Rat Somatosensory Cortex Research Articles

Size, Shape, and Distribution of Multivesicular Bodies in the Juvenile Rat Somatosensory Cortex: A 3D Electron Microscopy Study.

Multivesicular bodies (MVBs) are membrane-bound organelles that belong to the endosomal pathway. They participate in the transport, sorting, storage, recycling, degradation, and release of multiple substances. They interchange cargo with other organelles and participate in their renovation and degradation. We have used focused ion beam milling and scanning electron microscopy (FIB-SEM) to obtain stacks of serial sections from the neuropil of the somatosensory cortex of the juvenile rat. Using dedicated software, we have 3D-reconstructed 1618 MVBs. The mean density of MVBs was 0.21 per cubic micron. They were unequally distributed between dendrites (39.14%), axons (18.16%), and nonsynaptic cell processes (42.70%). About one out of five MVBs (18.16%) were docked on mitochondria, representing the process by which the endosomal pathway participates in mitochondrial maintenance. Other features of MVBs, such as the presence of tubular protrusions (6.66%) or clathrin coats (19.74%) can also be interpreted in functional terms, since both are typical of early endosomes. The sizes of MVBs follow a lognormal distribution, with differences across cortical layers and cellular compartments. The mean volume of dendritic MVBs is more than twice as large as the volume of axonic MVBs. In layer I, they are smaller, on average, than in the other layers.

Estimating the Readily-Releasable Vesicle Pool Size at Synaptic Connections in the Neocortex.

Previous studies based on the ‘Quantal Model’ for synaptic transmission suggest that neurotransmitter release is mediated by a single release site at individual synaptic contacts in the neocortex. However, recent studies seem to contradict this hypothesis and indicate that multi-vesicular release (MVR) could better explain the synaptic response variability observed in vitro. In this study we present a novel method to estimate the number of release sites per synapse, also known as the size of the readily releasable pool (NRRP), from paired whole-cell recordings of connections between layer 5 thick tufted pyramidal cell (L5_TTPC) in the juvenile rat somatosensory cortex. Our approach extends the work of Loebel et al. (2009) by leveraging a recently published data-driven biophysical model of neocortical tissue. Using this approach, we estimated NRRP to be between two to three for synaptic connections between L5_TTPCs. To constrain NRRP values for other connections in the microcircuit, we developed and validated a generalization approach using published data on the coefficient of variation (CV) of the amplitudes of post-synaptic potentials (PSPs) from literature and comparing them against in silico experiments. Our study predicts that transmitter release at synaptic connections in the neocortex could be mediated by MVR and provides a data-driven approach to constrain the MVR model parameters in the microcircuit.

Behavioral and Cortical Correlates of Self-Suppression, Anticipation, and Ambivalence in Rat Tickling.

The relationship between tickling, sensation, and laughter is complex. Tickling or its mere anticipation makes us laugh, but not when we self-tickle. We previously showed rat somatosensory cortex drives tickling-evoked vocalizations and now investigated self-tickle suppression and tickle anticipation. We recorded somatosensory cortex activity while tickling and touching rats and while rats touched themselves. Allo-touch and tickling evoked somatotopic cortical excitation and vocalizations. Self-touch induced wide-ranging inhibition and vocalization suppression. Self-touch also suppressed vocalizations and cortical responses evoked by allo-touch or cortical microstimulation. We suggest a global-inhibition model of self-tickle suppression, which operates without the classically assumed self versus other distinction. Consistent with this inhibition hypothesis, blocking cortical inhibition with gabazine abolished self-tickle suppression. We studied anticipation in a nose-poke-for-tickling paradigm. Although rats nose poked for tickling, they also showed escaping, freezing, and alarm calls. Such ambivalence ("Nervenkitzel") resembles tickle behaviors in children. We conclude that self-touch-induced GABAergic cortical inhibition prevents self-tickle, whereas anticipatory layer 5 activity drives anticipatory laughter. VIDEO ABSTRACT.

Stereological Analysis of Early Gene Expression Using Egr-1 Immunolabeling After Spreading Depression in the Rat Somatosensory Cortex.

