Many networks generate distinct rhythms with multiple frequency and amplitude characteristics. The respiratory network in the pre-Bötzinger complex (pre-Böt) generates both the low-frequency, large-amplitude sigh rhythm and a faster, smaller-amplitude eupneic rhythm. Could the same set of pacemakers generate both rhythms? Here we used an in vitro respiratory brainslice preparation. We describe a subset of synaptically isolated pacemakers that spontaneously generate two distinct bursting patterns. These two patterns resemble network activity including sigh-like bursts that occur at low frequencies and have large amplitudes and eupneic-like bursts with higher frequency and smaller amplitudes. Cholinergic neuromodulation altered the network and pacemaker bursting: fictive sigh frequency is increased dramatically, whereas fictive eupneic frequency is drastically lowered. The data suggest that timing and amplitude characteristics of fictive eupneic and sigh rhythms are set by the same set of pacemakers that are tuned by changes in the neuromodulatory state.
Mammals have circadian variation in blood pressure, heart rate, vascular tone, thrombotic tendency, and cerebral blood flow (CBF). These changes may be in part orchestrated by circadian variation in clock gene expression within cells comprising the vasculature that modulate blood flow (e.g., fibroblasts, cerebral vascular smooth muscle cells, astrocytes, and endothelial cells). However, the downstream mechanisms that underlie circadian changes in blood flow are unknown. Cytochrome P450 epoxygenases (Cyp4x1 and Cyp2c11) are expressed in the brain and vasculature and metabolize arachidonic acid (AA) to form epoxyeicosatrienoic acids (EETs). EETs are released from astrocytes, neurons, and vascular endothelial cells and act as potent vasodilators, increasing blood flow. EETs released in response to increases in neural activity evoke a corresponding increase in blood flow known as the functional hyperemic response. We examine the hypothesis that Cyp2c11 and Cyp4x1 expression and EETs production vary in a circadian manner in the rat brain and cerebral vasculature. RT-PCR revealed circadian/diurnal expression of clock and clock-controlled genes as well as Cyp4x1 and Cyp2c11, within the rat hippocampus, middle cerebral artery, inferior vena cava, hippocampal astrocytes and rat brain microvascular endothelial cells. Astrocyte and endothelial cell culture experiments revealed rhythmic variation in Cyp4x1 and Cyp2c11 gene and protein expression with a 12-h period and parallel rhythmic production of EETs. Our data suggest there is circadian regulation of Cyp4x1 and Cyp2c11 gene expression. Such rhythmic EETs production may contribute to circadian changes in blood flow and alter risk of adverse cardiovascular events throughout the day.
To test the hypothesis that focal and parafocal neocortical tissue from pediatric patients with intractable epilepsy exhibits cellular and synaptic differences, the authors characterized the propensity of these neurons to generate (a) voltage-dependent bursting and (b) synaptically driven paroxysmal depolarization shifts. Neocortical slices were prepared from tissue resected from patients with intractable epilepsy. Multiunit network activity and simultaneous whole-cell patch recordings were made from neurons from three patient groups: (1) those with normal histology; (2) those with mild and severe cortical dysplasia; and (3) those with abnormal pathology but without cortical dysplasia. Seizure-like activity was characterized by population bursting with concomitant bursting in intracellularly recorded cortical neurons (n = 59). The authors found significantly more N-methyl-d-aspartic acid-driven voltage-dependent bursting neurons in focal versus parafocal tissue in patients with severe cortical dysplasia (P < 0.01). Occurrence of paroxysmal depolarization shifts and burst amplitude and burst duration were significantly related to tissue type: focal or parafocal (P < 0.05). The authors show that functional differences between focal and parafocal tissue in patients with severe cortical dysplasia exist. There are functional differences between patient groups with different histology, and bursting properties can be significantly associated with the distinction between focal and parafocal tissue.
Abstract Infraslow activity (ISA) is a biomarker that has recently become of interest in the characterization of seizure recordings. Recent data from a small number of studies have suggested that the epileptogenic zone may be identified by the presence of ISA. Investigation of low frequency activity in clinical seizure recordings, however, has been hampered by technical limitations. EEG systems necessarily include a high-pass filter early in the measurement chain to remove large artifactual drifts that can saturate recording elements such as the amplifier. This filter, unfortunately, attenuates legitimately seizure-related low frequencies, making ISA difficult to study in clinical EEG recordings. In this study, we present a deconvolution-based digital inverse filter that allows recovery of attenuated low frequency activity in intracranial recordings of temporal lobe epilepsy patients. First, we show that the unit impulse response (UIR) of an EEG system can be characterized by differentiation of the system’s step response. As proof of method, we present several examples that show that the low frequency component of a high-pass filtered signal can be restored by deconvolution with the UIR. We then demonstrate that this method can be applied to biologically relevant signals including clinical EEG recordings obtained from seizure patients. Finally, we discuss how this method can be applied to study ISA to identify and assess the seizure onset zone.
