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1.  A Toolbox for Spatiotemporal Analysis of Voltage-Sensitive Dye Imaging Data in Brain Slices 
PLoS ONE  2014;9(9):e108686.
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.
PMCID: PMC4178182  PMID: 25259520
2.  Dietary Therapy Mitigates Persistent Wake Deficits Caused by Mild Traumatic Brain Injury 
Science translational medicine  2013;5(215):215ra173.
Sleep disorders are highly prevalent in patients with traumatic brain injury (TBI) and can significantly impair cognitive rehabilitation. No proven therapies exist to mitigate the neurocognitive consequences of TBI. We show that mild brain injury in mice causes a persistent inability to maintain wakefulness and decreases orexin neuron activation during wakefulness. We gave mice a dietary supplement of branched-chain amino acids (BCAAs), precursors for de novo glutamate synthesis in the brain. BCAA therapy reinstated activation of orexin neurons and improved wake deficits in mice with mild brain injury. Our data suggest that dietary BCAA intervention, acting in part through orexin, can ameliorate injury-induced sleep disturbances and may facilitate cognitive rehabilitation after brain injury.
PMCID: PMC3951738  PMID: 24337480
3.  Augmented Inhibition from Cannabinoid-Sensitive Interneurons Diminishes CA1 Output after Traumatic Brain Injury 
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.
PMCID: PMC4271495  PMID: 25565968
interneuron; traumatic brain injury; CA1; cholecystokinin; cannabinoid type 1 receptor; action potential; lateral fluid percussion; voltage-sensitive dye
4.  Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury 
Short Abstract
A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.
Long Abstract
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 impairments (1). Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction (2,3). In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation (4–6).
Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits.
The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.
PMCID: PMC3523419  PMID: 23183856
hippocampus; traumatic brain injury; electrophysiology; patch clamp; voltage sensitive dye; extracellular recording; high-performance liquid chromatography; gas chromatography-mass spectrometry
5.  GABA and Glutamate are not colocalized in mossy fiber terminals of developing rodent hippocampus 
Brain research  2012;1474:40-49.
It has been hypothesized that, in the developing rodent hippocampus, mossy fiber terminals release GABA together with glutamate. Here, we used transgenic glutamic acid decarboxylase-67 (GAD67)-GFP expressing mice and multi-label immunohistochemistry to address whether glutamatergic and GABAergic markers are colocalized. We demonstrate that in the dentate gyrus, interneurons positive for GABA/GAD are sparsely distributed along the edge of the hilus, in a different pattern than the densely packed granule cells. Co-staining for synaptophysin and vesicular glutamate transporter1 (VGLUT1) in postnatal day 14 brain sections from both mice and rats identified mossy fiber terminals as a group of large (2 – 5μm in diameter) VGLUT1-positive excitatory presynaptic terminals in the stratum lucidum of area CA3a/b. Furthermore, co-staining for synaptophysin and vesicular GABA transporter (VGAT) revealed a group of small-sized (~0.5μm in diameter) inhibitory presynaptic terminals in the same area where identified mossy fiber terminals were present. The two types of terminals appeared to be mutually exclusive, and showed no colocalization. Thus, our results do not support the hypothesis that GABA is released as a neurotransmitter from mossy fiber terminals during development.
PMCID: PMC3695404  PMID: 22842523
granule cell; vesicular glutamate transporter; vesicular GABA transporter; immunofluorescence; synaptic button
6.  RNAi targeting μ-calpain increases neuron survival and preserves hippocampal function after global brain ischemia 
Experimental neurology  2010;224(1):170-177.
The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following acute brain injury. However, the relative contribution of calpain isoforms has not been determined in in vivo models. Identification of the calpain isoform responsible for neuronal injury is particularly important given the differential role of calpain isoforms in normal physiology. This study evaluates the role of m-calpain and μ-calpain in an in vivo model of global brain ischemia. Adeno-associated viral vectors expressing short hairpin RNAs targeting the catalytic subunits of μ- or m-calpain were used to knockdown expression of the targeted isoforms in adult rat hippocampal CA1 pyramidal neurons. Knockdown of μ-calpain, but not m-calpain, prevented calpain activity 72 hours after 6-minute transient forebrain ischemia, increased long-term survival and protected hippocampal electrophysiological function. These findings represent the first in vivo evidence that reducing expression of an individual calpain isoform can decrease post-ischemic neuronal death and preserve hippocampal function.
PMCID: PMC2885584  PMID: 20298691
calpain; ischemia; hippocampus; RNA interference; adeno-associated virus
7.  Harvested human neurons engineered as live nervous tissue constructs: implications for transplantation 
Journal of neurosurgery  2008;108(2):343-347.