Early growth response-1 (Egr-1), defined as a zinc finger transcription factor, is an upstream master switch of the inflammatory response, and its expression can be used to investigate the spatial and temporal extent of inflammatory changes in the brain. Cortical spreading depression (CSD) is characterized as a slowly propagating (2–5 mm/min) depolarization wave through neurons and astrocytes in humans that contributes to migraines and possibly to other brain pathologies. In rodents, CSD can be induced experimentally, which involves unilateral depolarization that is associated with microglial and astrocyte responses. The impact of CSD on structures beyond the affected hemisphere has not been explored. Here, we used an optical fractionator method to investigate potential correlations between the number of and period of the eletrophysiologic record of CSD phenomena and Egr-1 expression in ipsilateral and contralateral hemispheres. CSD was elicited by the restricted application of a 2% KCl solution over the left premotor cortex. Electrophysiological events were recorded using a pair of Ag/AgCl agar-Ringer electrodes for 2 or 6 h. An optical fractionator was applied to count the Egr-1 positive cells. We found that CSD increased Egr-1 expression in a time- and event-dependent manner in the ipsilateral/left hemisphere. Although CSD did not cross the midline, multiple CSD inductions were associated with an increased number of Egr-1 positive cells in the contralateral/right hemisphere. Thus, repeated CSD waves may have far reaching effects that are more global than previously considered possible. The mechanism of contralateral expression is unknown, but we speculate that callosal projections from the depolarized hemisphere may be related to this phenomenon.

Development of Efficient AAV2/DJ-Based Viral Vectors to Selectively Downregulate the Expression of Neuronal or Astrocytic Target Proteins in the Rat Central Nervous System.

Viral vectors have become very popular to overexpress or downregulate proteins of interest in different cell types. They conveniently allow the precise targeting of well-defined tissue areas, which is particularly useful in complex organs like the brain. In theory, each vector should have its own cell specificity that can be obtained by using different strategies (e.g., using a cell-specific promoter). For the moment, there is few vectors that have been developed to alternatively target, using the same capsid, neurons and astrocytes in the central nervous system. There is even fewer examples of adeno-associated viral vectors able to efficiently transduce cells both in vitro and in vivo. The development of viral vectors allowing the cell-specific downregulation of a protein in cultured cells of the central nervous system as well as in vivo within a large brain area would be highly desirable to address several important questions in neurobiology. Here we report that the use of the AAV2/DJ viral vector associated to an hybrid CMV/chicken β-actin promoter (CBA) or to a modified form of the glial fibrillary acidic protein promoter (G1B3) allows a specific transduction of neurons or astrocytes in more than half of the barrel field within the rat somatosensory cortex. Moreover, the use of the miR30E-shRNA technology led to an efficient downregulation of two proteins of interest related to metabolism both in vitro and in vivo. Our results demonstrate that it is possible to downregulate the expression of different protein isoforms in a cell-specific manner using a common serotype. It is proposed that such an approach could be extended to other cell types and used to target several proteins of interest within the same brain area.

Telemetry-controlled simultaneous stimulation-and-recording device (SRD) to study interhemispheric cortical circuits in rat primary somatosensory (SI) cortex

BackgroundA growing need exists for neuroscience platforms that can perform simultaneous chronic recording and stimulation of neural tissue in animal models in a telemetry-controlled fashion with signal processing for analysis of the chronic recording data and external triggering capability. We describe the system design, testing, evaluation, and implementation of a wireless simultaneous stimulation-and-recording device (SRD) for modulating cortical circuits in physiologically identified sites in primary somatosensory (SI) cortex in awake-behaving and freely-moving rats. The SRD was developed using low-cost electronic components and open-source software. The function of the SRD was assessed by bench and in-vivo testing.ResultsThe SRD recorded spontaneous spiking and bursting neuronal activity, evoked responses to programmed intracortical microstimulation (ICMS) delivered internally by the SRD, and evoked responses to external peripheral forelimb stimulation.ConclusionsThe SRD is capable of wireless stimulation and recording on a predetermined schedule or can be wirelessly synchronized with external input as would be required in behavioral testing prior to, during, and following ICMS.

A Derived Positional Mapping of Inhibitory Subtypes in the Somatosensory Cortex.

