Brain function relies on coordinated activity across spatial and temporal scales. Single neurons integrate local and long-range connectivity and reflect activity across brain-wide networks. Understanding integrated brain function requires tools capable of recording from anatomically connected populations in distributed brain areas to bridge local and global dynamics. Here, we present high-density, micro-electrocorticography arrays that facilitate multi-scale studies of brain activity. The hybrid arrays integrate the desirable features of silicone elastomers and polyimide films: silicone provides optical transparency and permits repeated penetration with intracortical arrays, while polyimide enables fine photolithographic feature definition. This combination facilitates high-throughput functional mapping to identify targets and insertion of intracortical arrays for dense local sampling. We demonstrate functional mapping in rats, cats, and marmosets, showing how functional maps guide multi-area laminar recordings. Finally, we demonstrate local and feedforward optogenetic stimulation to investigate cortico-cortical interactions. These capabilities establish the hybrid μECoG as a compelling tool for systems neuroscience.

Measuring fast neuronal signals is the domain of electrophysiology and magnetophysiology. Although electrophysiology is easier to perform, magnetophysiology avoids tissue-based distortions and measures a signal with directional information. At the macroscale, magnetoencephalography (MEG) is established, and at the mesoscale, visually evoked magnetic fields have been reported. At the microscale however, while benefits of recording magnetic counterparts of electric spikes would be numerous, they are also highly challenging in vivo. Here, we combine magnetic and electric recordings of neuronal action potentials in anesthetized, male rats using miniaturized giant magnetoresistance (GMR) sensors. We reveal the magnetic signature of action potentials of well-isolated single units. The recorded magnetic signals showed a distinct waveform and considerable signal strength. This demonstration of in vivo magnetic action potentials opens a wide field of possibilities to profit from the combined power of magnetic and electric recordings and thus to significantly advance the understanding of neuronal circuits.NEW & NOTEWORTHY Electrophysiological tools allow the measurement of single-neuron action potentials with high temporal resolution. Magnetophysiological measurements add valuable information but are hard to achieve for single neurons. Established technology for noninvasive magnetic measurements cannot be used in vivo. We demonstrate that miniaturized giant magnetoresistance sensors enable measuring magnetic counterparts of action potentials in vivo. This proof-of-principle shows a way toward integrating magnetic and electric recordings to profit from the complementary information measured by each modality.

The common marmoset has emerged as a key model in neuroscience. Marmosets are small in size, show great potential for genetic modification and exhibit complex behaviors. Thus, it is necessary to develop technology that enables monitoring and manipulation of the underlying neural circuits. Here, we describe a novel approach to record and optogenetically manipulate neural activity in awake, behaving marmosets. Our design utilizes a light-weight, 3D printed titanium chamber that can house several high-density silicon probes for semi-chronic recordings, while enabling simultaneous optogenetic stimulation. We demonstrate the application of our method in male marmosets by recording multi- and single-unit data from areas V1 and V6 with 192 channels simultaneously, and show that optogenetic activation of excitatory neurons in area V6 can influence behavior in a detection task. This method may enable future studies to investigate the neural basis of perception and behavior in the marmoset.

Datasets to reproduce figures from our manuscript "Multi-area recordings and optogenetics in the awake, behaving marmoset". https://www.biorxiv.org/content/10.1101/2021.10.30.466578v1 Analysis code can be found at https://github.com/PJendritza/MultiAreaOptoMarmo.

The marmoset has emerged as a promising primate model system, in particular for visual neuroscience. Many common experimental paradigms rely on head fixation and an extended period of eye fixation during the presentation of salient visual stimuli. Both of these behavioral requirements can be challenging for marmosets. Here, we present two methodological developments, each addressing one of these difficulties. First, we show that it is possible to use a standard eye tracking system without head fixation to assess visual behavior in the marmoset. Eye tracking quality from head-free animals is sufficient to obtain precise psychometric functions from a visual acuity task. Secondly, we introduce a novel method for efficient receptive field mapping that does not rely on moving stimuli but uses fast flashing annuli and wedges. We present data recorded during head-fixation in areas V1 and V6 and show that receptive field locations are readily obtained within a short period of recording time. Thus, the methodological advancements presented in this work will contribute to establish the marmoset as a valuable model in neuroscience.

Synchronization has been implicated in neuronal communication, but causal evidence remains indirect, We use optogenetics to generate depolarizing currents in pyramidal neurons of the cat visual cortex, emulating excitatory synaptic inputs under precise temporal control, while measuring spike output. The cortex transforms constant excitation into strong gamma-band synchronization, revealing the well-known cortical resonance. Increasing excitation with ramps increases the strength and frequency of synchronization. Slow, symmetric excitation profiles reveal hysteresis of power and frequency. White-noise input sequences enable causal analysis of network transmission, establishing that the cortical gamma-band resonance preferentially transmits coherent input components. Models composed of recurrently coupled excitatory and inhibitory units uncover a crucial role of feedback inhibition and suggest that hysteresis can arise through spike-frequency adaptation, The presented approach provides a powerful means to investigate the resonance properties of local circuits and probe how these properties transform input and shape transmission.

