Layer-specific stimulations of parvalbumin-positive cortical interneurons in mice entrain brain rhythms to different frequencies.

Neocortical interneurons provide inhibition responsible for organizing neuronal activity into brain oscillations that subserve cognitive functions such as memory, attention, or prediction. However, the interneuronal contribution to the entrainment of neocortical oscillations within and across different cortical layers was not described. Here, using layer-specific optogenetic stimulations with micro-Light-Emitting Diode arrays, directed toward parvalbumin-expressing (PV) interneurons in non-anesthetized awake mice, we found that supragranular layer stimulations of PV neurons were most efficient at entraining supragranular local field potential (LFP) oscillations at gamma frequencies (γ: 25-80 Hz), whereas infragranular layer stimulation of PV neurons better entrained the LFP at delta (δ: 2-5 Hz) and theta (θ: 6-10 Hz) frequencies. At the level of neuronal action potential activity, we observed that supragranular neurons better followed the imposed PV stimulation rhythm than their infragranular counterparts at most frequencies when the stimulation was delivered in their respective layer. Moreover, the neuronal entrainment evoked by local stimulation could propagate across layers, though with a lesser impact when the stimulation occurs in deep layers, suggesting a direction-specific laminar propagation. These results establish a layer-based framework for oscillations to entrain the primary somatosensory cortex in awake conditions.

Vigilance and behavioral state-dependent modulation of cortical neuronal activity throughout the sleep/wake cycle.

GABAergic inhibitory neurons, through their molecular, anatomic and physiological diversity, provide a substrate for the modulation of ongoing cortical circuit activity throughout the sleep-wake cycle. Here, we investigated neuronal activity dynamics of parvalbumin (PV), vasoactive intestinal polypeptide (VIP) and somatostatin (SST) neurons in naturally-sleeping head-restrained mice at the level of layer 2/3 of the primary somatosensory barrel cortex of mice. Through calcium-imaging and targeted single-unit loose-patch or whole-cell recordings, we found that PV action potential (AP) firing activity was largest during both NREM (non-rapid eye movement) and REM sleep stages, that VIP neurons were most active during REM sleep and that the overall activity of SST neurons remained stable throughout the sleep/wake cycle. Analysis of neuronal activity dynamics uncovered rapid decreases in PV cell firing at wake onset followed by a progressive recovery during wake. Simultaneous local field potential (LFP) recordings further revealed that, except for SST neurons, a large proportion of neurons were modulated by ongoing delta and theta oscillations. During NREM sleep spindles, PV and SST activity increased and decreased, respectively. Finally, we uncovered the presence of whisking behavior in mice during REM sleep and show that the activity of VIP and SST is differentially modulated during awake and sleeping whisking bouts, which may provide a neuronal substrate for internal brain representations occurring during sleep.SIGNIFICANCE STATEMENTIn the sensory cortex, the balance between excitation and inhibition is believed to be highly dynamic throughout the sleep/wake cycle, shaping the response of cortical circuits to external stimuli, while allowing the formation of newly encoded memory. Using in vivo two-photon calcium imaging or targeted single-unit recordings combined with local field potential recordings, we describe the vigilance state and whisking-behavior -dependent activity of excitatory pyramidal and inhibitory GABAergic neurons in the supragranular layers of mouse somatosensory cortex. Interneuronal activity was found to be differentially modulated by ongoing delta and theta waves, sleep spindles and a novel type of whisking observed during Rapid Eye Movement (REM sleep), potentially providing a neuronal substrate for internal brain representations occurring during sleep.

Compact Optical Neural Probes With Up to 20 Integrated Thin-Film µLEDs Applied in Acute Optogenetic Studies.

