investigations-on-alterations-hippocampal-circuit-function-following

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

maintenance-of-a-lateral-fluid-percussion-injury-device

Maintenance of a Lateral Fluid Percussion Injury Device

Traumatic brain injury (TBI) accounts for roughly 2.5 million emergency room visits and hospitalizations annually and is a leading cause of death and ...

An active microvalve that uses a meltable piston in place of a conventional solid material to obstruct fluid flow in a microfluidic channel has been developed. This phase change valve is simple to operate and requires no additional fabrication steps. The valve is inherently latched, reusable, and leak-proof (to at least 250 psi) and can be electronically addressed using resistive heaters. The valve has been characterized for a range of operational parameters that will serve as a design guide. For the designs tested, piston displacements of 5 mm or more in 1 s have been achieved. Valves 1.4 mm in length in a 50 microm x 200 microm channel have been integrated on a biochemical reaction device, and successful DNA amplification using PCR has been achieved. The phase change valve can be easily implemented in an array format that can be used to realize complex microfluidic circuits.

Phase change microvalve for integrated devices.

Eine aktive Mikroventil, die einen schmelzbaren Kolben anstelle einer konventionellen Vollmaterial verwendet, um Fl&uuml;ssigkeit in einem Kanal mikrofluidische behindert wurde entwickelt. Diese Phase &Auml;nderung Ventil ist einfach zu bedienen und erfordert keine zus&auml;tzliche Fabrikation-Schritte. Das Ventil ist von Natur aus verriegelt, wiederverwendbare und auslaufsicheren (bis mindestens 250 Psi) und elektronisch mit resistive Heizungen adressiert werden. Das Ventil war f&uuml;r eine Reihe von operativen Parametern charakterisiert, die als Entwurf Leitfaden dienen wird. F&uuml;r die Entw&uuml;rfe getestet, Kolben Verschiebungen von 5 mm oder mehr in 1 s erreicht worden. Ventile 1,4 mm L&auml;nge in einem 50 Microm X 200 Microm Kanal wurden auf eine biochemische Reaktion-Ger&auml;t integriert und erfolgreich mit PCR DNA-Amplifikation erreicht wurde. Das Phase-&auml;ndern-Ventil kann leicht in einem Array-Format implementiert werden, die verwendet werden k&ouml;nnen, komplexe mikrofluidische Schaltungen zu realisieren.

Phase &Auml;nderung Mikroventil f&uuml;r integrierte Ger&auml;te.

Loss of Function-Mutationen in Kv7.2 oder Kv7.3 K(+) Kanal-Untereinheiten zugrunde liegt, die neonatale Epilepsie benigne famili&auml;re neonatale Kr&auml;mpfe (BFNC). Diese zwei Untereinheiten zu interagieren, um einen funktionalen K(+)-Kanal zu bilden, der den M-Strom (I(M)), eine Spannung-abh&auml;ngigen noninactivating K(+) aktuellen. zugrunde liegt In BFNC Anf&auml;lle beginnen kurz nach der Geburt und spontan in den ersten Lebensmonaten &uuml;berweisen. Die Natur dieses Fensters Schwachstelle ist unklar. Wir begegnen diesem Problem mit einem hippocampal Segment-Modell, um die Auswirkungen der Blockade mit I(M) oder Brustvergr&ouml;&szlig;erung Epileptiform T&auml;tigkeit zu studieren.

K + M-Strom regelt den &Uuml;bergang zu Sicherstellungen unreif und Erwachsenen Hippocampus.

Loss-of-function mutations in Kv7.2 or Kv7.3 K(+) channel subunits underlies the neonatal epilepsy benign familial neonatal convulsions (BFNC). These two subunits interact to form a functional K(+) channel that underlies the M-current (I(M)), a voltage-dependent noninactivating K(+) current. In BFNC, seizures begin shortly after birth, and spontaneously remit in the first few months of life. The nature of this window of vulnerability is unclear. We address this issue using a hippocampal slice model, to study the effects of I(M) blockade or augmentation on epileptiform activity.

K+ M-current regulates the transition to seizures in immature and adult hippocampus.