Neuronal electrical activity is widely studied in vivo, and the ability to measure its magnetic equivalent to obtain an undisturbed signal with both amplitude and direction information leading to neuronal signal mapping would be a promising tool for neuroscience. To provide such a tool, a probe with spin-electronics-based magnetic sensors with orthogonal axes of sensitivity for two directions of measurement is realized, thanks to a local magnetization re-orientation technique induced by Joule heating. This probe is tested under in vivo measurement conditions in the brain of an anesthetized rat. To be as close as possible to neurons and to create minimal damage during the probe's insertion, the tip thickness has been drastically decreased using a silicon-on-insulator substrate. Our probes provide the ability to perform in vivo magnetic measurements on two orthogonal axes on a 25 μm thick silicon tip with a sensitivity of 1.7%/mT along one axis and 0.9%/mT along the perpendicular axis in the sensor plane, for a limit of detection at 1 kHz of 1.0 and 1.3 nT, respectively. These probes have been tested through a phantom study and during an in vivo experiment. The robustness and stability over one year are demonstrated.

The gamma rhythm has been implicated in neuronal communication, but causal evidence remains indirect. We measured spike output of local neuronal networks and emulated their synaptic input through optogenetics. Opsins provide currents through somato-dendritic membranes, similar to synapses, yet under experimental control with high temporal precision. We expressed Channelrhodopsin-2 in excitatory neurons of cat visual cortex and recorded neuronal responses to light with different temporal characteristics. Sine waves of different frequencies entrained neuronal responses with a reliability that peaked for input frequencies in the gamma band. Crucially, we also presented white-noise sequences, because their temporal unpredictability enables analysis of causality. Neuronal spike output was caused specifically by the input’s gamma component. This gamma-specific transfer function is likely an emergent property of in-vivo networks with feedback inhibition. The method described here could reveal the transfer function between the input to any one and the output of any other neuronal group.

SummarySynchronization has been implicated in neuronal communication, but causal evidence remains indirect. We used optogenetics to generate depolarizing currents in pyramidal neurons of cat visual cortex, emulating excitatory synaptic inputs under precise temporal control, while measuring spike output. Cortex transformed constant excitation into strong gamma-band synchronization, revealing the well-known cortical resonance. Increasing excitation with ramps increased the strength and frequency of synchronization. Slow, symmetric excitation profiles revealed hysteresis of power and frequency. Crucially, white-noise input sequences enabled causal analysis of network transmission, establishing that cortical resonance selectively transmits coherent input components. Models composed of recurrently coupled excitatory and inhibitory units uncovered a crucial role of feedback inhibition and suggest that hysteresis can arise through spike-frequency adaptation. The presented approach provides a powerful means to investigate the resonance properties of local circuits and probe how these properties transform input and shape transmission.

Activated visual cortex typically engages in neuronal synchronization in the gamma-frequency band (30-90 Hz). Gamma-band synchronization is related to cognitive functioning, and its mechanisms have been extensively investigated, predominantly through in-vitro studies. To further elucidate its mechanisms in-vivo, we performed simultaneous optogenetic stimulation and electrophysiological recordings of visual cortical areas 17 and 21a in the anesthetized cat. Viral transfection with AAV1 or AAV9 under a CamKIIα promoter led to robust Channelrhodopsin-2 (ChR2) expression. Immunohistochemical analysis showed that all ChR2-expressing neurons were negative for Parvalbumin, consistent with predominant or exclusive expression in excitatory neurons. Optogenetic stimulation used primarily surface illumination directly above the transfected and recorded cells. Stimulation with constant light led to strong and sustained gamma-band synchronization with strength and bandwidth similar to visually induced gamma. Rhythmic stimulation with light-pulse trains or sinusoidal light modulation revealed strongest resonance for gamma-band frequencies. Gamma resonance was confirmed by optogenetic white-noise stimulation. White-noise stimulation allowed the quantification of the transfer function between the optogenetic stimulation and the local field potential response. This transfer function showed a dominant peak in the gamma band. Thus, we find that visual cortical circuits resonate most strongly to gamma-band components in their input. This resonance renders both the sensitivity to input, and the output of these circuits, selectively tuned to gamma.Significance Statement Activated groups of cortical neurons often display rhythmic synchronization in the gamma-frequency band (30-90 Hz). Gamma-band synchronization is particularly well studied in visual cortex. We used optogenetics to control visual cortex neurons with light. Different optogenetic stimulation protocols, using constant light, rhythmically modulated light or white-noise modulated light, all demonstrated that the investigated circuits predominantly resonate to stimulation in the gamma band. The observed gamma-band resonance renders visual cortical circuits most sensitive to gamma-rhythmic synaptic inputs. This in turn renders their spike output and the ensuing interareal synchronization gamma rhythmic.This work was supported by DFG (SPP 1665, FOR 1847, FR2557/5-1-CORNET to P.F.; EXC 1086, DI 1908/5-1, DI 1908/6-1 to I.D.), BMBF (01GQ1301 to I.D.), EU (HEALTH-F2-2008-200728-BrainSynch, FP7-604102-HBP, FP7-600730-Magnetrodes to P.F.; ERC Starting Grant OptoMotorPath to I.D.), a European Young Investigator Award to P.F., the FENS-Kavli Network of Excellence to I.D., National Institutes of Health (1U54MH091657-WU-Minn-Consortium-HCP to P.F.), the LOEWE program (NeFF to P.F. and I.D.). Present address of I.D.: Optophysiology, Bernstein Center and BrainLinks-BrainTools, University of Freiburg, Albertstrase 23, 79104 Freiburg, Germany.Author contributions J.N, C.M.L., T.W., P.F. designed research; J.N, C.M.L., T.W., P.J., I.D., P.F. performed experiments; J.N., C.M.L., T.W. analyzed data; J.N., P.F. wrote the paper.

