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V, power of 0.58 0.12 mV 2, and frequency of 2.1 0.3 Hz. Delta rhythms were also generated in this experimental condition in other cortical association areas temporal association area and frontal cortex. Only weak, slower events could be detected in primary sensory areas S1 and Au1 data not shown. To further uncover the origin of the delta rhythm, we quantified the current source densities across laminae in parietal cortex. As with data from human invasive electrode studies Csercsa et al., 2010, dominant sourcesink pairs were seen in superficial layers in Utah recordings Fig. 1A. This was associated with an abrupt phase reversal when comparing pairwise glass electrode recordings, with reference electrode in layer 5, between layers 2 and 1 18 20 o vs 175 8 o, n 5 Fig. 1A. However, both phase reversal and sourcesink pair location were displaced from the layers in which peak delta power was seen. In all association areas, delta power was clearly largest in layer 5 see Discussion. To determine whether this dominance of the delta rhythm power in deep layers was translatable to human neocortex in this experimental model, we used voltagesensitive dye recordings from nonepileptic human frontal cortical tissue. In the neuromodulatory environment used for rat studies, human association cortex also generated persistent delta rhythms Fig. 1C. Mean frequency was not different from rat 2.4 0.5 Hz, n 6 epochs over 3 h. As with the rodent model, peak power was clearly localized to deep layers, with additional, smaller peaks in power around layers 2 and 3. Pharmacological clues to the mechanism underlying the delta rhythm suggested a complex interaction of multiple factors Fig. 1Bii. The dependence on cholinergic drive appeared to be predominantly via muscarinic receptors pirenzipine 10 M reduced delta power to below 10 of control values. In terms of synaptic excitation, blockade of either NMDARs alone with 50 M DAP5 or both AMPARs and kainate receptors 20 M NBQX also reduced delta power to 10 of control values. However, blockade of AMPARs or kainate receptors alone did not have such a dramatic effect on delta power, suggesting that a general reduction in excitation was required. There was a clear divergence in the effects of synaptic inhibition on the delta rhythm. Reduced GABAA receptormediated excitation increased mean delta power, whereas reduced GABAB receptormediated inhibition reduced delta power to 17 5 of control n 5 Fig. 1Bii. In addition, a range of drugs reducing gap junction conductance each with different nonspecific effects all nearly abolished the delta rhythm. Together, these pharmacological manipulations pointed to multiple mechanisms combining to generate the rhythm seen. To attempt to identify the key mechanisms, we next examined the outputs and synaptic inputs of different association neocortical neurons. Neuronal subtypes in layer 5 involved in the delta rhythm Both a subset of FS interneurons and all intrinsically bursting principal cells were seen to generate bursts of spike outputs on almost every delta period phase locked to concurrently recorded layer 5 field potentials Fig. 2. In each case, bursts of spikes were seen to ride on slow compound EPSPs. Mean spike incidence per delta period for deltalocked FS cells was 6 2 100 periods each in n 4 neurons Fig. 2B, FS1. Excitatory inputs were characterized by nearcontinuous occurrence of small, fast EPSPs 1.6 0.8 mV, d 2.7 0.3 ms. Superimposed on this lowlevel noise were large compound EPSPs that appeared to be made up of intense barrages of the small single EPSPs see during the quiescent part of each period. Mean peak amplitude of these delta phaselocked compound events was 13 3 mV from 70mV membrane potential. Interestingly, pooled power spectral analysis of FS EPSP traces 60s long, n 4 Fig. 2B revealed a second, smaller more spectrally spread peak in addition to the dominant delta frequency. Peak frequency of this additional rhythmic component was within the theta range 4.8 1.0 Hz. Bursts of spiking from layer 5 IB neurons were also seen on each delta period Fig. 2C. Mean spike incidence per delta period was greater than that seen in FS cells 10 3, 100 periods each in n 6 neurons, and spikes arose from large compound somatic EPSPs 9.4 2.0 mV amplitude from 70 mV. Although the overall active period of IB neurons coincided with a plateau depolarization, EPSP shapes were characteristically ramped, increasing in amplitude as the active period progressed, suggesting that initial spiking was related to the intrinsic burst ability