule Cell Spines.

In the mammalian olfactory bulb, the inhibitory axonless granule cells (GCs) feature reciprocal synapses that interconnect them with the principal neurons of the bulb, mitral, and tufted cells. These synapses are located within large excitable spines that can generate local action potentials (APs) upon synaptic input ("spine spike"). Moreover, GCs can fire global APs that propagate throughout the dendrite. Strikingly, local postsynaptic Ca2+ entry summates mostly linearly with Ca2+ entry due to coincident global APs generated by glomerular stimulation, although some underlying conductances should be inactivated. We investigated this phenomenon by constructing a compartmental GC model to simulate the pairing of local and global signals as a function of their temporal separation Δt. These simulations yield strongly sublinear summation of spine Ca2+ entry for the case of perfect coincidence Δt = 0 ms. Summation efficiency (SE) sharply rises for both positive and negative Δt. The SE reduction for coincident signals depends on the presence of voltage-gated Na+ channels in the spine head, while NMDARs are not essential. We experimentally validated the simulated SE in slices of juvenile rat brain (both sexes) by pairing two-photon uncaging of glutamate at spines and APs evoked by somatic current injection at various intervals Δt while imaging spine Ca2+ signals. Finally, the latencies of synaptically evoked global APs and EPSPs were found to correspond to Δt ≈ 10 ms, explaining the observed approximately linear summation of synaptic local and global signals. Our results provide additional evidence for the existence of the GC spine spike.SIGNIFICANCE STATEMENT Here we investigate the interaction of local synaptic inputs and global activation of a neuron by a backpropagating action potential within a dendritic spine with respect to local Ca2+ signaling. Our system of interest, the reciprocal spine of the olfactory bulb granule cell, is known to feature a special processing mode, namely, a synaptically triggered action potential that is restricted to the spine head. Therefore, coincidence detection of local and global signals follows different rules than in more conventional synapses. We unravel these rules using both simulations and experiments and find that signals coincident within ≈±7 ms around 0 ms result in sublinear summation of Ca2+ entry because of synaptic activation of voltage-gated Na+ channels within the spine.

Suppression of SNARE-dependent exocytosis in retinal glial cells and its effect on ischemia-induced neurodegeneration.

Nervous tissue is characterized by a tight structural association between glial cells and neurons. It is well known that glial cells support neuronal functions, but their role under pathologic conditions is less well understood. Here, we addressed this question in vivo using an experimental model of retinal ischemia and transgenic mice for glia-specific inhibition of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent exocytosis. Transgene expression reduced glutamate, but not ATP release from single Müller cells, impaired glial volume regulation under normal conditions and reduced neuronal dysfunction and death in the inner retina during the early stages of ischemia. Our study reveals that the SNARE-dependent exocytosis in glial cells contributes to neurotoxicity during ischemia in vivo and suggests glial exocytosis as a target for therapeutic approaches.

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.

An arterially perfused nose-olfactory bulb preparation of the rat.

A main feature of the mammalian olfactory bulb network is the presence of various rhythmic activities, in particular, gamma, beta, and theta oscillations, with the latter coupled to the respiratory rhythm. Interactions between those oscillations as well as the spatial distribution of network activation are likely to determine olfactory coding. Here, we describe a novel semi-intact perfused nose-olfactory bulb-brain stem preparation in rats with both a preserved olfactory epithelium and brain stem, which could be particularly suitable for the study of oscillatory activity and spatial odor mapping within the olfactory bulb, in particular, in hitherto inaccessible locations. In the perfused olfactory bulb, we observed robust spontaneous oscillations, mostly in the theta range. Odor application resulted in an increase in oscillatory power in higher frequency ranges, stimulus-locked local field potentials, and excitation or inhibition of individual bulbar neurons, similar to odor responses reported from in vivo recordings. Thus our method constitutes the first viable in situ preparation of a mammalian system that uses airborne odor stimuli and preserves these characteristic features of odor processing. This preparation will allow the use of highly invasive experimental procedures and the application of techniques such as patch-clamp recording, high-resolution imaging, and optogenetics within the entire olfactory bulb.

Blood pressure pulsations modulate central neuronal activity via mechanosensitive ion channels.

The transmission of the heartbeat through the cerebral vascular system causes intracranial pressure pulsations. We discovered that arterial pressure pulsations can directly modulate central neuronal activity. In a semi-intact rat brain preparation, vascular pressure pulsations elicited correlated local field oscillations in the olfactory bulb mitral cell layer. These oscillations did not require synaptic transmission but reflected baroreceptive transduction in mitral cells. This transduction was mediated by a fast excitatory mechanosensitive ion channel and modulated neuronal spiking activity. In awake animals, the heartbeat entrained the activity of a subset of olfactory bulb neurons within ~20 milliseconds. Thus, we propose that this fast, intrinsic interoceptive mechanism can modulate perception-for example, during arousal-within the olfactory bulb and possibly across various other brain areas.