Most neurons possess a single, nonmotile cilium that projects out from the cell surface. These microtubule-based organelles are important in brain development and neurogenesis; however, their function in mature neurons is unknown. Cilia express a complement of proteins distinct from other neuronal compartments, one of which is the somatostatin receptor subtype SST(3). We show here that SST(3) is critical for object recognition memory in mice. sst3 knock-out mice are severely impaired in discriminating novel objects, whereas they retain normal memory for object location. Further, systemic injection of an SST(3) antagonist (ACQ090) disrupts recall of familiar objects in wild-type mice. To examine mechanisms of SST(3), we tested synaptic plasticity in CA1 hippocampus. Electrically evoked long-term potentiation (LTP) was normal in sst3 knock-out mice, while adenylyl cyclase/cAMP-mediated LTP was impaired. The SST(3) antagonist also disrupted cAMP-mediated LTP. Basal cAMP levels in hippocampal lysate were reduced in sst3 knock-out mice compared with wild-type mice, while the forskolin-induced increase in cAMP levels was normal. The SST(3) antagonist inhibited forskolin-stimulated cAMP increases, whereas the SST(3) agonist L-796,778 increased basal cAMP levels in hippocampal slices but not hippocampal lysate. Our results show that somatostatin signaling in neuronal cilia is critical for recognition memory and suggest that the cAMP pathway is a conserved signaling motif in cilia. Neuronal cilia therefore represent a novel nonsynaptic compartment crucial for signaling involved in a specific form of synaptic plasticity and in novelty detection.

Somatostatin signaling in neuronal cilia is critical for object recognition memory.

Die meisten Neuronen besitzen eine einzige, bildet Zilie, die heraus von der Zellenoberfl&auml;che projiziert. Diese Microtubule-basierte Organellen sind in der Entwicklung des Gehirns und Neurogenese wichtig; Ihre Funktion in Reife Neuronen ist jedoch unbekannt. Zilien schnelles Erg&auml;nzung Proteine unterscheiden andere neuronale F&auml;cher, von denen der Somatostatin-Rezeptor-Subtyp SST(3) ist. Wir zeigen hier, dass SST(3) f&uuml;r Objekt Anerkennung Speicher bei M&auml;usen kritisch ist. sst3 Knock-out-M&auml;use sind in unterscheidende neuartige Objekte, stark beeintr&auml;chtigt, w&auml;hrend normale Speicher f&uuml;r die Position eines Objektes zu behalten. Weitere, systemische Injektion von ein SST(3) Antagonist (ACQ090) st&ouml;rt R&uuml;ckruf von vertraute Gegenst&auml;nde in den Wildtyp M&auml;usen. Um Mechanismen der SST(3) zu untersuchen, testeten wir synaptische Plastizit&auml;t CA1 Hippocampus. Elektrisch evozierte langfristige Erm&ouml;glichung (LTP) war normal in sst3 Knock-out-M&auml;use, war w&auml;hrend der Fehlfunktion Guanylatcyclase/Lager-vermittelte LTP beeintr&auml;chtigt. Der SST(3)-Antagonist gest&ouml;rt auch cAMP-vermittelten LTP. CAMP Basalebenen in hippocampal lysate reduzierten sich in sst3 Knock-out-M&auml;use im Vergleich zu Wildtyp M&auml;usen, w&auml;hrend der Forskolin-induzierten Anstieg cAMP normal war. Der SST(3)-Antagonist gehemmt Forskolin stimuliert cAMP erh&ouml;ht, w&auml;hrend die SST(3)-Agonist L-796.778 cAMP Basalebenen in hippocampal Scheiben aber nicht hippocampal lysate gestiegen. Unsere Ergebnisse zeigen, dass Somatostatin signalisieren in neuronalen Zilien ist entscheidend f&uuml;r die Anerkennung Speicher und schlagen vor, da&szlig; die Lager Pathway ein konservierten Signalling Motiv in Zilien ist. Neuronale Zilien stellen somit ein neuartiges nonsynaptic Fach entscheidend f&uuml;r die Signalisierung einer spezifischen Form der synaptischen Plastizit&auml;t und der Neuheit Erkennung dar.

Somatostatin signalisieren in neuronalen Zilien ist entscheidend f&uuml;r die Anerkennung Speicher Objekts.

The recent shift among developers of microfluidic technologies toward modularized "plug and play" construction reflects the steadily increasing realization that, for many would-be users of microfluidic tools, traditional clean-room microfabrication is prohibitively complex and/or expensive. In this work, we present an advanced modular microfluidic construction scheme in which pre-fabricated microfluidic assembly blocks (MABs) can be quickly fashioned, without expertise or specialized facilities, into sophisticated microfluidic devices for a wide range of applications. Specifically, we describe three major advances to the MAB concept: (1) rapid production and extraction of MABs using flexible casting trays, (2) use of pre-coated substrates for simultaneous assembly and bonding, and (3) modification of block design to include automatic alignment and sealing structures. Finally, several exemplary applications of these MABs are demonstrated in chemical gradient synthesis, droplet generation, and total internal reflection fluorescence microscopy.