of these neurons and the later spikes directly arising from the network EPSP. Synaptic inhibition in IB neurons consisted of two components. During the active phase of each delta period, IB cells received a barrage of fast IPSPs that weakly temporally summed until spike termination. In contrast, the quiescent part of each delta cycle was associated with a slow hyperpolarizing potential resembling GABABmediated IPSPs in these cells. Mean power spectra of these synaptic events recorded at a mean membrane potential of 30 mV also showed two components a dominant, sharp peak at theta frequency 4.2 0.4 Hz, see above for comparison with FS EPSPs and a weaker, more spectrally spread peak at delta frequency. Neuronal subtypes involved in layer 5 in the nested theta rhythm Not all FS cells showed the tight temporal relationship between spiking and the field delta rhythm described above. A subset of neurons with nearidentical spike shapes and response to depolarizing current steps to those described above were found to generate outputs dominated by single action potentials at theta frequency 4.5 0.5 Hz, n 3 neurons from 3 slices Fig. 3B, FS2. Occasional intense bursts of action potentials were also generated in this neuron subtype phase locked to the field delta rhythm mean burst incidence, 0.4 per delta period. The contrasting outputs of FS1 and FS2 interneurons appeared to be a consequence of their respective synaptic excitation profiles. Slow excitatory events phase locked to the field delta rhythm were much weaker and more erratic in FS2 cells. Mean amplitude was 5.2 1.4 mV p 0.05 compared with FS1 deltafrequency EPSPs. The thetafrequency spike incidence matched a different set of synaptic inputs fast EPSPs 4.4 0.8 mV, d 2.5 0.4 ms occurred in a highly rhythmic manner in each FS2 cell recorded. The combination of these two types of input resulted in a broad mean spectrum with modal peaks at delta frequency 2.0 0.4 Hz and theta frequency 4.6 0.8 Hz. Layer 5 principal cells showed a remarkable divergence in spike behavior and inputs. Unlike the intense bursting of IB cells with the field delta rhythm, layer 5 RS neurons did not burst. Instead, a range of spiking patterns from single spikes on each delta period to nearcontinuous thetafrequency spiking was seen Fig. 3C. Mean spike incidence histograms revealed a modal peak at 4.4 1.0 Hz n 9. This pattern of outputs did not correspond to EPSP inputs seen in RS neurons. As with IB neurons, compound excitatory inputs occurred phase locked to the field delta rhythm. However, they were significantly smaller 4.1 0.6 mV, p 0.05 compared with IB neuron EPSPs and showed a different ramped profile. Although IB EPSPs ramped up in amplitude during the active phase of each delta period, RS EPSPs ramped down. IPSP inputs also differed between the two10754 J. Neurosci., June 26, 2013 332610750 10761Carracedo et al. Dynamic Cortical Interlaminar InteractionsABCcell types. No trains of fast IPSPs were seen in RS neurons during the active phase of the delta period, and the quiescent phase was often interrupted by additional IPSP inputs. IPSPs seen had kinetics intermediate to the fast and slow events recorded in IB neurons 6.7 1.1 mV, d 21.2 1.3 ms. This complex pattern of different outputs from layer 5 FS neurons and principal cells was captured by the revised cortical column computational model used in this study see Materials and Methods. The dominant, deltalocked spike outputs occurred in model IB and FS2 neurons. IB neuron spiking was initiated by predominantly NMDARmediated IBIB recurrent excitatory connections and terminated by a combination of the strong intrinsic afterhyperpolarization in this neuron subtype and a phasic GABAB receptormediated slow IPSP Fig. 4. This latter network component came from FS2 interneurons modeled as neurogliaformlike cells that generated intense bursts on each delta period a burst incidence seen in experimental FS2 cells interspersed with nearcontinuous thetafrequency single spikes compare Figs. 3B, 4. In contrast, basketlike FS1 interneurons generated weaker spike trains at delta frequency, and layer 5 RSlike cells generated deltanested single spikes or spike pairs, or continuous thetafrequency spike outputs. This model confirmed the pharmacological data Fig. 1Bii implicating NMDARmediated excitation between IB neurons4 the concurrently recorded field A. Top histogram shows mean burst incidence at delta frequency. Bottom trace shows membrane potential at 70 mV mean revealing large, slow, regular depolarizations interspersed with more rapid but smaller, faster EPSPs. Bottom