Obtaining a catalog of cell types is a fundamental building block for understanding the brain. The ideal classification of cell-types is based on the profile of molecules expressed by a cell, in particular, the profile of genes expressed. One strategy is, therefore, to obtain as many single-cell transcriptomes as possible and isolate clusters of neurons with similar gene expression profiles. In this study, we explored an alternative strategy. We explored whether cell-types can be algorithmically derived by combining protein tissue stains with transcript expression profiles. We developed an algorithm that aims to distribute cell-types in the different layers of somatosensory cortex of the developing rat constrained by the tissue- and cellular level data. We found that the spatial distribution of major inhibitory cell types can be approximated using the available data. The result is a depth-wise atlas of inhibitory cell-types of the rat somatosensory cortex. In principle, any data that constrains what can occur in a particular part of the brain can also strongly constrain the derivation of cell-types. This draft inhibitory cell-type mapping is therefore dynamic and can iteratively converge towards the ground truth as further data is integrated.

Probing activation-induced neurochemical changes using optogenetics combined with functional magnetic resonance spectroscopy: a feasibility study in the rat primary somatosensory cortex.

Knowledge about biochemical processes is a prerequisite for a better understanding of mechanisms underlying brain activity. 1 H functional magnetic resonance spectroscopy (1 H-fMRS) at high magnetic fields allows for the non-invasive measurement of metabolic changes during brain activation. Optogenetics, on the other hand, has revolutionized the field of neuroscience. It was previously coupled with functional magnetic resonance imaging (fMRI) techniques in rodents enabling population-specific targeting of cells, investigating brain networks with unprecedented in vivo precision. The coupling of optogenetics and 1 H-fMRS is expected to enhance the specificity of metabolic readouts to validate neuro-energetic theories underlying brain activity. To date, the feasibility of combining optogenetic stimulation with fMRS has not been explored. We used green laser stimulation delivered through an optical fiber implanted superficially above the primary somatosensory forelimb cortex (S1FL) of rats expressing the C1V1 opsin in excitatory neurons. A protocol for the acquisition of functional 1 H MR spectra upon optogenetic stimulation without craniotomy-induced artefacts was established. Quantification of metabolite concentrations in S1FL upon optogenetic and electrical forepaw stimulation demonstrated significantly increased glutamate levels (+8.8% and +9.9%, p<0.05 respectively), which were compensated by decreased glutamine levels (-17% and -18%, p<0.05 respectively). Our results demonstrate for the first time the feasibility of combining optogenetic control and functional MR spectroscopy (o-fMRS) in the rat somatosensory cortex, opening new possibilities for monitoring the energetic demands of specific cell populations and for exploring the underpinnings of energy metabolism during brain activity. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Cortical stroke affects activity and stability of theta/delta states in remote hippocampal regions.

Cognitive impairment is a common outcome of ischemic stroke. Our previous work has shown that an experimental stroke in the cortex reduces activity in remote hippocampal layers in rats. This study seeks to uncover the underlying functional connections between these areas by analyzing changes to oscillatory activity, signal power, and communication. We induced an ischemic stroke in the left somatosensory cortex of rats and used linear micro-electrode arrays to simultaneously record from cortex and hippocampus under urethane anesthesia at two weeks and one month after stroke. We found significant increase in signal power, as well as an increase in the number of brain state changes in response to stroke. Our results suggest that the cortex modulates the activity and stability of hippocampal oscillations, which is disrupted following cortical stroke that can lead to cognitive impairment.

The lower expression of parvalbumin in the primary somatosensory cortex of WAG/Rij rats may facilitate the occurrence of absence seizures

Absence epilepsy (AE) is classified as a genetic generalized epilepsies. WAG/Rij strain of rats are regarded one of the most validated models of absence epilepsy. Studies point out the existence of hyperexcitable focus in somatosensory cortex of these rats, which has been attributed to the deficits in the GABAergic system. In the current study, we studied the changes of calcium binding proteins (CaBPs) in somatosensory cortex (S1) of the 2 and 8 month-old WAG/Rij rats and their age-matched Wistar Albino controls by investigating the expression levels of CaBPs (calbindin, calretinin and parvalbumin) in western blotting. Since WAG/Rij rats showed the low expression level of parvalbumin (PV) in western blots in comparison to Wistar Albino rats, we selectively investigated the number of PV positive neurons using the immunofluorescence staining method in order to confirm this decrement in the perioral region of somatosensory cortex (S1po). The most critical finding of this study was the age- independent reduction in the expression level of PV in the somatosensory cortex of epileptic rats as demonstrating western blotting. Nevertheless, no significant difference was found among numbers of PV + neuron in the S1po region by immunofluorescence staining concerning both of age and strain dependency. These results suggest that the disruption in the activity of the PV-expressing GABAergic interneurons might be involved in the generation of rather than the age-dependent increase in the SWDs in WAG/Rij rats.