Flexible casting of modular self-aligning microfluidic assembly blocks.

Die j&uuml;ngste Schicht unter Entwicklern mikrofluidische Technologien modularisiert &quot;Plug And Play&quot; Bau spiegelt die st&auml;ndig zunehmende Erkenntnis, dass, f&uuml;r viele M&ouml;chtegern-Anwender mikrofluidische Tools, traditionellen Reinraum Microfabrication &uuml;berm&auml;&szlig;ig komplexe und/oder teuer ist. In dieser Arbeit pr&auml;sentieren wir eine erweiterte modulare mikrofluidische-Bau-Regelung in die vorgefertigten mikrofluidische Baugruppe blockiert (MABs) schnell gestaltet werden k&ouml;nnen, ohne Fachwissen oder spezialisierte Einrichtungen, in anspruchsvolle Microfluidic Vorrichtungen f&uuml;r eine Vielzahl von Anwendungen. Im einzelnen beschreiben wir drei gro&szlig;e Forderungen an das MAB-Konzept: (1) schnelle Produktion und Gewinnung von MABs mit flexiblen Gie&szlig;en Tabletts, (2) verwenden vorbeschichtete Substrate f&uuml;r gleichzeitige Montage und Verklebung und (3) &Auml;nderung von Block-Design f&uuml;r automatische Ausrichtung und Abdichtung Strukturen enthalten. Schlie&szlig;lich werden einige beispielhafte Anwendungen von diesen MABs in chemische gradient Synthese, Tr&ouml;pfchen Generierung und Totalreflexion Fluoreszenzmikroskopie veranschaulicht.

Flexible Gie&szlig;en von Modular Pendelkugellagern mikrofluidische Montage Bl&ouml;cke.

Voltage-sensitive dye imaging (VSDI) can simultaneously monitor the spatiotemporal electrical dynamics of thousands of neurons and is often used to identify functional differences in models of neurological disease. While the chief advantage of VSDI is the ability to record spatiotemporal activity, there are no tools available to visualize and statistically compare activity across the full spatiotemporal range of the VSDI dataset. Investigators commonly analyze only a subset of the data, and a majority of the dataset is routinely excluded from analysis. We have developed a software toolbox that simplifies visual inspection of VSDI data, and permits unaided statistical comparison across spatial and temporal dimensions. First, the three-dimensional VSDI dataset (x,y,time) is geometrically transformed into a two-dimensional spatiotemporal map of activity. Second, statistical comparison between groups is performed using a non-parametric permutation test. The result is a 2D map of all significant differences in both space and time. Here, we used the toolbox to identify functional differences in activity in VSDI data from acute hippocampal slices obtained from epileptic Arx conditional knock-out and control mice. Maps of spatiotemporal activity were produced and analyzed to identify differences in the activity evoked by stimulation of each of two axonal inputs to the hippocampus: the perforant pathway and the temporoammonic pathway. In mutant hippocampal slices, the toolbox identified a widespread decrease in spatiotemporal activity evoked by the temporoammonic pathway. No significant differences were observed in the activity evoked by the perforant pathway. The VSDI toolbox permitted us to visualize and statistically compare activity across the spatiotemporal scope of the VSDI dataset. Sampling error was minimized because the representation of the data is standardized by the toolbox. Statistical comparisons were conducted quickly, across the spatiotemporal scope of the data, without a priori knowledge of the character of the responses or the likely differences between them.

A toolbox for spatiotemporal analysis of voltage-sensitive dye imaging data in brain slices.

The neurological impairments associated with traumatic brain injury include learning and memory deficits and increased risk of seizures. The hippocampus is critically involved in both of these phenomena and highly susceptible to damage by traumatic brain injury. To examine network activity in the hippocampal CA1 region after lateral fluid percussion injury, we used a combination of voltage-sensitive dye, field potential, and patch clamp recording in mouse hippocampal brain slices. When the stratum radiatum (SR) was stimulated in slices from injured mice, we found decreased depolarization in SR and increased hyperpolarization in stratum oriens (SO), together with a decrease in the percentage of pyramidal neurons firing stimulus-evoked action potentials. Increased hyperpolarization in SO persisted when glutamatergic transmission was blocked. However, we found no changes in SO responses when the alveus was stimulated to directly activate SO. These results suggest that the increased SO hyperpolarization evoked by SR stimulation was mediated by interneurons that have cell bodies and/or axons in SR, and form synapses in stratum pyramidale and SO. A low concentration (100 nM) of the synthetic cannabinoid WIN55,212-2, restored CA1 output in slices from injured animals. These findings support the hypothesis that increased GABAergic signaling by cannabinoid-sensitive interneurons contributes to the reduced CA1 output following traumatic brain injury.