spectrogram shows that mean power of EPSPs onto FS cells had a modal peak at delta frequency but with a smaller additional peak in the theta 5 Hz band. C, Example recordings from an IB neuron in layer 5. Step depolarization with 0.2 nA 200 ms reveals the intrinsic bursting behavior of this cell type. Top trace shows spontaneous bursts of spike generation at resting membrane potential phase locked to the layer 5 field delta rhythm note that these traces were not concurrently recorded with the example field in A. Top histogram demonstrates mean burst incidence at delta frequency. Middle trace shows a recording from the same neuron held at 70 mV mean revealing large, ramped EPSPs underlying the bursting behavior. Mean power spectra again show peak incidence of EPSPs at delta frequency. Bottom trace shows activity in the same cell held at 30 mV to reveal IPSP inputs. IPSPs were complex, consisting of deltafrequency bursts of higher frequency, fast IPSPs interleaved with single, slow hyperpolarizations. Mean spectra bottom graph of such behavior in n 5 neurons exposed a bimodal power distribution with peaks at delta and theta frequencies. Calibration 200 mV field, 20 mV resting membrane potential, 10 mV 70 and 30 mV recordings, 0.5 s.Figure 2. Delta rhythms are generated by layer 5 IB neurons. A, Example trace showing a layer 5 LFP recording of delta activity. Spectrum shows a tight, single modal peak at 2 Hz. Data plotted as mean black line and SEM gray lines. B, Example recordings from a nonaccommodating, FS interneuron in layer 5. Response to step injection of 0.2 nA current for 200 ms demonstrates the intrinsic spiking behavior. Top trace shows spontaneous bursts of spike generation at resting membrane potential phase locked toCarracedo et al. Dynamic Cortical Interlaminar InteractionsJ. Neurosci., June 26, 2013 332610750 10761 10755Aand GABAB receptormediated inhibition as the primary features of the layer 5 delta rhythm generator. It also suggested the layer 5 theta generator was mediated by a combination of RS neuron intrinsic properties and synaptic inputs. Layer 5 theta outputs are highly labile and manifest in the layer 23 LFP The model data predicted that transient, thetafrequency epochs of spikes from RS neurons would be highly sensitive to tonic excitatory drive to this cell type, with these cells being only weakly influenced by the layer 5 delta rhythm. A change in tonic drive of 0.2 nA to model RS neurons was sufficient to change outputs from single, deltalocked spikes to this continuous thetafrequency output Fig. 5A. During delta rhythms in association cortical slices, RS cell spike outputs could be modified across this range by injection of a small amount of tonic current 0.1 nA. A mean membrane potential difference of 5.2 0.5 mV was sufficient in each case to transform single, deltalocked spikes to continuous thetafrequency spiking. Over an even narrower range of membrane potentials, a stable theta burst of output two to three spikes could be generated. Interspike intervals within these brief bursts and during continuous spike were the same 170 15 vs 185 20 ms, respectively, p 0.1. To quantify the extent of the influence of the deltafrequency EPSPs in these neurons Fig. 3C, spike probability relative to peak positivity of the layer 5 delta LFP was calculated. Despite the difference in spike output patterns, there was a peak in spike incidence 150 180 ms into these wavetriggered avBC4 but smaller, faster EPSPs with large events occurring at theta frequency. Bottom spectrogram shows mean power of EPSPs onto FS cells had a bimodal peak at delta and theta frequency. Lines plotted show means of each of the three cells recorded. C, Example recordings from an RS neuron in layer 5. Step depolarization with 0.2 nA 200 ms reveals the RS behavior of this cell type. Top trace shows spontaneous spike generation at resting membrane potential is dominated by single or double spikes per delta period note that these traces were not concurrently recorded with the example field in A. Top histogram demonstrates mean spike incidence approximately at theta frequency. Middle trace shows a recording from the same neuron held at 70 mV mean revealing small compound EPSPs occurring at delta frequency from mean power spectrum on the left. Bottom trace shows activity in the same cell held at 30 mV to reveal IPSP inputs. IPSPs were complex, consisting of both delta and thetafrequency components. Note the absence of the runs of fast IPSPs seen in the FS cell in Figure 2. Calibration 200 V field, 20 mV resting membrane potential, 10 mV 70 and 