Limited Excessive Voluntary Alcohol Drinking Leads to Liver Dysfunction in Mice.

BACKGROUND: Liver damage is a serious and sometimes fatal consequence of long-term alcohol intake, which progresses from early-stage fatty liver (steatosis) to later-stage steatohepatitis with inflammation and fibrosis/necrosis. However, very little is known about earlier stages of liver disruption that may occur in problem drinkers, those who drink excessively but are not dependent on alcohol. METHODS: We examined how repeated binge-like alcohol drinking in C57BL/6 mice altered liver function, as compared with a single binge-intake session and with repeated moderate alcohol consumption. We measured a number of markers associated with early- and later-stage liver disruption, including liver steatosis, measures of liver cytochrome P4502E1 (CYP2E1) and alcohol dehydrogenase (ADH), alcohol metabolism, expression of cytokine mRNA, accumulation of 4-hydroxynonenal (4-HNE) as an indicator of oxidative stress, and alanine transaminase/aspartate transaminase as a measure of hepatocyte injury. RESULTS: Importantly, repeated binge-like alcohol drinking increased triglyceride levels in the liver and plasma, and increased lipid droplets in the liver, indicators of steatosis. In contrast, a single binge-intake session or repeated moderate alcohol consumption did not alter triglyceride levels. In addition, alcohol exposure can increase rates of alcohol metabolism through CYP2E1 and ADH, which can potentially increase oxidative stress and liver dysfunction. Intermittent, excessive alcohol intake increased liver CYP2E1 mRNA, protein, and activity, as well as ADH mRNA and activity. Furthermore, repeated, binge-like drinking, but not a single binge or moderate drinking, increased alcohol metabolism. Finally, repeated, excessive intake transiently elevated mRNA for the proinflammatory cytokine IL-1B and 4-HNE levels, but did not alter markers of later-stage liver hepatocyte injury. CONCLUSIONS: Together, we provide data suggesting that even relatively limited binge-like alcohol drinking can lead to disruptions in liver function, which might facilitate the transition to more severe forms of liver damage.

Protein tyrosine phosphatase α in the dorsomedial striatum promotes excessive ethanol-drinking behaviors.

We previously found that excessive ethanol drinking activates Fyn in the dorsomedial striatum (DMS) (Wang et al., 2010; Gibb et al., 2011). Ethanol-mediated Fyn activation in the DMS leads to the phosphorylation of the GluN2B subunit of the NMDA receptor, to the enhancement of the channel's activity, and to the development and/or maintenance of ethanol drinking behaviors (Wang et al., 2007, 2010). Protein tyrosine phosphatase α (PTPα) is essential for Fyn kinase activation (Bhandari et al., 1998), and we showed that ethanol-mediated Fyn activation is facilitated by the recruitment of PTPα to synaptic membranes, the compartment where Fyn resides (Gibb et al., 2011). Here we tested the hypothesis that PTPα in the DMS is part of the Fyn/GluN2B pathway and is thus a major contributor to the neuroadaptations underlying excessive ethanol intake behaviors. We found that RNA interference (RNAi)-mediated PTPα knockdown in the DMS reduces excessive ethanol intake and preference in rodents. Importantly, no alterations in water, saccharine/sucrose, or quinine intake were observed. Furthermore, downregulation of PTPα in the DMS of mice significantly reduces ethanol-mediated Fyn activation, GluN2B phosphorylation, and ethanol withdrawal-induced long-term facilitation of NMDAR activity without altering the intrinsic features of DMS neurons. Together, these results position PTPα upstream of Fyn within the DMS and demonstrate the important contribution of the phosphatase to the maladaptive synaptic changes that lead to excessive ethanol intake.

Inhibition of striatal-enriched tyrosine phosphatase 61 in the dorsomedial striatum is sufficient to increased ethanol consumption.

The STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61 ) inhibits the activity of the tyrosine kinase Fyn and dephosphorylates the GluN2B subunit of the NMDA receptor, whereas the protein kinase A phosphorylation of STEP61 inhibits the activity of the phosphatase (Pharmacol. Rev., 64, , p. 65). Previously, we found that ethanol activates Fyn in the dorsomedial striatum (DMS) leading to GluN2B phosphorylation, which, in turn, underlies the development of ethanol intake (J. Neurosci., 30, , p. 10187). Here, we tested the hypothesis that inhibition of STEP61 by ethanol is upstream of Fyn/GluN2B. We show that exposure of mice to ethanol increased STEP61 phosphorylation in the DMS, which was maintained after withdrawal and was not observed in other striatal regions. Specific knockdown of STEP61 in the DMS of mice enhanced ethanol-mediated Fyn activation and GluN2B phosphorylation, and increased ethanol intake without altering the level of water, saccharine, quinine consumption or spontaneous locomotor activity. Together, our data suggest that blockade of STEP61 activity in response to ethanol is sufficient for the activation of the Fyn/GluN2B pathway in the DMS. Being upstream of Fyn and GluN2B, inactive STEP61 in the DMS primes the induction of ethanol intake. We show that ethanol-mediated inhibition of STEP61 in the DMS leads to Fyn activation and GluN2B phosphorylation. (a) Under basal conditions, active STEP61 inhibits Fyn activity and dephosphorylates GluN2B. (b) Ethanol leads to the phosphorylation of STEP61 on a specific inhibitory site. The inhibition of STEP61 activity contributes to the activation of Fyn in response to ethanol, which, in turn, phosphorylates GluN2B. These molecular adaptations in the DMS promote ethanol drinking.

The small G protein H-Ras in the mesolimbic system is a molecular gateway to alcohol-seeking and excessive drinking behaviors.

Uncontrolled consumption of alcohol is a hallmark of alcohol abuse disorders; however, the central molecular mechanisms underlying excessive alcohol consumption are still unclear. Here, we report that the GTP binding protein, H-Ras in the nucleus accumbens (NAc) plays a key role in neuroadaptations that underlie excessive alcohol-drinking behaviors. Specifically, acute (15 min) systemic administration of alcohol (2.5 g/kg) leads to the activation of H-Ras in the NAc of mice, which is observed even 24 h later. Similarly, rat operant self-administration of alcohol (20%) also results in the activation of H-Ras in the NAc. Using the same procedures, we provide evidence suggesting that the exchange factor GRF1 is upstream of H-Ras activation by alcohol. Importantly, we show that infection of mice NAc with lentivirus expressing a short hairpin RNA that targets the H-Ras gene produces a significant reduction of voluntary consumption of 20% alcohol. In contrast, knockdown of H-Ras in the NAc of mice did not alter water, quinine, and saccharin intake. Furthermore, using two-bottle choice and operant self-administration procedures, we show that inhibiting H-Ras activity by intra-NAc infusion of the farnesyltransferase inhibitor, FTI-276, produced a robust decrease of rats' alcohol drinking; however, sucrose consumption was unaltered. Finally, intra-NAc infusion of FTI-276 also resulted in an attenuation of seeking for alcohol. Together, these results position H-Ras as a central molecular mediator of alcohol's actions within the mesolimbic system and put forward the potential value of the enzyme as a novel target to treat alcohol use disorders.

Alk is a transcriptional target of LMO4 and ERα that promotes cocaine sensitization and reward.

Previously, we showed that the mouse LIM-domain only 4 (Lmo4) gene, which encodes a protein containing two zinc-finger LIM domains that interact with various DNA-binding transcription factors, attenuates behavioral sensitivity to repeated cocaine administration. Here we show that transcription of anaplastic lymphoma kinase (Alk) is repressed by LMO4 in the striatum and that Alk promotes the development of cocaine sensitization and conditioned place preference, a measure of cocaine reward. Since LMO4 is known to interact with estrogen receptor α (ERα) at the promoters of target genes, we investigated whether Alk expression might be controlled by a similar mechanism. We found that LMO4 and ERα are associated with the Alk promoter by chromatin immunoprecipitation and that Alk is an estrogen-responsive gene in the striatum. Moreover, we show that ERα knock-out mice exhibit enhanced cocaine sensitization and conditioned place preference and an increase in Alk expression in the nucleus accumbens. These data define a novel regulatory network involved in behavioral responses to cocaine. Interestingly, sex differences in several behavioral responses to cocaine in humans and rodents have been described, and estrogen is thought to mediate some of these differences. Our data suggest that estrogen regulation of Alk may be one mechanism responsible for sexually dimorphic responses to cocaine.

A versatile toolbox for studying cortical physiology in primates.

