John A. Wolf

Accumulation of Amyloid β and Tau and the Formation of Neurofilament Inclusions Following Diffuse Brain Injury in the Pig

Brain trauma in humans increases the risk for developing Alzheimer disease (AD) and may induce the acute formation of AD-like plaques containing amyloid beta (A beta). To further explore the potential link between brain trauma and neurodegeneration, we conducted neuropathological studies using a pig model of diffuse brain injury. Brain injury was induced in anesthetized animals via nonimpact head rotational acceleration of 110 degrees over 20 ms in the coronal plane (n = 15 injured, n = 3 noninjured). At 1, 3, 7, and 10 days post-trauma, control and injured animals were euthanized and immunohistochemical analysis was performed on brain sections using antibodies specific for A beta, beta-amyloid precursor protein (betaPP), tau, and neurofilament (NF) proteins. In addition to diffuse axonal pathology, we detected accumulation of A beta and tau that colocalized with immunoreactive betaPP and NF in damaged axons throughout the white matter in all injured animals at 3-10 days post-trauma. In a subset of brain injured animals, diffuse A beta-containing plaque-like profiles were found in both the gray and white matter, and accumulations of tau and NF rich inclusions were observed in neuronal perikarya. These results show that this pig model of diffuse brain injury is characterized by accumulations of proteins that also form pathological aggregates in AD and related neurodegenerative diseases.

Traumatic Axonal Injury Induces Proteolytic Cleavage of the Voltage-Gated Sodium Channels Modulated by Tetrodotoxin and Protease Inhibitors

We demonstrated previously that dynamic stretch injury of cultured axons induces structural changes and Ca2+ influx modulated by tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (NaChs). In the present study, we evaluated potential damage to the NaCh α-subunit, which can cause noninactivation of NaChs. In addition, we explored the effects of pre-injury and post-injury treatment with TTX and protease inhibition on proteolysis of the NaCh α-subunit and intra-axonal calcium levels ([Ca2+]i) over 60 min after trauma. After stretch injury, we found that [Ca2+]i continued to increase in untreated axons for at least 60 min. We also observed that the III-IV intra-axonal loop of the NaCh α-subunit was proteolyzed between 5 and 20 min after trauma. Pre-injury treatment of the axons with TTX completely abolished the posttraumatic increase in [Ca2+]i and proteolysis of the NaCh α-subunit. In addition, both pre-injury and post-injury inhibition of protease activity attenuated long-term increases in [Ca2+]i as well as mitigating degradation of the NaCh α-subunit. These results suggest a unique “feed-forward” deleterious process initiated by mechanical trauma of axons. Na+ influx through NaChs resulting from axonal deformation triggers initial increases in [Ca2+]i and subsequent proteolysis of the NaChα-subunit. In turn, degradation of the α-subunit promotes persistent elevations in [Ca2+]i, fueling additional pathologic changes. These observations may have important implications for developing therapeutic strategies for axonal trauma.

NMDA/AMPA Ratio Impacts State Transitions and Entrainment to Oscillations in a Computational Model of the Nucleus Accumbens Medium Spiny Projection Neuron

We describe a computational model of the principal cell in the nucleus accumbens (NAcb), the medium spiny projection (MSP) neuron. The model neuron, constructed in NEURON, includes all of the known ionic currents in these cells and receives synaptic input from simulated spike trains via NMDA, AMPA, and GABAA receptors. After tuning the model by adjusting maximal current conductances in each compartment, the model cell closely matched whole-cell recordings from an adult rat NAcb slice preparation. Synaptic inputs in the range of 1000-1300 Hz are required to maintain an “up” state in the model. Cell firing in the model required concurrent depolarization of several dendritic branches, which responded independently to afferent input. Depolarization from action potentials traveled to the tips of the dendritic branches and increased Ca2+ influx through voltage-gated Ca2+ channels. As NMDA/AMPA current ratios were increased, the membrane showed an increase in hysteresis of “up” and “down” state dwell times, but intrinsic bistability was not observed. The number of oscillatory inputs required to entrain the model cell was determined to be ∼20% of the “up” state inputs. Altering the NMDA/AMPA ratio had a profound effect on processing of afferent input, including the ability to entrain to oscillations in afferent input in the theta range (4-12 Hz). These results suggest that afferent information integration by the NAcb MSP cell may be compromised by pathology in which the NMDA current is altered or modulated, as has been proposed in both schizophrenia and addiction.