30 mV recordings, 0.5 s.Figure 3. Layer 5 RS neurons produce thetafrequency spike outputs. A, Example trace showing a layer 5 LFP recording of delta activity. Spectrum shows a tight, single modal peak at 2 Hz as in Figure 2. B, Example recordings from a nonaccommodating, FS interneuron in layer 5. Response to step injection of 0.2 nA current for 200 ms demonstrates the intrinsic spiking behavior. Top trace shows sporadic bursts of spike generation at resting membrane potential phase locked to the concurrently recorded field A. Note also predominant thetafrequency spike generation. Top histogram shows mean spike incidence at theta frequency. Bottom trace shows membrane potential at 70 mV mean revealing small, slow, regular depolarizations interspersed with more rapid10756 J. Neurosci., June 26, 2013 332610750 10761Carracedo et al. Dynamic Cortical Interlaminar InteractionsABerages at the beginning of the active phase of each delta period Fig. 5A. Despite the clear presence of this thetagenerating subcircuit within layer 5, the LFP was dominated by the IB neuronmediated delta rhythm. However, a field manifestation of this deltanested theta activity was seen in superficial layers Fig. 5B. Concurrent LFP recordings from layer 5 and layer 2 showed a phase reversal of the delta component of the rhythm Fig. 1A. However, additional detail in the superficial layer LFP suggested higherfrequency components. Spectrograms of activity in the two layers revealed a clear, iterative presence of power within the theta band only in superficial recordings. Therefore, we examined cellular activity patterns in layers 23 in more detail. Cellular responses in superficial layers RS neurons in layer 23 spiked sparsely during the delta rhythm. Mean spike incidence per delta period was 0.6 0.2 n 14 neurons from 11 slices. Spike timing relative to the LFP delta rhythm was highly variable with two clear maxima spread across the active phase data not shown. Examination of EPSP profiles revealed a possible source for this most delta periods were associated with compound EPSPs in superficial RS neurons with clear dual components Fig. 6B. Peak EPSP amplitude was 5.5 0.6 mV from 70 mV mean membrane potential, and the profile of these events looked remarkably similar to the spike incidence profile in layer 5 RS neurons in the narrow range of dual spike generation Fig. 5A, black line. Spectral analysis of these superficial layer RS EPSPs also revealed dual peaks at delta and theta frequency. In contrast, IPSPs received by superficial RS neurons had only a relatively weak delta component to their spectra and almost no power in the theta band. The source of these compound IPSPs within the superficial layer consisted of at least local FS and LTS interneurons Fig. 6C,D. Both interneuron subtypes generated spike bursts phase locked to the field delta rhythm, with outputs from FS cells more intense than LTS cells 7.2 2.0 vs 2.2 1.0 spikes per delta period, respectively, n 3 and 4. LTS cell EPSP recordings showed almost no evidence for thetafrequency inputs, but superficial FS cell excitatory inputs were more complex. As with layer 23 RS cells, FS cells in these layers exhibited dualcompound EPSPs approximately on every other delta period Fig. 6D. Spikefield coherence reveals a reciprocal laminar interaction afforded by deltanested theta rhythms The above demonstration of overt thetafrequency inputs onto superficial RS neurons despite a near absence of local theta generation in other superficial neurons suggested a degree of interplay between the theta source in layer 5 and the pattern of activity seen in superficial layers. To address this, we used Utah electrode arrays to concurrently record unit activity from all layers simultaneously. As with intracellular data, layer 5 units could be divided into two clearly different output patterns. Of the 52 layer 5 units recorded from seven slices, 23 demonstrated intense bursts on each delta period 7.8 0.5 spikes per period in a manner similar to that seen for intracellular recordings from IB4 FS2, modeled as neurogliaformlike cells demonstrated more intense spike bursts interspersed with thetafrequency single spikes compare with Fig. 3B. D, Example trace of activity in nontufted, layer 5 RS cells from the same simulation as AC. Note the absence of deltafrequency bursts, being replaced by doublet spikes with interspike interval reflecting theta period, occurring every field potential delta period. Calibration arbitrary field and IB GABAB 50 mV spike behavior examples, 0.5 s.CD
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