This paper reports on the development, characterization and in vivo validation of compact optical neural probes. These novel intracerebral devices comprise micro light-emitting diodes ( µLEDs) integrated along their slender probe shanks with up to 20 µLEDs per device. Blue light with a peak wavelength of 455 nm is emitted from circular apertures 100 µm in diameter. The µLEDs are structured on GaN-on-sapphire wafers and subsequently transferred onto silicon (Si) carrier wafers. The wafer-scale transfer process provides the opportunity to process the functional GaN layer stack from both sides and hence enables maximizing the efficiency of the µLEDs. Combined with standard MEMS fabrication processes for Si, linear µLED arrays with small inter- µLED distances are achieved on thin probe shanks with cross-sections measuring [Formula: see text]. Devices are interconnected using highly flexible polyimide cables in order to mechanically decouple them from the peripheral electronics during in vivo experiments. Assembled probes emit a peak optical radiant flux of 440 µW (emittance 56 mW mm -2) at 5 mA driving current. Thermal characterization of test probes reveals a temperature increase of 1.5 K measured using an integrated thermistor. Electrical functionality stress tests have been carried out to evaluate the device passivation against the physiological environment. It is estimated to endure at least 48 h during continuously pulsed µLED operation. A compact driving circuitry enables low-noise µLED operation in in vivo optogenetic experiments. The radiant flux necessary to elicit an acceptable neuronal response is determined between 1.36 µW and 17.5 µW. Probe validation successfully demonstrates the layer-specific stimulation in the cortex in multiple in vivo trials.

Hybrid intracerebral probe with integrated bare LED chips for optogenetic studies.

This article reports on the development, i.e., the design, fabrication, and validation of an implantable optical neural probes designed for in vivo experiments relying on optogenetics. The probes comprise an array of ten bare light-emitting diode (LED) chips emitting at a wavelength of 460 nm and integrated along a flexible polyimide-based substrate stiffened using a micromachined ladder-like silicon structure. The resulting mechanical stiffness of the slender, 250-µm-wide, 65-µm-thick, and 5- and 8-mm-long probe shank facilitates its implantation into neural tissue. The LEDs are encapsulated by a fluropolymer coating protecting the implant against the physiological conditions in the brain. The electrical interface to the external control unit is provided by 10-µm-thick, highly flexible polyimide cables making the probes suitable for both acute and chronic in vivo experiments. Optical and electrical properties of the probes are reported, as well as their in vivo validation in acute optogenetic studies in transgenic mice. The depth-dependent optical stimulation of both excitatory and inhibitory neurons is demonstrated by altering the brain activity in the cortex and the thalamus. Local network responses elicited by 20-ms-long light pulses of different optical power (20 µW and 1 mW), as well as local modulation of single unit neuronal activity to 1-s-long light pulses with low optical intensity (17 µW) are presented. The ability to modulate neural activity makes these devices suitable for a broad variety of optogenetic experiments.

Dopamine Neuron-Specific Optogenetic Stimulation in Rhesus Macaques.

Optogenetic studies in mice have revealed new relationships between well-defined neurons and brain functions. However, there are currently no means to achieve the same cell-type specificity in monkeys, which possess an expanded behavioral repertoire and closer anatomical homology to humans. Here, we present a resource for cell-type-specific channelrhodopsin expression in Rhesus monkeys and apply this technique to modulate dopamine activity and monkey choice behavior. These data show that two viral vectors label dopamine neurons with greater than 95% specificity. Infected neurons were activated by light pulses, indicating functional expression. The addition of optical stimulation to reward outcomes promoted the learning of reward-predicting stimuli at the neuronal and behavioral level. Together, these results demonstrate the feasibility of effective and selective stimulation of dopamine neurons in non-human primates and a resource that could be applied to other cell types in the monkey brain.

Stem cells expanded from the human embryonic hindbrain stably retain regional specification and high neurogenic potency.

Stem cell lines that faithfully maintain the regional identity and developmental potency of progenitors in the human brain would create new opportunities in developmental neurobiology and provide a resource for generating specialized human neurons. However, to date, neural progenitor cultures derived from the human brain have either been short-lived or exhibit restricted, predominantly glial, differentiation capacity. Pluripotent stem cells are an alternative source, but to ascertain definitively the identity and fidelity of cell types generated solely in vitro is problematic. Here, we show that hindbrain neuroepithelial stem (hbNES) cells can be derived and massively expanded from early human embryos (week 5-7, Carnegie stage 15-17). These cell lines are propagated in adherent culture in the presence of EGF and FGF2 and retain progenitor characteristics, including SOX1 expression, formation of rosette-like structures, and high neurogenic capacity. They generate GABAergic, glutamatergic and, at lower frequency, serotonergic neurons. Importantly, hbNES cells stably maintain hindbrain specification and generate upper rhombic lip derivatives on exposure to bone morphogenetic protein (BMP). When grafted into neonatal rat brain, they show potential for integration into cerebellar development and produce cerebellar granule-like cells, albeit at low frequency. hbNES cells offer a new system to study human cerebellar specification and development and to model diseases of the hindbrain. They also provide a benchmark for the production of similar long-term neuroepithelial-like stem cells (lt-NES) from pluripotent cell lines. To our knowledge, hbNES cells are the first demonstration of highly expandable neuroepithelial stem cells derived from the human embryo without genetic immortalization.

