A simplified electronic circuit for combined single-cell stimulation and recording using loose cell-attached electrodes

While tight-seal patch clamp recordings have found wide use in neuroscience and in other fields, the requirement to replace the glass pipette after every attempted recording represents an impediment to high throughput studies such as searching for monosynaptically connected pairs of neurons. Loose cell-attached recording was introduced in 2000 to circumvent this problem since it enabled combined recording and stimulation of visually-identified neurons without necessitating a tight (gigaohm) seal. Since the stimulus voltages required to evoke action potentials through low resistance seals are beyond the capacity of most commercial amplifiers, Barbour and Isope introduced a variation of classic patch clamp amplifier circuit that is able to deliver stimulus voltages that are effective in triggering action potentials under the loose cell-attached patch clamp configuration. The present report presents the design and operation of a simpler amplifier that contains only two integrated circuits and is able to effectively stimulate and record action potentials in mitral cells in rodent olfactory bulb slices. The addition of an accessory analog gating circuit enables manual control of the stimulus voltage with pulse timing controlled by a digital output from a computer. This system may be useful in studies that require surveying many potential pairs of neurons for synaptic connections and for sampling and manipulating single-cell activity in in vivo electrophysiology experiments.

Spatial structure of synchronized inhibition in the olfactory bulb

Olfactory sensory input is detected by receptor neurons in the nose which then send information to the olfactory bulb, the first brain region for processing olfactory information. Within the olfactory bulb, many local circuit interneurons, including axonless granule cells, function to facilitate fine odor discrimination. How interneurons interact with principal cells to affect bulbar processing is not known though the mechanism is likely to be different than in sensory cortical regions since the olfactory bulb lacks an obvious topographical organization; neighboring glomerular columns, representing inputs from different receptor neuron subtypes, typically have different odor tuning. Determining the spatial scale over which interneurons such as granule cells can affect principal cells is a critical step towards understanding how the olfactory bulb operates. We addressed this question by assaying inhibitory synchrony using intracellular recordings from pairs of principal cells with different inter-somatic spacing. We find that in acute rat olfactory bulb slices, inhibitory synchrony is evident in the spontaneous synaptic input in mitral cells separated up to 300 μm. At all inter-somatic spacing assayed, inhibitory synchrony was dependent on fast Na+ channels, suggesting that action potentials in granule cells function to coordinate GABA release at relatively distant dendrodendritic synapses formed throughout the the dendritic arbor. Our results suggest that individual granule cells are able to influence relatively large groups of mitral and tufted cells belonging to clusters of at least 15 glomerular modules, providing a potential mechanism to integrate signals reflecting a wide variety of odorants.

Metabotropic glutamate receptors promote disinhibition of olfactory bulb glomeruli that scales with input strength

Increasing evidence indicates that the neural circuitry within glomeruli of the olfactory bulb plays a major role in affecting information flow between olfactory sensory neurons (OSNs) and output mitral cells (MCs). Glutamatergic external tufted (ET) cells, located at glomeruli, can act as intermediary cells in excitation between OSNs and MCs, whereas activation of MCs by OSNs is, in turn, suppressed by inhibitory synapses onto ET cells. In this study, we used patch-clamp recordings in rat olfactory bulb slices to examine the function of metabotropic glutamate receptors (mGluRs) in altering these glomerular signaling mechanisms. We found that activation of group II mGluRs profoundly reduced inhibition onto ET cells evoked by OSN stimulation. The mGluRs that mediated disinhibition were located on presynaptic GABAergic periglomerular cells and appeared to be activated by glutamate transients derived from dendrites in glomeruli. In terms of glomerular output, the mGluR-mediated reduction in GABA release led to a robust increase in the number of action potentials evoked by OSN stimulation in both ET cells and MCs. Importantly, however, the enhanced excitation was specific to when a glomerulus was strongly activated by OSN inputs. By being selective for strong vs. weak glomerular activation, mGluR-mediated disinhibition provides a mechanism to enhance the contrast in odor signals that activate OSN inputs into a single glomerulus at varying intensities.