Lesioning and neurophysiological studies have facilitated the elucidation of cortical functions and mechanisms of functional recovery following injury. Clinical translation of such studies is contingent on their employment in non-human primates (NHPs), yet tools for monitoring and modulating cortical physiology are incompatible with conventional lesioning techniques. To address these challenges, we developed a toolbox validated in seven macaques. We introduce the photothrombotic method for inducing focal cortical lesions, a quantitative model for designing experiment-specific lesion profiles and optical coherence tomography angiography (OCTA) for large-scale (~5 cm2) monitoring of vascular dynamics. We integrate these tools with our electrocorticographic array for large-scale monitoring of neural dynamics and testing stimulation-based interventions. Advantageously, this versatile toolbox can be incorporated into established chronic cranial windows. By combining optical and electrophysiological techniques in the NHP cortex, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, integrate physiological and behavioral findings, and develop neurorehabilitative treatments. MOTIVATION The primate neocortex encodes for complex functions and behaviors, the physiologies of which are yet to be fully understood. Such an understanding in both healthy and diseased states can be crucial for the development of effective neurorehabilitative strategies. However, there is a lack of a comprehensive and adaptable set of tools that enables the study of multiple physiological phenomena in healthy and injured brains. Therefore, we developed a toolbox with the capability to induce targeted cortical lesions, monitor dynamics of underlying cortical microvasculature, and record and stimulate neural activity. With this toolbox, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, test stimulation-based interventions, and integrate physiological and behavioral findings.

High-density single-unit human cortical recordings using the Neuropixels probe.

The action potential is a fundamental unit of neural computation. Even though significant advances have been made in recording large numbers of individual neurons in animal models, translation of these methodologies to humans has been limited because of clinical constraints and electrode reliability. Here, we present a reliable method for intraoperative recording of dozens of neurons in humans using the Neuropixels probe, yielding up to ∼100 simultaneously recorded single units. Most single units were active within 1 min of reaching target depth. The motion of the electrode array had a strong inverse correlation with yield, identifying a major challenge and opportunity to further increase the probe utility. Cell pairs active close in time were spatially closer in most recordings, demonstrating the power to resolve complex cortical dynamics. Altogether, this approach provides access to population single-unit activity across the depth of human neocortex at scales previously only accessible in animal models.

Protein Phosphatase 2a and glycogen synthase kinase 3 signaling modulate prepulse inhibition of the acoustic startle response by altering cortical M-Type potassium channel activity.

There is considerable interest in the regulation of sensorimotor gating, since deficits in this process could play a critical role in the symptoms of schizophrenia and other psychiatric disorders. Sensorimotor gating is often studied in humans and rodents using the prepulse inhibition of the acoustic startle response (PPI) model, in which an acoustic prepulse suppresses behavioral output to a startle-inducing stimulus. However, the molecular and neural mechanisms underlying PPI are poorly understood. Here, we show that a regulatory pathway involving protein phosphatase 2A (PP2A), glycogen synthase kinase 3 beta (GSK3beta), and their downstream target, the M-type potassium channel, regulates PPI. Mice (Mus musculus) carrying a hypomorphic allele of Ppp2r5delta, encoding a regulatory subunit of PP2A, show attenuated PPI. This PPP2R5delta reduction increases the phosphorylation of GSK3beta at serine 9, which inactivates GSK3beta, indicating that PPP2R5delta positively regulates GSK3beta activity in the brain. Consistently, genetic and pharmacological manipulations that reduce GSK3beta function attenuate PPI. The M-type potassium channel subunit, KCNQ2, is a putative GSK3beta substrate. Genetic reduction of Kcnq2 also reduces PPI, as does systemic inhibition of M-channels with linopirdine. Importantly, both the GSK3 inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)1H-pyrrole-2,5-dione (SB216763) and linopirdine reduce PPI when directly infused into the medial prefrontal cortex (mPFC). Whole-cell electrophysiological recordings of mPFC neurons show that SB216763 and linopirdine have similar effects on firing, and GSK3 inhibition occludes the effects of M-channel inhibition. These data support a previously uncharacterized mechanism by which PP2A/GSK3beta signaling regulates M-type potassium channel activity in the mPFC to modulate sensorimotor gating.

Nucleus accumbens-derived glial cell line-derived neurotrophic factor is a retrograde enhancer of dopaminergic tone in the mesocorticolimbic system.

