Genetic Activation, Inactivation, and Deletion Reveal a Limited And Nuanced Role for Somatostatin-Containing Basal Forebrain Neurons in Behavioral State Control

Recent studies have identified an especially important role for basal forebrain GABAergic (BF(VGAT)) neurons in the regulation of behavioral waking and fast cortical rhythms associated with cognition. However, BF(VGAT) neurons comprise several neurochemically and anatomically distinct subpopulations, including parvalbumin-containing BF(VGAT) neurons and somatostatin-containing BF(VGAT) neurons (BF(SOM) neurons), and it was recently reported that optogenetic activation of BF(SOM) neurons increases the probability of a wakefulness to non-rapid-eye movement (NREM) sleep transition when stimulated during the rest period of the animal. This finding was unexpected given that most BF(SOM) neurons are not NREM sleep active and that central administration of the synthetic somatostatin analog, octreotide, suppresses NREM sleep or increases REM sleep. Here we used a combination of genetically driven chemogenetic and optogenetic activation, chemogenetic inhibition, and ablation approaches to further explore the in vivo role of BF(SOM) neurons in arousal control. Our findings indicate that acute activation or inhibition of BF(SOM) neurons is neither wakefulness nor NREM sleep promoting and is without significant effect on the EEG, and that chronic loss of these neurons is without effect on total 24 h sleep amounts, although a small but significant increase in waking was observed in the lesioned mice during the early active period. Our in vitro cell recordings further reveal electrophysiological heterogeneity in BF(SOM) neurons, specifically suggesting at least two distinct subpopulations. Together, our data support the more nuanced view that BF(SOM) neurons are electrically heterogeneous and are not NREM sleep or wake promoting per se, but may exert, in particular during the early active period, a modest inhibitory influence on arousal circuitry.SIGNIFICANCE STATEMENT The cellular basal forebrain (BF) is a highly complex area of the brain that is implicated in a wide range of higher-level neurobiological processes, including regulating and maintaining normal levels of electrocortical and behavioral arousal. The respective in vivo roles of BF cell populations and their neurotransmitter systems in the regulation of electrocortical and behavioral arousal remains incompletely understood. Here we seek to define the neurobiological contribution of GABAergic somatostatin-containing BF neurons to arousal control. Understanding the respective contribution of BF cell populations to arousal control may provide critical insight into the pathogenesis of a host of neuropsychiatric and neurodegenerative disorders, including Alzheimer\u27s disease, Parkinson\u27s disease, schizophrenia, and the cognitive impairments of normal aging

Recovering Arrhythmic EEG Transients from Their Stochastic Interference

Traditionally, the neuronal dynamics underlying electroencephalograms (EEG) have been understood as arising from \textit{rhythmic oscillators with varying degrees of synchronization}. This dominant metaphor employs frequency domain EEG analysis to identify the most prominent populations of neuronal current sources in terms of their frequency and spectral power. However, emerging perspectives on EEG highlight its arrhythmic nature, which is primarily inferred from broadband EEG properties like the ubiquitous $1/f$ spectrum. In the present study, we use an \textit{arrhythmic superposition of pulses} as a metaphor to explain the origin of EEG. This conceptualization has a fundamental problem because the interference produced by the superpositions of pulses generates colored Gaussian noise, masking the temporal profile of the generating pulse. We solved this problem by developing a mathematical method involving the derivative of the autocovariance function to recover excellent approximations of the underlying pulses, significantly extending the analysis of this type of stochastic processes. When the method is applied to spontaneous mouse EEG sampled at $5$ kHz during the sleep-wake cycle, specific patterns -- called $\Psi$-patterns -- characterizing NREM sleep, REM sleep, and wakefulness are revealed. $\Psi$-patterns can be understood theoretically as \textit{power density in the time domain} and correspond to combinations of generating pulses at different time scales. Remarkably, we report the first EEG wakefulness-specific feature, which corresponds to an ultra-fast ($\sim 1$ ms) transient component of the observed patterns. By shifting the paradigm of EEG genesis from oscillators to random pulse generators, our theoretical framework pushes the boundaries of traditional Fourier-based EEG analysis, paving the way for new insights into the arrhythmic components of neural dynamics.Comment: Original research manuscript in PDF format, 46 pages long, with 13 figures and one tabl

Natural (∆9-THC) and synthetic (JWH-018) cannabinoids induce seizures by acting through the cannabinoid CB1 receptor

Natural cannabinoids and their synthetic substitutes are the most widely used recreational drugs. Numerous clinical cases describe acute toxic symptoms and neurological consequences following inhalation of the mixture of synthetic cannabinoids known as “Spice.” Here we report that an intraperitoneal administration of the natural cannabinoid Δ9-tetrahydrocannabinol (10 mg/kg), one of the main constituent of marijuana, or the synthetic cannabinoid JWH-018 (2.5 mg/kg) triggered electrographic seizures in mice, recorded by electroencephalography and videography. Administration of JWH-018 (1.5, 2.5 and 5 mg/kg) increased seizure spikes dose-dependently. Pretreatment of mice with AM-251 (5 mg/kg), a cannabinoid receptor 1-selective antagonist, completely prevented cannabinoid-induced seizures. These data imply that abuse of cannabinoids can be dangerous and represents an emerging public health threat. Additionally, our data strongly suggest that AM-251 could be used as a crucial prophylactic therapy for cannabinoid-induced seizures or similar life-threatening conditions

Role of electrical activity in horizontal axon growth in the developing cortex: a time-lapse study using optogenetic stimulation.