Deep brain stimulation in the treatment of obesity

Obesity is a growing global health problem frequently intractable to current treatment options. Recent evidence suggests that deep brain stimulation (DBS) may be effective and safe in the management of various, refractory neuropsychiatric disorders, including obesity. The authors review the literature implicating various neural regions in the pathophysiology of obesity, as well as the evidence supporting these regions as targets for DBS, in order to explore the therapeutic promise of DBS in obesity. The lateral hypothalamus and ventromedial hypothalamus are the appetite and satiety centers in the brain, respectively. Substantial data support targeting these regions with DBS for the purpose of appetite suppression and weight loss. However, reward sensation associated with highly caloric food has been implicated in overconsumption as well as obesity, and may in part explain the failure rates of conservative management and bariatric surgery. Thus, regions of the brains reward circuitry, such as the nucleus accumbens, are promising alternatives for DBS in obesity control. The authors conclude that deep brain stimulation should be strongly considered as a promising therapeutic option for patients suffering from refractory obesity.

Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue

Transcriptome profiling of single cells resident in their natural microenvironment depends upon RNA capture methods that are both noninvasive and spatially precise. We engineered a transcriptome in vivo analysis (TIVA) tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA tag in combination with RNA sequencing (RNA-seq), we analyzed transcriptome variance among single neurons in culture and in mouse and human tissue in vivo. Our data showed that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology is, to our knowledge, the first noninvasive approach for capturing mRNA from live single cells in their natural microenvironment.

Evolution of Neurofilament Subtype Accumulation in Axons Following Diffuse Brain Injury in the Pig

Although accumulation of neurofilament (NF) proteins in axons has been recognized as a prominent feature of brain trauma, the temporal course of the accumulation of specific NF subtypes has not been well established. In the present study, 17 miniature swine were subjected to nonimpact inertial brain injury. At 3 hours (h), 6 h, 24 h, 3 days, 7 days, and 10 days post-trauma, immunohistochemical analysis was performed to determine axonal accumulation of NF-light (NF-L), the rod and sidearm domains and sidearm phosphorylation states of NF-medium (NF-M), and heavy (NF-H). We found that NF-L accumulation was easily identified in damaged axons by 6 h post-trauma, but NF-M and H accumulation was not clearly visualized until 3 days following injury. In addition, the axonal accumulation of NF-M and H appeared to be primarily comprised of the sidearm domains. While the accumulating NF was found to be predominantly dephosphorylated, we also detected accumulation of phosphorylated NF. Finally, we found that developing axonal pathology may proceed either towards axotomy with discrete terminal bulb formation or towards the development of varicose swellings encompassing long portions of axons. These findings suggest that there is a differential temporal course in NF subtype disassembly, dephosphorylation, and accumulation in axons following initial brain trauma and that these processes occur in morphologically distinct phenotypes of maturing axonal pathology.

Effect of acute calcium influx after mechanical stretch injury in vitro on the viability of hippocampal neurons

We use a new in vitro model to examine the effect of mechanical deformation on neurons. We examined acute changes in cytosolic calcium concentrations ([Ca(2+)](i)) caused by a rapid stretch of cultured hippocampal neurons, using mechanical loading conditions that mimic brain deformations during trauma. We found that stretch-injury of neurons induces a strain-dependent increase in [Ca(2+)](i). Remarkably, the extent of this calcium response exceeded the levels initiated by chemical toxicity with NMDA (100 microM) or glutamate (5 mM) exposure. Propidium iodide labeling at 24 h following stretch showed neuronal death occurred only at the most severe level of mechanical injury. Although NMDA-induced toxicity could be inhibited in calcium free media or by treatment with MK-801, stretch-induced neuronal death was not similarly reduced with either treatment. Unexpectedly, reduction of the acute stretch-induced calcium transient with calcium-free media or MK-801 resulted in an increase in neuronal death at lower stretch levels. These data suggest that mechanical stretch can initiate calcium influx in hippocampal neurons, but substantially modulating the early calcium flux from the extracellular space or through the NMDA channel does not provide an effective means for improving neuronal survival.

