Cholinergic activation and tonic excitation induce persistent gamma oscillations in mouse somatosensory cortex in vitro

Abstract

1. Concomitant application of the cholinergic agonist carbachol and nanomolar doses of kainate can elicit persistent gamma frequency oscillations in all layers of the mouse somatosensory cortex in vitro. Receptor pharmacology with bath-applied antagonists indicated that oscillatory network activity depended crucially on the participation of cholinergic muscarinic, (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate and GABAA receptors. 2. The timing of action potentials and the occurrence of excitatory as well as inhibitory postsynaptic events was highly correlated with the phasic change of extracellularly recorded population activity. Firing probability was lowest during the peak negativity of IPSPs and gradually increased during their ensuing decay. In conjunction with the effect of a barbiturate to decrease the frequency of gamma oscillations, this suggests a crucial role of IPSPs in phasing the suprathreshold activity of principal neurons. 3. At nearby (< 1 mm) sites contained within any given cortical layer, oscillatory extra- and intracellular activity was highly synchronous with no apparent phase lag. However, interlaminar mapping experiments demonstrated a phase reversal of both extra- and intracellularly recorded activity near the lower border of thalamo-recipient layer 4, thus corroborating findings that have been obtained in vivo. 4. In conclusion, a modest increase of tonic excitatory drive in conjunction with the activation of cholinergic muscarinic receptors can elicit persistent gamma frequency network oscillations in the rodent somatosensory cortex. These findings (re)emphasize the role of the cholinergic ascending system in the cortical processing of sensory information.

Figure 1. Pharmacological induction and receptor mechanisms of cortical gamma oscillations in vitro

A, extracellular recordings of two neighbouring layer 4 sites showing the induction of oscillatory network activity due to the conjoint action of kainate and carbachol. Cross-correlograms reveal a high degree of synchrony, whereas the power spectrum shows a prominent peak in the gamma frequency range. B, control experiment demonstrating that within layer 4, kainate alone failed to induce gamma oscillations. C, the muscarinic antagonist atropine potently blocked oscillatory activity elicited by carbachol in layer 5/6. D, likewise, the specific M1 receptor antagonist pirenzepine was also effective in reversibly antagonizing gamma frequency network oscillations. The interrupted lines in the corresponding power spectra (bottom panels) denote baseline activity (kainate only); continuous lines (addition of carbachol) reveal a clear increase of power in the gamma frequency spectrum, whereas both muscarinic antagonists (thick lines) reduce spectral activity back to baseline levels. Calibration bars in B are for panels A and B, those in D are for C and D.

Figure 2. The effect of glutamate receptor antagonists on neocortical gamma oscillations in vitro

A, oscillatory activity was induced by superfusing mouse somatosensory cortex (layer 5/6) with both kainate and carbachol. Neither the metabotropic glutamate antagonist (S)-MCPG ((S)-α-methyl-4-carboxyphenylglycine) nor the NMDA receptor antagonist D-AP5 (D-2-amino-5-phosphonopentanoic acid) were effective in diminishing gamma oscillations, whereas the AMPA/kainate receptor blocker NBQX (6-nitro-7-sulphamoylbenzo (f) quinoxaline-2, 3-dione) eliminated all rhythmic activity. B, the corresponding power spectra reveal modest changes in spectral amplitude following the addition of (S)-MCPG and D-AP5, whereas neither of the drugs had an effect on oscillatory peak frequency.

Figure 3. A prominent role of GABAergic mechanisms in the generation of gamma oscillations

A, non-saturating doses of the competitive GABA_A receptor antagonist bicuculline abolished gamma oscillatory activity in layer 2/3 (thick line in power spectrum). Note that the corresponding spectra reveal a disinct notch at 50 Hz, resulting from a modest degree of contamination with mains hum. During washout, oscillatory activity reappeared at the same frequency as during the control period (thin line). B, in contrast, the GABA_A receptor modulator pentobarbitone was effective in dramatically decreasing the oscillation frequency (thick line in spectrum). The effect was reversible following washout (interrupted line). C, intracellular recording of a layer 2/3 cell at a depolarized holding potential (−40 mV) revealed fast hyperpolarizing events, presumably IPSPs that were tightly correlated, albeit in antiphase, with concomitantly recorded extracellular field activity. D, dual intracellular impalements of regular spiking neurons in layer 2/3 revealed synchronous activity in both recordings.

Figure 4. The relationship of oscillatory field activity with the sequential timing of intracellularly recorded sub- and suprathreshold events

A, rheobasic action potentials were elicited in a regular spiking neuron of an oscillating slice. Spike-triggered averages of the concomitantly recorded extracellular field activity revealed action potentials to be located near the peak negativity of an oscillatory cycle. B, when using field triggered data acquisition and constructing spike latency histograms, it was apparent that action potentials could occur during any part of an oscillatory cycle, but with the peak of their probability distribution overlapping with the trough of the averaged extracellular field. C, when adjusting the membrane potential during intracellular recordings to the apparent point of IPSP reversal (−70 mV), it was feasible to discriminate depolarizing events, presumably EPSPs, which were clearly correlated with the extracellular population activity. D, field triggered averages of such recordings were nearly matching, albeit with a slight precession of the intracellular waveform. E, field triggered acquisition of intracellular recordings displaying numerous IPSPs at depolarized holding potentials showed the peak positivity of the intracellular trace to coincide with the trough of the averaged field. All recordings in A-E were obtained within layer 5/6.

Figure 5. Interlaminar correlation of extra- and intracellular oscillatory activity

A, two-site mapping experiments along vertical strips of cortex revealed gamma oscillations in layers (L) 2/3 and 4 to occur without appreciable phase-lag. B, however, when leaving a reference electrode in layer 4 and shifting the position of a roving second electrode across the layer 4/5 border, a near-complete phase reversal became apparent. C, consequently, oscillatory activity in layers 2/3 and 5/6 was also found to be in antiphase. In contrast to their antiphasic relationship within a given cortical layer (panel D; for upper layers, see Fig. 3C) extra- and intracellular activity across the layer 4/5 border were found to be near synchronous (E). F, the prominent extracellular phase shift was also corroborated with dual intracellular recordings at depolarized holding potentials, here showing IPSPs in layers 2/3 and 5/6 being in antiphase (for dual impalements within a given layer, see Fig. 3D).