Augmented Inhibition from Cannabinoid-Sensitive Interneurons Diminishes CA1 Output after Traumatic Brain Injury.

Traumatic brain injury (TBI) results in long-lasting cognitive impairments for which there is currently no accepted treatment. A well-established mouse model of mild to moderate TBI, lateral fluid percussion injury (FPI), shows changes in network excitability in the hippocampus including a decrease in net synaptic efficacy in area CA1 and an increase in net synaptic efficacy in dentate gyrus. Previous studies identified a novel therapy consisting of branched chain amino acids (BCAAs), which restored normal mouse hippocampal responses and ameliorated cognitive impairment following FPI. However, the optimal BCAA dose and length of treatment needed to improve cognitive recovery is unknown. In the current study, mice underwent FPI then consumed 100 mM BCAA supplemented water ad libitum for 2, 3, 4, 5, and 10 days. BCAA therapy ameliorated cognitive impairment at 5 and 10 days duration. Neither BCAA supplementation at 50 mM nor BCAAs when dosed 5 days on then 5 days off was sufficient to ameliorate cognitive impairment. These results suggest that brain injury causes alterations in hippocampal function, which underlie and contribute to hippocampal cognitive impairment, which are reversible with at least 5 days of BCAA treatment, and that sustaining this effect is dependent on continuous treatment. Our findings have profound implications for the clinical investigation of TBI therapy.

Efficacy, dosage, and duration of action of branched chain amino Acid therapy for traumatic brain injury.

Hippocampal-dependent deficits in learning and memory formation are a prominent feature of traumatic brain injury (TBI); however, the role of the hippocampus in cognitive dysfunction after concussion (mild TBI) is unknown. We therefore investigated functional and structural changes in the swine hippocampus following TBI using a model of head rotational acceleration that closely replicates the biomechanics and neuropathology of closed-head TBI in humans. We examined neurophysiological changes using a novel ex vivo hippocampal slice paradigm with extracellular stimulation and recording in the dentate gyrus and CA1 occurring at 7 days following non-impact inertial TBI in swine. Hippocampal neurophysiology post-injury revealed reduced axonal function, synaptic dysfunction, and regional hyperexcitability at one week following even "mild" injury levels. Moreover, these neurophysiological changes occurred in the apparent absence of intra-hippocampal neuronal or axonal degeneration. Input-output curves demonstrated an elevated excitatory post-synaptic potential (EPSP) output for a given fiber volley input in injured versus sham animals, suggesting a form of homeostatic plasticity that manifested as a compensatory response to decreased axonal function in post-synaptic regions. These data indicate that closed-head rotational acceleration-induced TBI, the common cause of concussion in humans, may induce significant alterations in hippocampal circuitry function that have not resolved at 7 days post-injury. This circuitry dysfunction may underlie some of the post-concussion symptomatology associated with the hippocampus, such as post-traumatic amnesia and ongoing cognitive deficits.

Concussion Induces Hippocampal Circuitry Disruption in Swine.