Homeostatic Synapse-Driven Membrane Plasticity in Nucleus Accumbens Neurons

Stable brain function relies on homeostatic maintenance of the functional output of individual neurons. In general, neurons function by converting synaptic input to output as action potential firing. To determine homeostatic mechanisms that balance this input–output/synapse–membrane interaction, we focused on nucleus accumbens (NAc) neurons and demonstrated a novel form of synapse-to-membrane homeostatic regulation, homeostatic synapse-driven membrane plasticity (hSMP). Through hSMP, NAc neurons adjusted their membrane excitability to functionally compensate for basal shifts in excitatory synaptic input. Furthermore, hSMP was triggered by synaptic NMDA receptors (NMDARs) and expressed by the modification of SK-type Ca2+-activated potassium channels. Moreover, hSMP in NAc neurons was abolished in rats during a short- (2 d) or long- (21 d) term withdrawal from repeated intraperitoneal injections of cocaine (15 mg/kg/d, 5 d). These results suggest that hSMP is a novel form of synapse-to-membrane homeostatic plasticity and dysregulation of hSMP may contribute to cocaine-induced cellular alterations in the NAc.

Amelioration of binge eating by nucleus accumbens shell deep brain stimulation in mice involves D2 receptor modulation.

Hedonic overconsumption contributing to obesity involves altered activation within the mesolimbic dopamine system. Dysregulation of dopamine signaling in the nucleus accumbens shell (NAS) has been implicated in reward-seeking behaviors, such as binge eating, which contributes to treatment resistance in obesity (Wise, 2012). Direct modulation of the NAS with deep brain stimulation (DBS), a surgical procedure currently under investigation in humans for the treatment of major depression, obsessive–compulsive disorder, and addiction, may also be effective in ameliorating binge eating. Therefore, we examined the ability of DBS of the NAS to block this behavior in mice. c-Fos immunoreactivity was assessed as a marker of DBS-mediated neuronal activation. NAS DBS was found to reduce binge eating and increased c-Fos levels in this region. DBS of the dorsal striatum had no influence on this behavior, demonstrating anatomical specificity for this effect. The dopamine D2 receptor antagonist, raclopride, attenuated the action of DBS, whereas the D1 receptor antagonist, SCH-23390, was ineffective, suggesting that dopamine signaling involving D2 receptors underlies the effect of NAS DBS. To determine the potential translational relevance to the obese state, chronic NAS DBS was also examined in diet-induced obese mice and was found to acutely reduce caloric intake and induce weight loss. Together, these findings support the involvement of the mesolimbic dopamine pathways in the hedonic mechanisms contributing to obesity, and the efficacy of NAS DBS to modulate this system.

Development of transplantable nervous tissue constructs comprised of stretch-grown axons.

Pursuing a new approach to nervous system repair, fasciculated axon tracts grown in vitro were developed into nervous tissue constructs designed to span peripheral nerve or spinal cord lesions. We optimized the newfound process of extreme axon stretch growth to maximize the number and length of axon tracts, reach an unprecedented axon growth-rate of 1cm/day, and create 5cm long axon tracts in 8 days to serve as the core component of a living nervous tissue construct. Immunocytochemical analysis confirmed that elongating fibers were axons, and that all major cytoskeletal constituents were present across the stretch-growth regions. We formed a transplantable nervous tissue construct by encasing the elongated cells in an 80% collagen hydrogel, removing them from culture, and inserting them into a synthetic conduit. Alternatively, we induced axon stretch growth directly on a surgical membrane that could be removed from the elongation device, and formed into a cylindrical construct suitable for transplant. The ability to rapidly create living nervous tissue constructs that recapitulates the uniaxial orientations of the original nerve offers an unexplored and potentially complimentary direction in nerve repair. Ideally, bridging nerve damage with living axon tracts may serve to establish or promote new functional connections.