During development, layer 2/3 neurons in the neocortex extend their axons horizontally, within the same layers, and stop growing at appropriate locations to form branches and synaptic connections. Firing and synaptic activity are thought to be involved in this process, but how neuronal activity regulates axonal growth is not clear. Here, we studied axonal growth of layer 2/3 neurons by exciting cell bodies or axonal processes in organotypic slice cultures of the rat cortex. For neuronal stimulation and morphological observation, plasmids encoding channelrhodopsin-2 (ChR2) and DsRed were coelectroporated into a small number of layer 2/3 cells. Firing activity induced by photostimulation (475 nm) was confirmed by whole-cell patch recording. Axonal growth was observed by time-lapse confocal microscopy, using a different excitation wavelength (560 nm), at 10-20-min intervals for several hours. During the first week in vitro, when spontaneous neuronal activity is low, DsRed- and ChR2-expressing axons grew at a constant rate. When high-frequency photostimulation (4 or 10 Hz) for 1 min was applied to the soma or axon, most axons paused in their growth. In contrast, lower-frequency stimulation did not elicit this pause behavior. Moreover, in the presence of tetrodotoxin, even high-frequency stimulation did not cause axonal growth to pause. These results indicate that increasing firing activity during development suppresses axon growth, suggesting the importance of neuronal activity for the formation of horizontal connections

Horizontal axon growth before and after high-frequency stimulation to the axon.

<p><b>A,</b> High-frequency photostimulation (4 Hz) elicited retraction of a representative growth cone. Scale bar, 5 µm. <b>B,</b> Growth of the horizontal axon shown in A. The arrow indicates the time of photostimulation. <b>C,</b> Growth of individual axons before and after photostimulation (arrow). <b>D,</b> Horizontal axon growth in the presence of TTX after high-frequency photostimulation. TTX (1 µM) was added to the culture medium 1 h prior to the observation. Photostimulation was applied at 4 Hz for 1 min to the cell body (n = 2).</p

Horizontal axon growth in cortical slice culture.

<p><b>A,</b> DsRed and ChR2-EYFP plasmids were introduced into several layer 2/3 cells at 1 DIV, and labeled axons were observed by confocal microscopy (560 nm excitation) at 4 DIV. This excitation does not activate ChR2. Note that a horizontally elongating axon is clearly labeled. The interrupted lines indicate the pial surface and the presumed layer 2/3 borders. Scale bar, 100 µm. <b>B</b>, Laminar localizations of the cells projecting horizontal axons which were used for the time-lapse study. Red dots indicate the locations of the cell bodies. Arrows indicate the presumed borders of layer 1, 2/3, 4, 5 and 6. <b>C,</b> Growth of the horizontal axon revealed by time-lapse study. The axon was followed every 10 min. Scale bar, 5 µm. <b>D,</b> Growth curve of the horizontal axon shown in <b>C</b>. <b>E,</b> Average growth of horizontal axons observed in cortical slice cultures from 3 to 10 DIV (n = 32).</p

Axonal growth after low-frequency stimulation of the soma.

<p><b>A, B,</b> Photostimulation at 1(<b>A</b>) or 0.1 Hz (<b>B</b>) for 1 min was applied (arrows) during the time-lapse study. <b>C,</b> A total of 120 pulses was applied to a single axon at low frequency (0.1 Hz) and subsequently at high frequency (4 Hz). Pause behavior was induced by high-frequency stimulation but not by low-frequency stimulation, even though the number of pulses was the same in both cases.</p

Electrophysiological responses of ChR2-expressing cortical neurons to photostimulation.

<p><b>A</b> and <b>B</b>, ChR2-expressing cells among layer 2/3 neurons were subjected to patch-clamp recording at 4 DIV. Interrupted lines in <b>A</b> indicate the presumed layer 2/3 borders. The white line in <b>B</b> indicates the pial surface. Bar, 200 µm. <b>C,</b> Each flash with 475-nm wavelength light (50-msec duration in the upper trace and 200-msec duration in the lower trace) elicited depolarization and a single action potential.</p

Occurrence of pause behavior after photostimulation with different frequencies.

<p>Histograms show the percentage of axons displaying pause behavior after photostimulation with different frequencies. The numbers in parentheses are the numbers of samples examined for each frequency.</p