NMDA receptor-dependent synaptic activation of TRPC channels in olfactory bulb granule cells.

Canonical transient receptor potential (TRPC) channels are widely expressed throughout the nervous system including the olfactory bulb where their function is largely unknown. Here, we describe their contribution to central synaptic processing at the reciprocal mitral and tufted cell-granule cell microcircuit, the most abundant synapse of the mammalian olfactory bulb. Suprathreshold activation of the synapse causes sodium action potentials in mouse granule cells and a subsequent long-lasting depolarization (LLD) linked to a global dendritic postsynaptic calcium signal recorded with two-photon laser-scanning microscopy. These signals are not observed after action potentials evoked by current injection in the same cells. The LLD persists in the presence of group I metabotropic glutamate receptor antagonists but is entirely absent from granule cells deficient for the NMDA receptor subunit NR1. Moreover, both depolarization and Ca²âº rise are sensitive to the blockade of NMDA receptors. The LLD and the accompanying Ca²âº rise are also absent in granule cells from mice deficient for both TRPC channel subtypes 1 and 4, whereas the deletion of either TRPC1 or TRPC4 results in only a partial reduction of the LLD. Recordings from mitral cells in the absence of both subunits reveal a reduction of asynchronous neurotransmitter release from the granule cells during recurrent inhibition. We conclude that TRPC1 and TRPC4 can be activated downstream of NMDA receptor activation and contribute to slow synaptic transmission in the olfactory bulb, including the calcium dynamics required for asynchronous release from the granule cell spine.

Anatomical and Functional Connectivity at the Dendrodendritic Reciprocal Mitral Cell-Granule Cell Synapse: Impact on Recurrent and Lateral Inhibition.

In the vertebrate olfactory bulb, reciprocal dendrodendritic interactions between its principal neurons, the mitral and tufted cells, and inhibitory interneurons in the external plexiform layer mediate both recurrent and lateral inhibition, with the most numerous of these interneurons being granule cells. Here, we used recently established anatomical parameters and functional data on unitary synaptic transmission to simulate the strength of recurrent inhibition of mitral cells specifically from the reciprocal spines of rat olfactory bulb granule cells in a quantitative manner. Our functional data allowed us to derive a unitary synaptic conductance on the order of 0.2 nS. The simulations predicted that somatic voltage deflections by even proximal individual granule cell inputs are below the detection threshold and that attenuation with distance is roughly linear, with a passive length constant of 650 µm. However, since recurrent inhibition in the wake of a mitral cell action potential will originate from hundreds of reciprocal spines, the summated recurrent IPSP will be much larger, even though there will be substantial mutual shunting across the many inputs. Next, we updated and refined a preexisting model of connectivity within the entire rat olfactory bulb, first between pairs of mitral and granule cells, to estimate the likelihood and impact of recurrent inhibition depending on the distance between cells. Moreover, to characterize the substrate of lateral inhibition, we estimated the connectivity via granule cells between any two mitral cells or all the mitral cells that belong to a functional glomerular ensemble (i.e., which receive their input from the same glomerulus), again as a function of the distance between mitral cells and/or entire glomerular mitral cell ensembles. Our results predict the extent of the three regimes of anatomical connectivity between glomerular ensembles: high connectivity within a glomerular ensemble and across the first four rings of adjacent glomeruli, substantial connectivity to up to eleven glomeruli away, and negligible connectivity beyond. Finally, in a first attempt to estimate the functional strength of granule-cell mediated lateral inhibition, we combined this anatomical estimate with our above simulation results on attenuation with distance, resulting in slightly narrowed regimes of a functional impact compared to the anatomical connectivity.

Top-down acetylcholine signaling via olfactory bulb vasopressin cells contributes to social discrimination in rats.

Social discrimination in rats requires activation of the intrinsic bulbar vasopressin system, but it is unclear how this system comes into operation, as olfactory nerve stimulation primarily inhibits bulbar vasopressin cells (VPCs). Here we show that stimulation with a conspecific can activate bulbar VPCs, indicating that VPC activation depends on more than olfactory cues during social interaction. A series of in vitro electrophysiology, pharmacology and immunohistochemistry experiments implies that acetylcholine, probably originating from centrifugal projections, can enable olfactory nerve-evoked action potentials in VPCs. Finally, cholinergic activation of the vasopressin system contributes to vasopressin-dependent social discrimination, since recognition of a known rat was blocked by bulbar infusion of the muscarinic acetylcholine receptor antagonist atropine and rescued by additional bulbar application of vasopressin. Thus, our results implicate that top-down cholinergic modulation of bulbar VPC activity is involved in social discrimination in rats.

