Philip M. Heyward

Membrane Bistability in Olfactory Bulb Mitral Cells

Whole-cell patch-clamp recordings were used to investigate the electrophysiological properties of mitral cells in rat main olfactory bulb brain slice preparations. The majority of mitral cells are bistable. These cells spontaneously alternate between two membrane potentials, separated by ∼10 mV: a relatively depolarized potential (upstate), which is perithreshold for spike generation, and a relatively hyperpolarized potential (downstate), in which spikes do not occur. Bistability occurs spontaneously in the absence of ionotropic excitatory or inhibitory synaptic inputs. Bistability is voltage dependent; transition from the downstate to the upstate is a regenerative event activated by brief depolarization. A brief hyperpolarization can switch the membrane potential from the upstate to the downstate. In response to olfactory nerve (ON) stimulation, mitral cells in the upstate are more likely to fire an action potential than are those in the downstate. ON stimulation can switch the membrane potential from the downstate to the upstate, producing a prolonged and amplified depolarization in response to a brief synaptic input. We conclude that bistability is an intrinsic property of mitral cells that is a major determinant of their responses to ON input.

The antidepressant drug fluoxetine inhibits persistent sodium currents and seizure-like events

The antidepressant drug fluoxetine (FLX) has been shown to exert antiepileptic effects in several animal models, but mixed preclinical findings and occasional reports of proconvulsant effects have led to hesitation towards its use in epileptic people. Despite being developed as a selective serotonin reuptake inhibitor, FLX has numerous other targets in the brain. One of the proposed targets is the neuronal sodium channel, which is inhibited by many existing antiepileptic drugs. In this study, we used electrophysiological methods in a brain slice model of seizures to test for anticonvulsant and Na(+) channel-blocking effects of FLX. This approach allowed us to use a single biological system to study the effects of FLX on (1) epileptiform activity, (2) Na(+)-dependent action potential generation, and (3) the persistent Na(+) current (I(NaP)). We found that FLX was anticonvulsant in a dose- and time-dependent manner, and that this action was accompanied by strong I(NaP) inhibition and impairment of repetitive firing. These findings suggest that the effect of FLX on active membrane properties is similar to that of many antiepileptic drugs, and that this action may contribute to anticonvulsant effects.

Lithium modulates cortical excitability in vitro.

The sometimes devastating mood swings of bipolar disorder are prevented by treatment with selected antiepileptic drugs, or with lithium. Abnormal membrane ion channel expression and excitability in brain neurons likely underlie bipolar disorder, but explaining therapeutic effects in these terms has faced an unresolved paradox: the antiepileptic drugs effective in bipolar disorder reduce Na(+) entry through voltage-gated channels, but lithium freely enters neurons through them. Here we show that lithium increases the excitability of output neurons in brain slices of the mouse olfactory bulb, an archetypical cortical structure. Treatment in vitro with lithium (1 to 10mM) depolarizes mitral cells, blocks action potential hyperpolarization, and modulates their responses to synaptic input. We suggest that Na(+) entry through voltage-gated channels normally directly activates K(+) channels regulating neuron excitability, but that at therapeutic concentrations, lithium entry and accumulation reduces this K(+) channel activation. The antiepileptic drugs effective in bipolar disorder and lithium may thus share a membrane target consisting of functionally coupled Na(+) and K(+) channels that together control brain neuron excitability.

All-or-none population bursts temporally constrain surround inhibition between mouse olfactory glomeruli.

With each sniff, the olfactory bulbs of the brain generate a neural activity pattern representing the odour environment, transmitting this to higher brain centres in the form of mitral cell output. Inhibitory circuits in the olfactory bulb glomerular and external plexiform layers may amplify contrast in these patterns, through surround inhibition of mitral cells. These circuits may operate in series, but their respective roles are unclear. A single sniff is sufficient for odour discrimination, but is not clear that the inhibitory circuits act within this timeframe. We used microdissected slices of mouse olfactory bulb to study each circuit in isolation. We found that unlike surround inhibition mediated in the external plexiform layer, surround inhibition mediated in the glomerular layer was activated by sensory synaptic input, but not by mitral cell output. The results also suggest that interactions between olfactory glomeruli are exclusively inhibitory, unlike in antennal lobe, and that surround inhibition mediated within the external plexiform layer may involve neural circuit elements not preserved in slice preparations. Surround inhibition was effective only after an interval corresponding to a single sniff in vivo. Surplus excitation, initiated by sensory input but generated by collective all-or-none responses of mitral cells, may delay surround inhibition and allow the synchronous activation of multiple glomeruli without each suppressing the other. Surround inhibition in the glomerular layer may subsequently allow a fresh representation of the odour environment to be generated with each sniff. These findings are consistent with combinatorial odour coding based on all-or-none glomerular responses.