Although neuron transplantation to repair the nervous system has shown promise in animal models, there are few practical sources of viable neurons for clinical application and insufficient approaches to bridge extensive nerve damage in patients. Therefore, the authors sought a clinically relevant source of neurons that could be engineered into transplantable nervous tissue constructs. The authors chose to evaluate human dorsal root ganglion (DRG) neurons due to their robustness in culture.
Cervical DRGs were harvested from 16 live patients following elective ganglionectomies, and thoracic DRGs were harvested from 4 organ donor patients. Following harvest, the DRGs were digested in a dispase–collagenase treatment to dissociate neurons for culture. In addition, dissociated human DRG neurons were placed in a specially designed axon expansion chamber that induces continuous mechanical tension on axon fascicles spanning 2 populations of neurons originally plated ~ 100 μm apart.
The adult human DRG neurons, positively identified by neuronal markers, survived at least 3 months in culture while maintaining the ability to generate action potentials. Stretch-growth of axon fascicles in the expansion chamber occurred at the rate of 1 mm/day to a length of 1 cm, creating the first engineered living human nervous tissue constructs.
These data demonstrate the promise of adult human DRG neurons as an alternative transplant material due to their availability, viability, and capacity to be engineered. Also, these data show the feasibility of harvesting DRGs from living patients as a source of neurons for autologous transplant as well as from organ donors to serve as an allograft source of neurons.
PMCID: PMC3014262  PMID: 18240932
axon elongation; axon stretch growth; nervous tissue construct; peripheral nerve injury repair; spinal cord injury repair; tissue engineering
8.  Inflammation-induced Preterm Birth Alters Neuronal Morphology in the Mouse Fetal Brain 
Journal of neuroscience research  2010;88(9):1872-1881.
Adverse neurological outcome is a major cause of long-term morbidity in ex-preterm children. To investigate the effect of parturition and inflammation on the fetal brain, we utilized two in vivo mouse models of preterm birth. To mimic the most common human scenario of preterm birth, we used a mouse model of intrauterine inflammation by intrauterine infusion of lipopolysaccharide (LPS). To investigate the effect of parturition on the immature fetal brain, in the absence of inflammation, we used a non-infectious model of preterm birth by administering RU486. Pro-inflammatory cytokines (IL-10, IL-1β, IL-6 and TNF-α) in amniotic fluid and inflammatory biomarkers in maternal serum and amniotic fluid were compared between the two models using ELISA. Pro-inflammatory cytokine expression was evaluated in the whole fetal brains from the two models. Primary neuronal cultures from the fetal cortex were established from the different models and controls in order to compare the neuronal morphology. Only the intrauterine inflammation model resulted in an elevation of inflammatory biomarkers in the maternal serum and amniotic fluid. Exposure to inflammation-induced preterm birth, but not non-infectious preterm birth, also resulted in an increase in cytokine mRNA in whole fetal brain and in disrupted fetal neuronal morphology. In particular, Microtubule-associated protein 2 (MAP2) staining was decreased and the number of dendrites was reduced (P < 0.001, ANOVA between groups). These results suggest that inflammation-induced preterm birth and not the process of preterm birth may result in neuroinflammation and alter fetal neuronal morphology.
PMCID: PMC2895318  PMID: 20155801
mouse model of preterm birth; neuroinflammation; neuronal injury
9.  Role of GluR1 in Activity-Dependent Motor System Development 
The Journal of Neuroscience  2008;28(40):9953-9968.
Activity-dependent specification of neuronal architecture during early postnatal life is essential for refining the precision of communication between neurons. In the spinal cord under normal circumstances, the AMPA receptor subunit GluR1 is expressed at high levels by motor neurons and surrounding interneurons during this critical developmental period, although the role it plays in circuit formation and locomotor behavior is unknown. Here, we show that GluR1 promotes dendrite growth in a non-cell-autonomous manner in vitro and in vivo. The mal-development of motor neuron dendrites is associated with changes in the pattern of interneuronal connectivity within the segmental spinal cord and defects in strength and endurance. Transgenic expression of GluR1 in adult motor neurons leads to dendrite remodeling and supernormal locomotor function. GluR1 expression by neurons within the segmental spinal cord plays an essential role in formation of the neural network that underlies normal motor behavior.
PMCID: PMC3844744  PMID: 18829953
motor neurons; spinal cord; synaptic activity; motor behavior; glutamate receptors; network activity
10.  Dentate granule cell GABAA receptors in epileptic hippocampus: enhanced synaptic efficacy and altered pharmacology 
The European journal of neuroscience  2003;17(8):1607-1616.
