Edward O. Mann

Perisomatic Feedback Inhibition Underlies Cholinergically Induced Fast Network Oscillations in the Rat Hippocampus In Vitro

Gamma frequency network oscillations are assumed to be important in cognitive processes, including hippocampal memory operations, but the precise functions of these oscillations remain unknown. Here, we examine the cellular and network mechanisms underlying carbachol-induced fast network oscillations in the hippocampus in vitro, which closely resemble hippocampal gamma oscillations in the behaving rat. Using a combination of planar multielectrode array recordings, imaging with voltage-sensitive dyes, and recordings from single hippocampal neurons within the CA3 gamma generator, active current sinks and sources were localized to the stratum pyramidale. These proximal currents were driven by phase-locked rhythmic inhibitory inputs to pyramidal cells from identified perisomatic-targeting interneurons. AMPA receptor-mediated recurrent excitation was necessary for the synchronization of interneuronal discharge, which strongly supports a synaptic feedback model for the generation of hippocampal gamma oscillations.

Which GABA(A) receptor subunits are necessary for tonic inhibition in the hippocampus

GABAA receptors (GABAARs) assembled of different subunits mediate tonic and phasic inhibition in hippocampal neurons. CA1/CA3 pyramidal cells (PCs) predominantly express α5 subunits whereas dentate gyrus granule cells (DGGCs) and molecular layer (ML) interneurons predominantly express δ subunits. Both α5- and δ-containing GABAARs mediate tonic inhibition. We have shown previously that mice lacking α5 subunits (Gabra5−/−) have a residual tonic current in CA1/CA3 PCs because of an upregulation of δ subunits, but the basis of the residual tonic current in DGGCs and ML interneurons of mice lacking the δ subunit (Gabrd−/−) is still unknown. We now show that wild-type DGGCs have a small tonic current mediated by α5 subunit-containing GABAARs responsible for ∼29% of the total tonic current. To better identify the GABAARs mediating tonic inhibition in hippocampal neurons, we generated mice lacking both α5 and δ subunits (Gabra5/Gabrd−/−). Recordings from CA1/CA3 PCs, DGGCs, and ML interneurons in these mice show an absence of tonic currents without compensatory changes in spontaneous IPSCs (sIPSCs), sEPSCs, and membrane resistance. The absence of tonic inhibition results in spontaneous gamma oscillations recordable in vitro in the CA3 pyramidal layer of these mice, which can be mimicked in wild-type mice by blocking α5 subunit-containing GABAARs with 50 nm L-655,708. In conclusion, depending on the cell type, the α5 and δ subunits are the principal GABAAR subunits responsible for mediating the lions share of tonic inhibition in hippocampal neurons.

Spike Timing of Distinct Types of GABAergic Interneuron during Hippocampal Gamma Oscillations In Vitro

Gamma frequency (30-100 Hz) network oscillations occur in the intact hippocampus during awake, attentive behavior. Here, we explored the underlying cellular mechanisms in an in vitro model of persistent gamma-frequency oscillations, induced by bath application of 20 μm carbachol in submerged hippocampal slices at 30 ± 1°C. Current-source density analysis of the field oscillation revealed a prominent alternating sink-source pair in the perisomatic and apical dendritic regions of CA3. To elucidate the active events generating these extracellular dipoles, we examined the firing properties of distinct neuron types. Visually guided unit recordings were obtained from individual CA3 neurons followed by intracellular labeling for anatomical identification. Pyramidal cells fired at 2.82 ± 0.7 Hz, close to the negative peak of the oscillation (0.03 ± 0.65 msec), and often in conjunction with a negative spike-like component of the field potential. In contrast, all phase-coupled interneurons fired after this negative peak. Perisomatic inhibitory interneurons fired at high frequency (18.1 ± 2.7 Hz), shortly after the negative peak (1.97 ± 0.95 msec) and were strongly phase-coupled. Dendritic inhibitory interneurons fired at lower frequency (8.4 ± 2.4 Hz) and with less fidelity and a longer delay after the negative peak (4.3 ± 1.1 msec), whereas interneurons with cell body in the stratum radiatum often showed no phase relationship with the field oscillation. The phase and spike time data of individual neurons, together with the current-source density analysis, support a synaptic feedback model of gamma oscillations primarily involving pyramidal cells and inhibitory cells targeting their perisomatic region.