The hippocampus plays a critical role in learning and memory and higher cognitive functions, and its dysfunction has been implicated in various neuropathological disorders. Electrophysiological recording undertaken in live brain slices is one of the most powerful tools for investigating hippocampal cellular and network activities. The plane for cutting the slices determines which afferent and/or efferent connections are best preserved, and there are three commonly used slices: hippocampal-entorhinal cortex (HEC), coronal and transverse. All three slices have been widely used for studying the major afferent hippocampal pathways including the perforant path (PP), the mossy fibers (MFs) and the Schaffer collaterals (SCs). Surprisingly, there has never been a systematic investigation of the anatomical and functional consequences of slicing at a particular angle. In the present study, we focused on how well fiber pathways are preserved from the entorhinal cortex (EC) to the hippocampus, and within the hippocampus, in slices generated by sectioning at different angles. The postmortem neural tract tracer 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) was used to label afferent fibers to hippocampal principal neurons in fixed slices or whole brains. Laser scanning confocal microscopy was adopted for imaging DiI-labeled axons and terminals. We demonstrated that PP fibers were well preserved in HEC slices, MFs in both HEC and transverse slices and SCs in all three types of slices. Correspondingly, field excitatory postsynaptic potentials (fEPSPs) could be consistently evoked in HEC slices when stimulating PP fibers and recorded in stratum lacunosum-moleculare (sl-m) of area CA1, and when stimulating the dentate granule cell layer (gcl) and recording in stratum lucidum (sl) of area CA3. The MF evoked fEPSPs could not be recorded in CA3 from coronal slices. In contrast to our DiI-tracing data demonstrating severely truncated PP fibers in coronal slices, fEPSPs could still be recorded in CA1 sl-m in this plane, suggesting that an additional afferent fiber pathway other than PP might be involved. The present study increases our understanding of which hippocampal pathways are best preserved in the three most common brain slice preparations, and will help investigators determine the appropriate slices to use for physiological studies depending on the subregion of interest.

A Comparison of Different Slicing Planes in Preservation of Major Hippocampal Pathway Fibers in the Mouse.

Immunostaining is a powerful technique and widely used to identify molecules in tissues and cells, although critical steps are necessary to block cross-reaction. Here we focused on an overlooked cross immunoreactivity issue where a secondary antibody (secondary) cross-reacts with a primary antibody (primary) from a different species. We first confirmed the previously reported cross-species binding of goat anti-mouse secondary to rat primary. This was accomplished by staining with a rat primary against glial fibrillary acidic protein (GFAP) and visualizing with goat (or donkey) anti-mouse secondary. We then further revealed the converse cross-species binding by staining with a mouse primary against neuronal nuclear protein (NeuN) and visualizing with anti-rat secondaries. We speculate that mouse and rat primaries share antigenicity, enabling either secondary to recognize either primary. To block this cross-species binding in double staining experiments, we compared three protocols using mouse anti-NeuN and rat anti-GFAP, two primaries whose antigens have non-overlapping distributions in brain tissues. Simultaneous staining resulted in cross-species astrocytic staining (anti-mouse secondary to rat anti-GFAP primary) but no cross-species neuronal staining (anti-rat secondary to mouse anti-NeuN primary). Cross-species astrocytic staining was missing after sequential same-species staining with mouse anti-NeuN primary, followed by rat anti-GFAP. However, cross-species astrocytic staining could not be diminished after sequential same-species staining with rat anti-GFAP primary, followed by mouse anti-NeuN. We thus hypothesize that a competition exists between anti-mouse and anti-rat secondaries in their binding to both primaries. Single staining for NeuN or GFAP visualized with dual secondaries at different dilution ratio supported this hypothesis.

ArticleTotal : 2	Year
Publication title Cited by 13	2012
Publication title Cited by 3	2023

Article	Year
Phase change microvalve for integrated devices. Analytical chemistry\| PubMed ID: 15228349	2004
K+ M-current regulates the transition to seizures in immature and adult hippocampus. Epilepsia\| PubMed ID: 17651418	2007
Somatostatin signaling in neuronal cilia is critical for object recognition memory. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 20335466	2010
Flexible casting of modular self-aligning microfluidic assembly blocks. Lab on a chip\| PubMed ID: 21412522	2011
A toolbox for spatiotemporal analysis of voltage-sensitive dye imaging data in brain slices. PloS one\| PubMed ID: 25259520	2014
Augmented Inhibition from Cannabinoid-Sensitive Interneurons Diminishes CA1 Output after Traumatic Brain Injury. Frontiers in cellular neuroscience\| PubMed ID: 25565968	2014
Efficacy, dosage, and duration of action of branched chain amino Acid therapy for traumatic brain injury. Frontiers in neurology\| PubMed ID: 25870584	2015
Concussion Induces Hippocampal Circuitry Disruption in Swine. Journal of neurotrauma\| PubMed ID: 28298170	2017
A Comparison of Different Slicing Planes in Preservation of Major Hippocampal Pathway Fibers in the Mouse. Frontiers in neuroanatomy\| PubMed ID: 29201002	2017
Blocking Cross-Species Secondary Binding When Performing Double Immunostaining With Mouse and Rat Primary Antibodies. Frontiers in neuroscience\| PubMed ID: 34113227	2021

Brian N. Johnson

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