Oxytocin and vasopressin within the ventral and dorsal lateral septum modulate aggression in female rats.

In contrast to male rats, aggression in virgin female rats has been rarely studied. Here, we established a rat model of enhanced aggression in females using a combination of social isolation and aggression-training to specifically investigate the involvement of the oxytocin (OXT) and arginine vasopressin (AVP) systems within the lateral septum (LS). Using neuropharmacological, optogenetic, chemogenetic as well as microdialysis approaches, we revealed that enhanced OXT release within the ventral LS (vLS), combined with reduced AVP release within the dorsal LS (dLS), is required for aggression in female rats. Accordingly, increased activity of putative OXT receptor-positive neurons in the vLS, and decreased activity of putative AVP receptor-positive neurons in the dLS, are likely to underly aggression in female rats. Finally, in vitro activation of OXT receptors in the vLS increased tonic GABAergic inhibition of dLS neurons. Overall, our data suggest a model showing that septal release of OXT and AVP differentially affects aggression in females by modulating the inhibitory tone within LS sub-networks.

A17 Amacrine Cells and Olfactory Granule Cells: Parallel Processors of Early Sensory Information.

Neurons typically receive synaptic input in their dendritic arbor, integrate inputs in their soma, and send output action potentials through their axon, following Cajal's law of dynamic polarization. Two notable exceptions are retinal amacrine cells and olfactory granule cells (GCs), which flout Cajal's edict by providing synaptic output from the same dendrites that collect synaptic input. Amacrine cells, a diverse cell class comprising >60 subtypes, employ various dendritic input/output strategies, but A17 amacrine cells (A17s) in particular share further interesting functional characteristics with GCs: both receive excitatory synaptic input from neurons in the primary glutamatergic pathway and return immediate, reciprocal feedback via GABAergic inhibitory synapses to the same synaptic terminals that provided input. Both neurons thereby process signals locally within their dendrites, shaping many parallels, signaling pathways independently. The similarities between A17s and GCs cast into relief striking differences that may indicate distinct processing roles within their respective circuits: First, they employ partially dissimilar molecular mechanisms to transform excitatory input into inhibitory output; second, GCs fire action potentials, whereas A17s do not. Third, GC signals may be influenced by cortical feedback, whereas the mammalian retina receives no such retrograde input. Finally, A17s constitute just one subtype within a diverse class that is specialized in a particular task, whereas the more homogeneous GCs may play more diverse signaling roles via multiple processing modes. Here, we review these analogies and distinctions between A17 amacrine cells and granule cells, hoping to gain further insight into the operating principles of these two sensory circuits.

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.

Presynaptic NMDARs cooperate with local spikes toward GABA release from the reciprocal olfactory bulb granule cell spine.

In the rodent olfactory bulb the smooth dendrites of the principal glutamatergic mitral cells (MCs) form reciprocal dendrodendritic synapses with large spines on GABAergic granule cells (GC), where unitary release of glutamate can trigger postsynaptic local activation of voltage-gated Na+-channels (Navs), that is a spine spike. Can such single MC input evoke reciprocal release? We find that unitary-like activation via two-photon uncaging of glutamate causes GC spines to release GABA both synchronously and asynchronously onto MC dendrites. This release indeed requires activation of Navs and high-voltage-activated Ca2+-channels (HVACCs), but also of NMDA receptors (NMDAR). Simulations show temporally overlapping HVACC- and NMDAR-mediated Ca2+-currents during the spine spike, and ultrastructural data prove NMDAR presence within the GABAergic presynapse. This cooperative action of presynaptic NMDARs allows to implement synapse-specific, activity-dependent lateral inhibition, and thus could provide an efficient solution to combinatorial percept synthesis in a sensory system with many receptor channels.

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.

Subcolumnar dendritic and axonal organization of spiny stellate and star pyramid neurons within a barrel in rat somatosensory cortex.

Excitatory neurons at the level of cortical layer 4 in the rodent somatosensory barrel field often display a strong eccentricity in comparison with layer 4 neurons in other cortical regions. In rat, dendritic symmetry of the 2 main excitatory neuronal classes, spiny stellate and star pyramid neurons (SSNs and SPNs), was quantified by an asymmetry index, the dendrite-free angle. We carefully measured shrinkage and analyzed its influence on morphological parameters. SSNs had mostly eccentric morphology, whereas SPNs were nearly radially symmetric. Most asymmetric neurons were located near the barrel border. The axonal projections, analyzed at the level of layer 4, were mostly restricted to a single barrel except for those of 3 interbarrel projection neurons. Comparing voxel representations of dendrites and axon collaterals of the same neuron revealed a close overlap of dendritic and axonal fields, more pronounced in SSNs versus SPNs and considerably stronger in spiny L4 neurons versus extragranular pyramidal cells. These observations suggest that within a barrel dendrites and axons of individual excitatory cells are organized in subcolumns that may confer receptive field properties such as directional selectivity to higher layers, whereas the interbarrel projections challenge our view of barrels as completely independent processors of thalamic input.