Maintaining network activity in submerged hippocampal slices: importance of oxygen supply.

Studies in brain slices have provided a wealth of data on the basic features of neurons and synapses. In the intact brain, these properties may be strongly influenced by ongoing network activity. Although physiologically realistic patterns of network activity have been successfully induced in brain slices maintained in interface‐type recording chambers, they have been harder to obtain in submerged‐type chambers, which offer significant experimental advantages, including fast exchange of pharmacological agents, visually guided patch‐clamp recordings, and imaging techniques. Here, we investigated conditions for the emergence of network oscillations in submerged slices prepared from the hippocampus of rats and mice. We found that the local oxygen level is critical for generation and propagation of both spontaneously occurring sharp wave–ripple oscillations and cholinergically induced fast oscillations. We suggest three ways to improve the oxygen supply to slices under submerged conditions: (i) optimizing chamber design for laminar flow of superfusion fluid; (ii) increasing the flow rate of superfusion fluid; and (iii) superfusing both surfaces of the slice. These improvements to the recording conditions enable detailed studies of neurons under more realistic conditions of network activity, which are essential for a better understanding of neuronal network operation.

Control of hippocampal gamma oscillation frequency by tonic inhibition and excitation of interneurons

Gamma-frequency oscillations depend on phasic synaptic GABAA receptor (GABAAR)-mediated inhibition to synchronize spike timing. The spillover of synaptically released GABA can also activate extrasynaptic GABAARs, and such tonic inhibition may also contribute to modulating network dynamics. In many neuronal cell types, tonic inhibition is mediated by δ subunit–containing GABAARs. We found that the frequency of in vitro cholinergically induced gamma oscillations in the mouse hippocampal CA3 region was increased by the activation of NMDA receptors (NMDARs) on interneurons. The NMDAR-dependent increase of gamma oscillation frequency was counteracted by the tonic inhibition of the interneurons mediated by δ subunit–containing GABAARs. Recordings of synaptic currents during gamma activity revealed that NMDAR-mediated increases in oscillation frequency correlated with a progressive synchronization of phasic excitation and inhibition in the network. Thus, the balance between tonic excitation and tonic inhibition of interneurons may modulate gamma frequency by shaping interneuronal synchronization.

Distinct roles of GABA(A) and GABA(B) receptors in balancing and terminating persistent cortical activity.

Cortical networks spontaneously fluctuate between persistently active Up states and quiescent Down states. The Up states are maintained by recurrent excitation within local circuits, and can be turned on and off by synaptic input. GABAergic inhibition is believed to be important for stabilizing such persistent activity by balancing the excitation, and could have an additional role in terminating the Up state. Here, we report that GABAA and GABAB receptor-mediated inhibition have distinct and complementary roles in balancing and terminating persistent activity. In a model of Up–Down states expressed in slices of rat entorhinal cortex, the GABAA receptor antagonist, gabazine (50–500 nm), concentration-dependently decreased Up state duration, eventually leading to epileptiform bursts. In contrast, the GABAB receptor antagonist, CGP55845 (50 nm to 1 μm), increased the duration of persistent network activity, and prevented stimulus-induced Down state transitions. These results suggest that while GABAA receptor-mediated inhibition is necessary for balancing persistent activity, activation of GABAB receptors contributes to terminating Up states.

Hippocampal gamma‐frequency oscillations: from interneurones to pyramidal cells, and back

GABAergic interneurones are necessary for the emergence of hippocampal gamma‐frequency network oscillations, during which they play a key role in the synchronization of pyramidal cell firing. However, it remains to be resolved how distinct interneurone subtypes contribute to gamma‐frequency oscillations, in what way the spatiotemporal pattern of interneuronal input affects principal cell activity, and by which mechanisms the interneurones themselves are synchronized. Here we summarize recent evidence from cholinergically induced gamma‐frequency network oscillations in vitro, showing that perisomatic‐targeting GABAergic interneurones provide prominent rhythmic inhibition in pyramidal cells, and that these interneurones are synchronized by recurrent excitation. We conclude by presenting a minimal integrate‐and‐fire network model which demonstrates that this excitatory‐inhibitory feedback loop is sufficient to explain the generation of intrahippocampal gamma‐frequency oscillations.