Neuronal activity generates ionic flows and thereby both magnetic fields and electric potential differences, i.e., voltages. Voltage measurements are widely used but suffer from isolating and smearing properties of tissue between source and sensor, are blind to ionic flow direction, and reflect the difference between two electrodes, complicating interpretation. Magnetic field measurements could overcome these limitations but have been essentially limited to magnetoencephalography (MEG), using centimeter-sized, helium-cooled extracranial sensors. Here, we report on in vivo magnetic recordings of neuronal activity from visual cortex of cats with magnetrodes, specially developed needle-shaped probes carrying micron-sized, non-cooled magnetic sensors based on spin electronics. Event-related magnetic fields inside the neuropil were on the order of several nanoteslas, informing MEG source models and efforts for magnetic field measurements through MRI. Though the signal-to-noise ratio is still inferior to electrophysiology, this proof of concept demonstrates the potential to exploit the fundamental advantages of magnetophysiology.

The mouse is receiving growing interest as a model organism for studying visual perception. However, little is known about how discrimination and learning interact to produce visual conditioned responses. Here, we adapted a two-alternative forced-choice visual discrimination task for mice and examined how training with equiprobable stimuli of varying similarity influenced conditioned response and discrimination performance as a function of learning. Our results indicate that the slope of the gradients in similarity during training determined the learning rate, the maximum performance and the threshold for successful discrimination. Moreover, the learning process obeyed an inverse relationship between discrimination performance and discriminative resolution, implying that sensitivity within a similarity range cannot be improved without sacrificing performance in another. Our study demonstrates how the interplay between discrimination and learning controls visual discrimination capacity and introduces a new training protocol with quantitative measures to study perceptual learning and visually-guided behavior in freely moving mice.

Excitatory neurotransmission mediated by N-methyl-d-aspartate receptors (NMDARs) is fundamental to learning and memory and, when impaired, causes certain neurological disorders. NMDARs are heterotetrameric complexes composed of two GluN1 [NR1] and two GluN2(A–D) [NR2(A–D)] subunits. The GluN2 subunit is responsible for subunit-specific channel activity and gating kinetics including activation (rise time), peak open probability (peak Po) and deactivation (decay time). The peak Po of recombinant NMDARs was recently described to be controlled by the extracellular GluN2 N-terminal domain (NTD). The cytoplasmic GluN2 C-terminal domain (CTD) could also be involved, because the Po of synaptic NMDARs is reduced in mice expressing C-terminally truncated GluN2 subunits. Here, we examined the role of the GluN2 cytoplasmic tail for NMDAR channel activity and gating in HEK-293 cells. C-terminal truncation of GluN2A, GluN2B or GluN2C did not change the subunit-specific rise time but accelerated the decay time of glutamate-activated currents. Furthermore, the peak Po was reduced by about 50% for GluN2A and GluN2B but not for GluN2C. These results indicated that the CTD of GluN2 has a modulating role in NMDAR gating even in the absence of interacting synaptic proteins. Reduction of peak Po and deactivation kinetics following GluN2 C-terminal truncation were reversed by re-introducing a CTD from a different GluN2 subunit. Thus, the CTDs of GluN2 subunits behave as constitutive structural elements required for normal functioning of NMDARs but are not involved in determining the subunit-specific gating properties of NMDARs.

In nature, sensory stimuli are organized in heterogeneous combinations. Salient items from these combinations 'stand-out' from their surroundings and determine what and how we learn. Yet, the relationship between varying stimulus salience and discrimination learning remains unclear.