Circular Bayesian classifiers using wrapped Cauchy distributions

Capturing the dependences among circular variables within supervised classification models is a challenging task. In this paper, we propose four different supervised Bayesian classification algorithms where the predictor variables follow all circular wrapped Cauchy distributions. For this purpose, we introduce four wrapped Cauchy classifiers. The bivariate wrapped Cauchy distribution is the only bivariate circular distribution whose marginals and conditionals are also wrapped Cauchy distributions, a property that makes it possible to define these models easily. Furthermore, the wrapped Cauchy tree-augmented naive Bayes classifier requires the definition of a conditional circular mutual information measure between variables that follow wrapped Cauchy distributions. Synthetic data is used to illustrate, compare and evaluate the classification algorithms (including a comparison with the Gaussian TAN classifier, decision tree, random forest, multinomial logistic regression, support vector machine and simple neural network), leading to satisfactory predictive results. We also use a real neuromorphological dataset obtained from juvenile rat somatosensory cortex cells, where we measure the bifurcation angles of the dendritic basal arbors.

Microelectrode array electrical impedance tomography for fast functional imaging in the thalamus

Electrical Impedance Tomography (EIT) has the potential to be able to observe functional tomographic images of neural activity in the brain at millisecond time-scales. Prior modelling and experimental work has shown that EIT is capable of imaging impedance changes from neural depolarisation in rat somatosensory cortex. Here, we investigate the feasibility of EIT for imaging impedance changes using a stereotaxically implanted microelectrode array in the thalamus. Microelectrode array EIT was simulated using an anatomically accurate marmoset brain model. Impedance imaging was validated and detectability estimated using physiological noise recorded from the marmoset visual thalamus. The results suggest that visual-input-driven impedance changes in visual subcortical bodies within 300 μm of the implanted array could be reliably reconstructed and localised, comparable to local field potential measurements. Furthermore, we demonstrated that microelectrode array EIT could reconstruct concurrent activity in multiple subcortical bodies simultaneously.

Time-resolved quantification of the dynamic extracellular space in the brain: study of cortical spreading depression.

Extracellular diffusion in the brain is customarily characterized by two parameters, the extracellular space (ECS) volume fraction α and the diffusion tortuosity λ. How these two parameters are temporarily modified and correlated in a physiological/pathological event remains unclear to date. Using tetramethylammonium (TMA+) as an ECS ion tracer in a newly updated iontophoretic sinusoidal method, we studied in this work the dynamic α(t) and λ(t) in rat somatosensory cortex during spreading depression (SD). Temporal variations of α(t) and λ(t), as evoked by SD, were obtained through analyses of the extracellular TMA+ diffusion waveform resulting from a sinusoidally modulated point source. Most of the time, cortical SD induced coordinated α(t) decreases and λ(t) increases. In rare occasions, SD induced sole decreases of α(t) with no changes in λ(t). The independent modulation of α(t) and λ(t) was neither associated with cortical anatomy nor with the specific shape of the SD field potential wave. Changes of α(t) and λ(t) often took place acutely at the onset of SD, followed by a more transient modulation. Compared with the prior iontophoretic methods of TMA+, the sinusoidal method provides time-resolved quantification of α(t) and λ(t) in relative terms but also raises a higher property requirement on the TMA+-selective microelectrode. The sinusoidal method could become a valuable tool in the studies of the dynamic ECS response in various brain events. NEW & NOTEWORTHY An iontophoretic sinusoidal method was applied to study the dynamic changes of two extracellular space parameters, the extracellular volume fraction α(t) and tortuosity λ(t), in the brain during cortical spreading depression. Both parameters showed coordinated (most often) and independent (rarely) modulations in spreading depression. The sinusoidal method is equally applicable to other acute pathological events and a valuable tool to study the functional role of extracellular space in brain events.