The dentate gyrus (DG) normally functions as a filter, preventing propagation of synchronized activity into the seizure-prone hippocampus. This filter or ‘gatekeeper’ attribute of the DG is compromised in various pathological states, including temporal lobe epilepsy (TLE). This study examines the role that altered inhibition may play in the deterioration of this crucial DG function. Using the pilocarpine animal model of TLE, we demonstrate that inhibitory synaptic function is altered in principal cells of the DG. Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in dentate granule cells (DGCs) from epileptic animals were larger, more sensitive to blockade by zinc and less sensitive to augmentation by the benzodiazepine type site 1 modulator zolpidem. Furthermore, mIPSCs examined during a quiescent period following injury but preceding onset of epilepsy were significantly smaller than those present either in control or in TLE DGCs, and had already acquired sensitivity to blockade by zinc prior to the onset of spontaneous seizures. Rapid agonist application experiments demonstrated that prolonged (>35 ms) exposure to zinc is required to block GABAA receptors (GABAARs) in patches pulled from epileptic DGCs. Therefore, zinc must be tonically present to block DGC GABAARs and alter DG function. This would occur only during repetitive activation of mossy fibres. Thus, in the pilocarpine animal model of TLE, an early, de novo, expression of zinc-sensitive GABAARs is coupled with delayed, epilepsy-induced development of a zinc delivery system provided by aberrant sprouting of zinc-containing mossy fibre recurrent collaterals. The temporal and spatial juxtaposition of these pathophysiological alterations may compromise normal ‘gatekeeper’ function of the DG through dynamic zinc-induced failure of inhibition, predisposing the hippocampal circuit to generate seizures.
PMCID: PMC2587416  PMID: 12752378
dentate gyrus; electrophysiology; pilocarpine; zinc; zolpidem
11.  Neurotrophin-mediated neuroprotection of hippocampal neurons following traumatic brain injury is not associated with acute recovery of hippocampal function 
Neuroscience  2007;148(2):359-370.
Traumatic brain injury (TBI) causes selective hippocampal cell death which is believed to be associated with the cognitive impairment observed in both clinical and experimental settings. The endogenous neurotrophin NT-4/5, a TrkB ligand, has been shown to be neuroprotective for vulnerable CA3 pyramidal neurons after experimental brain injury. In this study, infusion of recombinant NT-4/5 increased survival of CA2/3 pyramidal neurons to 71% after lateral fluid percussion injury in rats, compared to 55% in vehicle-treated controls. The functional outcome of this NT-4/5-mediated neuroprotection was examined using three hippocampal-dependent behavioral tests. Injury-induced impairment was evident in all three tests, but interestingly, there was no treatment-related improvement in any of these measures. Similarly, injury-induced decreased excitability in the Schaffer collaterals was not affected by NT-4/5 treatment. We propose that a deeper understanding of the factors that link neuronal survival to recovery of function will be important for future studies of potentially therapeutic agents.
PMCID: PMC2579330  PMID: 17681695
Neurotrophins; Rats; Hippocampal neurons; Neuroprotection; Neuropharmacology; NT-4/5; behavior; cognition; brain injury; Neurotrophin
12.  A Neuronal Glutamate Transporter Contributes to Neurotransmitter GABA Synthesis and Epilepsy 
The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring–freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.
PMCID: PMC2483507  PMID: 12151515
EAAC1; transport; antisense; GABA; metabolism; epilepsy
13.  Brain injury impairs dentate gyrus inhibitory efficacy 
Neurobiology of disease  2006;25(1):163-169.
Every 23 seconds a person sustains a traumatic brain injury in the United States leaving many patients with substantial cognitive impairment and epilepsy. Injury-induced alterations in the hippocampus underpin many of these disturbances of neurological function. Abnormalities in the dentate gyrus are likely to play a major role in the observed pathophysiology because this subregion functions as a filter impeding excessive or aberrant activity from propagating further into the circuit and following experimental brain injury, the dentate gyrus becomes more excitable. Although alteration in excitation or inhibition could mediate this effect in the dentate gyrus, we show a key role played by an impairment of GABAAergic inhibition. The efficacy of GABAA-mediated inhibition depends on a low [Cl−]i that is maintained by neuronal K-Cl co-transporter 2 (KCC2). Using fluid percussion injury (FPI) in the mouse, we demonstrate significant reductions in KCC2 protein and mRNA expression in the dentate gyrus that causes a depolarizing shift in GABAA reversal potential, due to impaired chloride clearance, resulting in reduced inhibitory efficiency. This study elucidates a novel mechanism underlying diminished dentate gyrus inhibitory efficacy and provides an innovative target for the development of potential therapeutics to restore the severe pathological consequences of traumatic brain injury.
PMCID: PMC1713625  PMID: 17045484

Results 1-13 (13)