Low-magnesium medium induces epileptiform activity in mouse olfactory bulb slices

Magnesium-free medium can be used in brain slice studies to enhance glutamate receptor function, but this manipulation causes seizure-like activity in many cortical areas. The rodent olfactory bulb (OB) slice is a popular preparation, and potentially ictogenic ionic conditions have often been used to study odor processing. We studied low Mg2+-induced epileptiform discharges in mouse OB slices using extracellular and whole cell electrophysiological recordings. Low-Mg2+ medium induced two distinct types of epileptiform activity: an intraglomerular delta-frequency oscillation resembling slow sniff-induced activity and minute-long seizure-like events (SLEs) consisting of large negative-going field potentials accompanied by sustained depolarization of output neurons. SLEs were dependent on N-methyl-d-aspartate receptors and sodium currents and were facilitated by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors. The events were initiated in the glomerular layer and propagated laterally through the external plexiform layer at a slow time scale. Our findings confirm that low-Mg2+ medium should be used with caution in OB slices. Furthermore, the SLEs resembled the so-called slow direct current (DC) shift of clinical and experimental seizures, which has recently been recognized as being of great clinical importance. The OB slice may therefore provide a robust and unique in vitro model of acute seizures in which mechanisms of epileptiform DC shifts can be studied in isolation from fast oscillations.

Transient Activity Induces a Long-Lasting Increase in the Excitability of Olfactory Bulb Interneurons

Little is known about the cellular mechanisms that underlie the processing and storage of sensory in the mammalian olfactory system. Here we show that persistent spiking, an activity pattern associated with working memory in other brain regions, can be evoked in the olfactory bulb by stimuli that mimic physiological patterns of synaptic input. We find that brief discharges trigger persistent activity in individual interneurons that receive slow, subthreshold oscillatory input in acute rat olfactory bulb slices. A 2- to 5-Hz oscillatory input, which resembles the synaptic drive that the olfactory bulb receives during sniffing, is required to maintain persistent firing. Persistent activity depends on muscarinic receptor activation and results from interactions between calcium-dependent afterdepolarizations and low-threshold Ca spikes in granule cells. Computer simulations suggest that intrinsically generated persistent activity in granule cells can evoke correlated spiking in reciprocally connected mitral cells. The interaction between the intrinsic currents present in reciprocally connected olfactory bulb neurons constitutes a novel mechanism for synchronized firing in subpopulations of neurons during olfactory processing.

Activation of Postsynaptic GABAB Receptors Modulates the Bursting Pattern and Synaptic Activity of Olfactory Bulb Juxtaglomerular Neurons

Olfactory bulb glomeruli are formed by a network of three major types of neurons collectively called juxtaglomerular (JG) cells, which include external tufted (ET), periglomerular (PG), and short axon (SA) cells. There is solid evidence that γ-aminobutyric acid (GABA) released from PG neurons presynaptically inhibits glutamate release from olfactory nerve terminals via activation of GABAB receptors (GABAB-Rs). However, it is still unclear whether ET cells have GABAB-Rs. We have investigated whether ET cells have functional postsynaptic GABAB-Rs using extracellular and whole cell recordings in olfactory bulb slices. In the presence of fast synaptic blockers (CNQX, APV, and gabazine), the GABAB-R agonist baclofen either completely inhibited the bursting or reduced the bursting frequency and increased the burst duration and the number of spikes/burst in ET cells. In the presence of fast synaptic blockers and tetrodotoxin, baclofen induced an outward current in ET cells, suggesting a direct postsynaptic effect. Baclofen reduced the frequency and amplitude of spontaneous EPSCs in PG and SA cells. In the presence of sodium and potassium channel blockers, baclofen reduced the frequency of miniature EPSCs, which were inhibited by the calcium channel blocker cadmium. All baclofen effects were reversed by application of the GABAB-R antagonist CGP55845 . We suggest that activation of GABAB-Rs directly inhibits ET cell bursting and decreases excitatory dendrodendritic transmission from ET to PG and SA cells. Thus the postsynaptic GABAB-Rs on ET cells may play an important role in shaping the activation pattern of the glomeruli during olfactory coding.

Endogenous GABA and Glutamate Finely Tune the Bursting of Olfactory Bulb External Tufted Cells

In rat olfactory bulb slices, external tufted (ET) cells spontaneously generate spike bursts. Although ET cell bursting is intrinsically generated, its strength and precise timing may be regulated by synaptic input. We tested this hypothesis by analyzing whether the burst properties are modulated by activation of ionotropic γ-aminobutyric acid (GABA) and glutamate receptors. Blocking GABAA receptors increased—whereas blocking ionotropic glutamate receptors decreased—the number of spikes/burst without changing the interburst frequency. The GABAA agonist (isoguvacine, 10 μM) completely inhibited bursting or reduced the number of spikes/burst, suggesting a shunting effect. These findings indicate that the properties of ET cell spontaneous bursting are differentially controlled by GABAergic and glutamatergic fast synaptic transmission. We suggest that ET cell excitatory and inhibitory inputs may be encoded as a change in the pattern of spike bursting in ET cells, which together with mitral/tufted cells constitute the output circuit of the olfactory bulb.