Objective Morphological Classification of Neocortical Pyramidal Cells.

A consensus on the number of morphologically different types of pyramidal cells (PCs) in the neocortex has not yet been reached, despite over a century of anatomical studies, due to the lack of agreement on the subjective classifications of neuron types, which is based on expert analyses of neuronal morphologies. Even for neurons that are visually distinguishable, there is no common ground to consistently define morphological types. The objective classification of PCs can be achieved with methods from algebraic topology, and the dendritic arborization is sufficient for the reliable identification of distinct types of cortical PCs. Therefore, we objectively identify 17 types of PCs in the rat somatosensory cortex. In addition, we provide a solution to the challenging problem of whether 2 similar neurons belong to different types or to a continuum of the same type. Our topological classification does not require expert input, is stable, and helps settle the long-standing debate on whether cell-types are discrete or continuous morphological variations of each other.

Abstract #6: Direct Evidence for Modulation of Single Unit Activity by tDCS in the Intact Somatosensory Cortex of Rats

Abstract #6: Direct Evidence for Modulation of Single Unit Activity by tDCS in the Intact Somatosensory Cortex of Rats

Functional Reorganization in the Primary Somatosensory Cortex of Rat Following Hind-Paw Amputation: A Study of Functional Imaging with 1.5 Tesla MRI.

To learn how rat primary somatosensory cortex (pSSC) responses to the loss of inputs from hind-paw, using fMRI of an inferior magnetic power (1.5 Tesla) with special designed high-powered rat coil. Ten adult male Sprague-Dawley rats were enrolled in this study. The rats were anesthetized with ketamine injection. Xylazine was intraperitoneally injected for analgesia and muscle relaxation with careful maintenance of spontaneous respiration. Either right or left hind-paws were amputated under aseptic conditions according to predefined random allocation of the rats. A 12-channel rat surface coil developed for proper image resolution in 1.5 Tesla MR was used. Functional magnetic resonance imaging was obtained before hind-paw amputation; 2, 15 and 30 days after the amputation. Activation signals were detected in 5 rats' contralateral pSSC before the hind-paw amputation with regression and cessation of the signal after the amputation. Signal re-appeared in the contralateral pSSC of only one rat (rat 9) 30 days after the amputation. This study showed that functional plasticity might occur in the pSSC following hind-paw amputation of rats. Further studies are necessary to understand the true nature of the plasticity observed in pSSC, with new and novel measurement techniques on cellular basis rather than gross anatomical one.

Chronic neural recording with probes of subcellular cross-section using 0.06 mm² dissolving microneedles as insertion device

Ultra-flexible electrode arrays with a cross-sectional area of only a few μm² show great promise for long-term, high resolution neural interfacing without detrimental scar tissue formation. However, due to their low stiffness, insertion is a challenge. In this work, we investigate microneedles consisting of quickly biodegradable, short-chained, acid terminated PLGA (50:50 lactide:glycolide ratio) as insertion device for a polyimide-based neural electrode array of 1 μm thickness. An upscalable, wafer-level fabrication process is presented. Both separate PLGA microneedles as well as complete, assembled neural probes were tested in vivo for up to 4 months. The arrays allowed to record spontaneous spike activity and evoked local field potentials in the somatosensory cortex of rats on all measured timepoints. Very limited lesion formation, measuring about 20% of the cross sectional area of the original microneedle, was observed. Neurons can be seen to infiltrate the area originally taken up by the dissolving PLGA microneedle. The results indicate that the presented electrode arrays and insertion method are well suitable for application in long-term, high resolution neural recording.

Biomimetic extracellular matrix coatings improve the chronic biocompatibility of microfabricated subdural microelectrode arrays.