Synaptic Currents in Anatomically Identified CA3 Neurons during Hippocampal Gamma Oscillations In Vitro

Gamma-frequency oscillations are prominent during active network states in the hippocampus. An intrahippocampal gamma generator has been identified in the CA3 region. To better understand the synaptic mechanisms involved in gamma oscillogenesis, we recorded action potentials and synaptic currents in distinct types of anatomically identified CA3 neurons during carbachol-induced (20–25 μm) gamma oscillations in rat hippocampal slices. We wanted to compare and contrast the relationship between excitatory and inhibitory postsynaptic currents in pyramidal cells and perisomatic-targeting interneurons, cell types implicated in gamma oscillogenesis, as well as in other interneuron subtypes, and to relate synaptic currents to the firing properties of the cells. We found that phasic synaptic input differed between cell classes. Most strikingly, the dominant phasic input to pyramidal neurons was inhibitory, whereas phase-coupled perisomatic-targeting interneurons often received a strong phasic excitatory input. Differences in synaptic input could account for some of the differences in firing rate, action potential phase precision, and mean action potential phase angle, both between individual cells and between cell types. There was a strong positive correlation between the ratio of phasic synaptic excitation to inhibition and firing rate over all neurons and between the phase precision of excitation and action potentials in interneurons. Moreover, mean action potential phase angle correlated with the phase of the peak of the net-estimated synaptic reversal potential in all phase-coupled neurons. The data support a recurrent mechanism of gamma oscillations, whereby spike timing is controlled primarily by inhibition in pyramidal cells and by excitation in interneurons.

Priming of Hippocampal Population Bursts by Individual Perisomatic-Targeting Interneurons

Hippocampal population bursts (“sharp wave–ripples”) occur during rest and slow-wave sleep and are thought to be important for memory consolidation. The cellular mechanisms involved are incompletely understood. Here we investigated the cellular mechanisms underlying the initiation of sharp waves using a hippocampal slice model. To this end, we used a combination of field recordings with planar multielectrode arrays and whole-cell patch-clamp recordings of individual anatomically identified pyramidal neurons and interneurons. We found that GABAA receptor-mediated inhibition is necessary for sharp wave generation. Moreover, the activity of individual perisomatic-targeting interneurons can both suppress, and subsequently enhance, the local generation of sharp waves. Finally, we show that this is achieved by the tight control of local excitation and inhibition by perisomatic-targeting interneurons. These results suggest that perisomatic-targeting interneurons assist in selecting the subset of pyramidal neurons that initiate each hippocampal sharp wave–ripple.

Novel modulatory mechanisms revealed by the sustained application of nicotine in the guinea‐pig hippocampus in vitro

The α7 nicotinic acetylcholine receptor (nAChR) has been implicated widely in behavioural functions and dysfunctions related to the hippocampus, but the detailed mechanisms by which this receptor contributes to these behavioural processes have yet to be elucidated. In the present study, sustained application (5 min) of nicotine significantly lowered the threshold for synaptic plasticity, and thus a long‐lasting potentiation was induced by a stimulus that would normally evoke only a short‐term potentiation. This effect appeared to be mediated by α7 nAChRs, as it was inhibited by the α7 nAChR‐specific antagonist α‐bungarotoxin (100 nm), but not by mecamylamine (50 μm) or dihydro‐β‐erythroidine (DHβE; 1 μm) at concentrations known to be selective for non‐α7 nAChRs. Further pharmacological dissection revealed that the effect was also abolished by the NMDA receptor antagonist, D‐(‐)‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5; 50 μm). This blockade, however, unmasked a slowly developing nicotine‐induced potentiation of field excitatory postsynaptic potential that appeared to be dependent on both α7 nAChR activation and non‐α7 nAChR desensitisation. This secondary effect of nicotine was blocked by a combination of picrotoxin (50 μm) and saclofen (100 μm), and thus appeared to be mediated via GABAergic interneurons. The important implication of this study was that the sustained application of α7 nAChR agonists could modulate the conditions for synaptic plasticity through multiple transduction pathways, and not simply the inactivation of α7 nAChRs. These α7‐nAChR‐dependent mechanisms could reconcile the discrepancies between the previously reported behavioural versus electrophysiological effects of nicotine in the hippocampus.