Vasopressin Cells in the Rodent Olfactory Bulb Resemble Non-Bursting Superficial Tufted Cells and Are Primarily Inhibited upon Olfactory Nerve Stimulation.

The intrinsic vasopressin system of the olfactory bulb is involved in social odor processing and consists of glutamatergic vasopressin cells (VPCs) located at the medial border of the glomerular layer. To characterize VPCs in detail, we combined various electrophysiological, neuroanatomical, and two-photon Ca2+ imaging techniques in acute bulb slices from juvenile transgenic rats with eGFP-labeled VPCs. VPCs showed regular non-bursting firing patterns, and displayed slower membrane time constants and higher input resistances versus other glutamatergic tufted cell types. VPC axons spread deeply into the external plexiform and superficial granule cell layer (GCL). Axonal projections fell into two subclasses, with either denser local columnar collaterals or longer-ranging single projections running laterally within the internal plexiform layer and deeper within the granule cell layer. VPCs always featured lateral dendrites and a tortuous apical dendrite that innervated a single glomerulus with a homogenously branching tuft. These tufts lacked Ca2+ transients in response to single somatically-evoked action potentials and showed a moderate Ca2+ increase upon prolonged action potential trains.Notably, electrical olfactory nerve stimulation did not result in synaptic excitation of VPCs, but triggered substantial GABAA receptor-mediated IPSPs that masked excitatory barrages with yet longer latency. Exogenous vasopressin application reduced those IPSPs, as well as olfactory nerve-evoked EPSPs recorded from external tufted cells. In summary, VPCs can be classified as non-bursting, vertical superficial tufted cells. Moreover, our findings imply that sensory input alone cannot trigger excitation of VPCs, arguing for specific additional pathways for excitation or disinhibition in social contexts.

A compact holographic projector module for high-resolution 3D multi-site two-photon photostimulation.

Patterned two-photon (2P) photolysis via holographic illumination is a powerful method to investigate neuronal function because of its capability to emulate multiple synaptic inputs in three dimensions (3D) simultaneously. However, like any optical system, holographic projectors have a finite space-bandwidth product that restricts the spatial range of patterned illumination or field-of-view (FOV) for a desired resolution. Such trade-off between holographic FOV and resolution restricts the coverage within a limited domain of the neuron's dendritic tree to perform highly resolved patterned 2P photolysis on individual spines. Here, we integrate a holographic projector into a commercial 2P galvanometer-based 2D scanning microscope with an uncaging unit and extend the accessible holographic FOV by using the galvanometer scanning mirrors to reposition the holographic FOV arbitrarily across the imaging FOV. The projector system utilizes the microscope's built-in imaging functions. Stimulation positions can be selected from within an acquired 3D image stack (the volume-of-interest, VOI) and the holographic projector then generates 3D illumination patterns with multiple uncaging foci. The imaging FOV of our system is 800×800 µm2 within which a holographic VOI of 70×70×70 µm3 can be chosen at arbitrary positions and also moved during experiments without moving the sample. We describe the design and alignment protocol as well as the custom software plugin that controls the 3D positioning of stimulation sites. We demonstrate the neurobiological application of the system by simultaneously uncaging glutamate at multiple spines within dendritic domains and consequently observing summation of postsynaptic potentials at the soma, eventually resulting in action potentials. At the same time, it is possible to perform two-photon Ca2+ imaging in 2D in the dendrite and thus to monitor synaptic Ca2+ entry in selected spines and also local regenerative events such as dendritic action potentials.

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.

Long-Term Plasticity at the Mitral and Tufted Cell to Granule Cell Synapse of the Olfactory Bulb Investigated with a Custom Multielectrode in Acute Brain Slice Preparations.

Single extracellular stimulation electrodes are a widespread means to locally activate synaptic inputs in acute brain slices. Here we describe the fabrication and application of a multielectrode stimulator that was developed for conditions under which independent stimulation of several nearby sites is desirable. For the construction of the multielectrode we have developed a method by which electrode wires can be spaced at minimal distances of 100 µm. This configuration increases the efficiency of stimulation paradigms, such as the comparison of proximal induced and control inputs for studies of synaptic plasticity.In our case the multielectrode was used for acute olfactory bulb slices to independently excite individual nearby glomeruli; the technique allowed us to demonstrate homosynaptic bidirectional long-term plasticity at the mitral/tufted cell to granule cell synapse. We also describe the determinants for successful recordings of long-term plasticity at this synapse, with mechanical and electrophysiological recording stability being tantamount. Finally, we briefly discuss data analysis procedures.