Intracranial electrodes are a vital component of implantable neurodevices, both for acute diagnostics and chronic treatment with open and closed-loop neuromodulation. Their performance is hampered by acute implantation trauma and chronic inflammation in response to implanted materials and mechanical mismatch between stiff synthetic electrodes and pulsating, natural soft host neural tissue. Flexible electronics based on thin polymer films patterned with microscale conductive features can help alleviate the mechanically induced trauma; however, this strategy alone does not mitigate inflammation at the device-tissue interface. In this study, we propose a biomimetic approach that integrates microscale extracellular matrix (ECM) coatings on microfabricated flexible subdural microelectrodes. Taking advantage of a high-throughput process employing micro-transfer molding and excimer laser micromachining, we fabricate multi-channel subdural microelectrodes primarily composed of ECM protein material and demonstrate that the electrochemical and mechanical properties match those of standard, uncoated controls. In vivo ECoG recordings in rodent brain confirm that the ECM microelectrode coatings and the protein interface do not alter signal fidelity. Astrogliotic, foreign body reaction to ECM coated devices is reduced, compared to uncoated controls, at 7 and 30 days, after subdural implantation in rat somatosensory cortex. We propose microfabricated, flexible, biomimetic electrodes as a new strategy to reduce inflammation at the device-tissue interface and improve the long-term stability of implantable subdural electrodes.

Structural Properties of Synaptic Transmission and Temporal Dynamics at Excitatory Layer 5B Synapses in the Adult Rat Somatosensory Cortex.

Cortical computations rely on functionally diverse and highly dynamic synapses. How their structural composition affects synaptic transmission and plasticity and whether they support functional diversity remains rather unclear. Here, synaptic boutons on layer 5B (L5B) pyramidal neurons in the adult rat barrel cortex were investigated. Simultaneous patch-clamp recordings from synaptically connected L5B pyramidal neurons revealed great heterogeneity in amplitudes, coefficients of variation (CVs), and failures (F%) of EPSPs. Quantal analysis indicated multivesicular release as a likely source of this variability. Trains of EPSPs decayed with fast and slow time constants, presumably representing release from small readily releasable (RRP; 5.40 ± 1.24 synaptic vesicles) and large recycling (RP; 74 ± 21 synaptic vesicles) pools that were independent and highly variable at individual synaptic contacts (RRP range 1.2–12.8 synaptic vesicles; RP range 3.4–204 synaptic vesicles). Most presynaptic boutons (~85%) had a single, often perforated active zone (AZ) with a ~2 to 5-fold larger pre- (0.29 ± 0.19 μm2) and postsynaptic density (0.31 ± 0.21 μm2) when compared with even larger CNS synaptic boutons. They contained 200–3400 vesicles (mean ~800). At the AZ, ~4 and ~12 vesicles were located within a perimeter of 10 and 20 nm, reflecting docked and readily releasable vesicles of a putative RRP. Vesicles (~160) at 60–200 nm constituting the structural estimate of the presumed RP were ~2-fold larger than our functional estimate of the RP although both with a high variability. The remaining constituted a presumed large resting pool. Multivariate analysis revealed two clusters of L5B synaptic boutons distinguished by the size of their resting pool. Our functional and ultrastructural analyses closely link stationary properties, temporal dynamics and endurance of synaptic transmission to vesicular content and distribution within the presynaptic boutons suggesting that functional diversity of L5B synapses is enhanced by their structural heterogeneity.

A Quantitative Study on the Distribution of Mitochondria in the Neuropil of the Juvenile Rat Somatosensory Cortex.

Mitochondria play a key role in energy production and calcium buffering, among many other functions. They provide most of the energy required by neurons, and they are transported along axons and dendrites to the regions of higher energy demands. We have used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the somatosensory cortex of the juvenile rat. We have estimated the volume fraction occupied by mitochondria and their distribution between dendritic, axonal, and nonsynaptic processes. The volume fraction of mitochondria increased from layer I (4.59%) to reach its maximum in layer IV (7.74%) and decreased to its minimum in layer VI (4.03%). On average, 44% of mitochondrial volume was located in dendrites, 15% in axons and 41% in nonsynaptic elements. Given that dendrites, axons, and nonsynaptic elements occupied 38%, 23%, and 39% of the neuropil, respectively, it can be concluded that dendrites are proportionally richer in mitochondria with respect to axons, supporting the notion that most energy consumption takes place at the postsynaptic side. We also found a positive correlation between the volume fraction of mitochondria located in neuronal processes and the density of synapses.