Frequency dependence of CA3 spike phase response arising from h-current properties.

The phase of firing of hippocampal neurons during theta oscillations encodes spatial information. Moreover, the spike phase response to synaptic inputs in individual cells depends on the expression of the hyperpolarization-activated mixed cation current (I h ), which differs between CA3 and CA1 pyramidal neurons. Here, we compared the phase response of these two cell types, as well as their intrinsic membrane properties. We found that both CA3 and CA1 pyramidal neurons show a voltage sag in response to negative current steps but that this voltage sag is significantly smaller in CA3 cells. Moreover, CA3 pyramidal neurons have less prominent resonance properties compared to CA1 pyramidal neurons. This is consistent with differential expression of I h by the two cell types. Despite their distinct intrinsic membrane properties, both CA3 and CA1 pyramidal neurons displayed bidirectional spike phase control by excitatory conductance inputs during theta oscillations. In particular, excitatory inputs delivered at the descending phase of a dynamic clamp-induced membrane potential oscillation delayed the subsequent spike by nearly 50 mrad. The effect was shown to be mediated by I h and was counteracted by increasing inhibitory conductance driving the membrane potential oscillation. Using our experimental data to feed a computational model, we showed that differences in I h between CA3 and CA1 pyramidal neurons could predict frequency-dependent differences in phase response properties between these cell types. We confirmed experimentally such frequency-dependent spike phase control in CA3 neurons. Therefore, a decrease in theta frequency, which is observed in intact animals during novelty, might switch the CA3 spike phase response from unidirectional to bidirectional and thereby promote encoding of the new context.

Hippocampal mossy fiber long-term depression in Grm2/3 double knockout mice.

Group II metabotropic glutamate receptors (mGluR2, encoded by Grm2, and mGluR3, encoded by Grm3) are inhibitory autoreceptors that negatively modulate the adenylate cyclase signaling cascade. Within the hippocampus, mGluR2 is believed to play a key role in the induction of long-term depression (LTD) at mossy fiber-CA3 synapses. Here, we used Grm2/3 double knockout (dko) mice to investigate to what extent group II mGluRs are necessary for mossy fiber LTD. Surprisingly, we found that these mice displayed prominent mossy fiber LTD. However, the induction of this form of LTD was sensitive to the external Ca(2+) concentration. Mossy fiber LTD in Grm2/3 dko mice was indistinguishable from that in wild-type mice at 4 mM Ca(2+) , but largely absent at 2 mM external Ca(2+) . Mossy fiber LTD in Grm2/3 dko mice was not blocked by the N-methyl-D-aspartic acid (NMDA) receptor antagonist D-AP5, confirming that the observed response did not reflect NMDA receptor-dependent LTD in contaminating associational-commissural fibers, and enabling us to use the NMDA receptor-mediated EPSC to monitor mossy fiber LTD. Using whole-cell recordings, we demonstrated that LTD of the NMDA receptor-mediated EPSC in Grm2/3 dko mice was not affected by intracellular application of BAPTA and CsF to block postsynaptic Ca(2+) and G-protein-mediated effects. This presynaptic LTD was, however, blocked by the AMPA/kainate receptor antagonist, NBQX. Thus, an activity-dependent, external Ca(2+) concentration-sensitive form of mossy fiber LTD can be induced in Grm2/3 dko mice. Two mGluR antagonists also failed to block mossy fiber LTD under 4 mM conditions in wild-type mice, strengthening the conclusion that group II mGluRs are not obligatory for mossy fiber LTD.