Inhibition of hippocampal excitability by citalopram

Purpose:  Preclinical data have suggested that selective serotonin reuptake inhibitors (SSRIs) may have anticonvulsant properties, and some SSRIs are known to modulate ion channels in vitro. We screened citalopram, fluoxetine, and sertraline for anticonvulsant actions in mouse hippocampal slices, and studied the effects of citalopram on active membrane properties and repetitive action potential firing.

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 Mg(2+)-induced epileptiform discharges in mouse OB slices using extracellular and whole cell electrophysiological recordings. Low-Mg(2+) 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-Mg(2+) 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.

A brain slice bath for physiology and compound microscopy, with dual-sided perifusion.

Contemporary in vitro brain slice studies can employ compound microscopes to identify individual neurons or their processes for physiological recording or imaging. This requires that the bath used to maintain the tissue fits within the working distances of a water‐dipping objective and microscope condenser. A common means of achieving this is to maintain thin tissue slices on the glass floor of a recording bath, exposing only one surface of the tissue to oxygenated bathing medium. Emerging evidence suggests that physiology can be compromised by this approach. Flowing medium past both sides of submerged brain slices is optimal, but recording baths utilizing this principle are not readily available for use on compound microscopes. This paper describes a tissue bath designed specifically for microscopy and physiological recording, in which temperature‐controlled medium flows past both sides of the slices. A particular feature of this design is the use of concentric mesh rings to support and transport the live tissue without mechanical disturbance. The design is also easily adapted for use with thin acute slices, cultured slices, and acutely dispersed or cultured cells maintained either on cover slips or placed directly on the floor of the bath. The low profile of the bath provides a low angle of approach for electrodes, and allows use of standard condensers, nosepieces and water‐dipping objective lenses. If visualization of individual neurons is not required, the bath can be mounted on a simple stand and used with a dissecting microscope. Heating is integral to the bath, and any temperature controller capable of driving a resistive load can be used. The bath is robust, readily constructed and requires minimal maintenance. Full construction and operation details are given.

A device for automated control of pipette internal pressure for patch-clamp recording.

Formation of a high-resistance seal between the tip of a glass recording pipette and the membrane of the recorded cell is the crucial step in patch clamping, or whole cell recording with patch pipettes. Formation of the seal, and subsequent rupture of the membrane for whole cell recording, requires a specific sequence of changes in pipette internal hydrostatic pressure. Generating this sequence of pressure changes adds to the complexity of setting up, gaining proficiency, and performing experiments. Automation of routine pipette pressure manipulations would simplify seal formation, and benefit productivity. Here we describe a device that automates control of patch pipette internal pressure. Solenoid valves sequentially operated by manual switching, or external electronic control, automatically provide the necessary sequence of connections to the pipette interior. This greatly simplifies the operations performed to obtain membrane seals and whole cell recordings and improves standardization and reproducibility in patch recording.

Transmembrane dye labeling and immunohistochemical staining of electrophysiologically characterized single neurons

Numerous studies have used whole-cell patch recording to characterize the electrophysiology of neurons and, via intracellular dye filling, the detailed morphology of the same cells. However, it has been difficult to demonstrate the presence of small soluble molecules within such cells, because washout of the soluble contents of the cell into the patch pipette precludes their later detection by immunohistochemistry. This leaves a major gap in our understanding of circuits made up of neurochemically heterogeneous neurons. To bridge this gap we have developed a transmembrane labeling approach, employing membrane-permeant dye in conjunction with perforated patch electrophysiology. Using this method we have successfully recorded from juxtaglomerular cells in the olfactory bulb, reconstructed the morphology of the cells, and demonstrated expression of soluble neurochemical markers within the same cells. This new technique provides a reliable means to link the physiology, morphology, and neurochemistry of single identified neurons studied using patch-clamp recording.