Many rhythmic behaviors are continuously modulated by endogenous peptides and amines, but whether neuromodulation is critical to the expression of a rhythmic behavior often remains unknown, particularly in mammals. Here, we address this issue in the respiratory network that was isolated in spontaneously rhythmic medullary slice preparations from mice. Under control conditions, the respiratory network generates fictive eupneic activity. We hypothesized previously that this activity depends on two types of pacemaker neurons. The bursting properties of one pacemaker rely on the persistent sodium current ( I Na(p) ) and are insensitive to blockade of calcium channels with cadmium (CI-pacemakers), whereas bursting mechanisms of a second pacemaker are sensitive to cadmium (CS-pacemakers) and the calcium-dependent nonspecific cation current blocker flufenamic acid. During hypoxia, fictive eupneic activity is supplanted by the neural correlate of gasping, which is proposed to depend only on CI-pacemakers. Because CI-pacemakers require endogenous activation of 5-HT 2A receptors, we tested the hypothesis that 5-HT 2A receptor activation is critical for gasping. Here, we demonstrate that fictive gasping and CI-pacemaker bursting were selectively eliminated by the 5-HT 2A receptor antagonist piperidine or ketanserin. Neither 5-HT 2A antagonist eliminated bursting by CS-pacemakers and ventral respiratory group (VRG) population activity. However, this VRG activity was very different from eupneic activity. In the presence of 5-HT 2A antagonists, VRG activity was eliminated by flufenamic acid and could not be reliably restored by adding substance P. These data support the hypothesis that two types of pacemaker bursting mechanisms underlie fictive eupnea, whereas only one burst mechanism is critical for gasping.
Abstract Recently developed methods for transforming 2D patterns of thin‐film materials into 3D mesostructures create many interesting opportunities in microsystems design. A growing area of interest is in multifunctional thermal, electrical, chemical, and optical interfaces to biological tissues, particularly 3D multicellular, millimeter‐scale constructs, such as spheroids, assembloids, and organoids. Herein, examples of 3D mechanical interfaces are presented, in which thin ribbons of parylene‐C form the basis of transparent, highly compliant frameworks that can be reversibly opened and closed to capture, envelop, and mechanically restrain fragile 3D tissues in a gentle, nondestructive manner, for precise measurements of viscoelastic properties using techniques in nanoindentation. Finite element analysis serves as a design tool to guide selection of geometries and material parameters for shape‐matching 3D architectures tailored to organoids of interest. These computational approaches also quantitate all aspects of deformations during the processes of opening and closing the structures and of forces imparted by them onto the surfaces of enclosed soft tissues. Studies of cerebral organoids by nanoindentation show effective Young's moduli in the range from 1.5 to 2.5 kPa depending on the age of the organoid. This collection of results suggests broad utility of compliant 3D mesostructures in noninvasive mechanical measurements of millimeter‐scale, soft biological tissues.
In a previous study, we combined joint kinematics and electromyograms (EMGs) to examine the change in the phase relationship of two principal leg joints during walking and searching. In this study, we recorded intracellularly from motor neurons in semi-intact behaving animals to examine mechanisms coordinating extension at these leg joints. In particular, we examined the change in the phase of the coxa-trochanter (CTr) and femur-tibia (FT) joint extension during walking and searching. In doing so, we discovered marked similarities in the activity of CTr and FT joint extensor motor neurons at the onset of extension during searching and at the end of stance during walking. The data suggest that the same interneurons may be involved in coordinating the CTr and FT extensor motor neurons during walking and searching. Previous studies in stick insects have suggested that extensor motor neuron activity during the stance phase of walking results from an increase in tonic excitation of the neuron leading to spiking that is periodically interrupted by centrally generated inhibition. However, the CTr and FT extensor motor neuron activity during walking consists of characteristic phasic modulations in motor neuron frequency within each step cycle. The phasic increases and decreases in extensor EMG frequency during stance are associated with kinematic events (i.e., foot set-down and joint cycle transitions) during walking. Sensory feedback associated with these events might be responsible for phasic modulation of the extensor motor neuron frequency. However, our data rule out the possibility that sensory cues resulting from foot set-down are responsible for a decline in CTr extensor activity that is characteristic of the Blaberusstep cycle. Our data also suggest that both phasic excitation and inhibition contribute to extensor motor neuron activity during the stance phase of walking.
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