Spontaneous firing of ventral tegmental area (VTA) dopamine (DA) neurons provides ambient levels of DA in target areas such as the nucleus accumbens (NAc) and the prefrontal cortex (PFC). Here we report that the glial cell line-derived neurotrophic factor (GDNF), produced in one target region, the NAc, is retrogradely transported by DA neurons to the VTA where the growth factor positively regulates the spontaneous firing activity of both NAc- and PFC-projecting DA neurons in a mechanism that requires the activation of the mitogen-activated protein kinase (MAPK) pathway. We also show that the consequence of GDNF-mediated activation of the MAPK signaling cascade in the VTA is an increase in DA overflow in the NAc. Together, these results demonstrate that NAc-produced GDNF serves as a retrograde enhancer that upregulates the activity of the mesocorticolimbic DA system.

Morphine-induced receptor endocytosis in a novel knockin mouse reduces tolerance and dependence.

Opioid drugs, such as morphine, are among the most effective analgesics available. However, their utility for the treatment of chronic pain is limited by side effects including tolerance and dependence. Morphine acts primarily through the mu-opioid receptor (MOP-R) , which is also a target of endogenous opioids. However, unlike endogenous ligands, morphine fails to promote substantial receptor endocytosis both in vitro, and in vivo. Receptor endocytosis serves at least two important functions in signal transduction. First, desensitization and endocytosis act as an "off" switch by uncoupling receptors from G protein. Second, endocytosis functions as an "on" switch, resensitizing receptors by recycling them to the plasma membrane. Thus, both the off and on function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether the low degree of endocytosis induced by morphine contributes to tolerance and dependence, we generated a knockin mouse that expresses a mutant MOP-R that undergoes morphine-induced endocytosis. Morphine remains an excellent antinociceptive agent in these mice. Importantly, these mice display substantially reduced antinociceptive tolerance and physical dependence. These data suggest that opioid drugs with a pharmacological profile similar to morphine but the ability to promote endocytosis could provide analgesia while having a reduced liability for promoting tolerance and dependence.

GDNF is a fast-acting potent inhibitor of alcohol consumption and relapse.

Previously, we demonstrated that the action of the natural alkaloid, ibogaine, to reduce alcohol (ethanol) consumption is mediated by the glial cell line-derived neurotrophic factor (GDNF) in the ventral tegmental area (VTA). Here we set out to test the actions of GDNF in the VTA on ethanol-drinking behaviors. We found that GDNF infusion very rapidly and dose-dependently reduced rat ethanol, but not sucrose, operant self-administration. A GDNF-mediated decrease in ethanol consumption was also observed in rats with a history of high voluntary ethanol intake. We found that the action of GDNF on ethanol consumption was specific to the VTA as infusion of the growth factor into the neighboring substantia nigra did not affect operant responses for ethanol. We further show that intra-VTA GDNF administration rapidly activated the MAPK signaling pathway in the VTA and that inhibition of the MAPK pathway in the VTA blocked the reduction of ethanol self-administration by GDNF. Importantly, we demonstrate that GDNF infused into the VTA alters rats' responses in a model of relapse. Specifically, GDNF application blocked reacquisition of ethanol self-administration after extinction. Together, these results suggest that GDNF, via activation of the MAPK pathway, is a fast-acting selective agent to reduce the motivation to consume and seek alcohol.

Endogenous BDNF in the dorsolateral striatum gates alcohol drinking.

We previously found that brain-derived neurotrophic factor (BDNF)-haplodeficient mice exhibit greater ethanol-induced place preference and psychomotor sensitization, and greater ethanol consumption after deprivation, than control mice. We further observed that, in mice, voluntary ethanol intake increases BDNF expression in the dorsal striatum (DS). Here, we determined whether BDNF within the DS regulates ethanol self-administration in Long-Evans rats trained to self-administer a 10% ethanol solution. We observed a greater increase in BDNF expression after ethanol self-administration in the dorsolateral striatum (DLS) than in the dorsomedial striatum (DMS). We further found that downregulation of endogenous BDNF using viral-mediated siRNA in the DLS, but not in the DMS, significantly increased ethanol self-administration. Infusion of exogenous BDNF (0.25 microg/microl/side into the DMS; 0.25 and 0.75 microg/microl/side into the DLS) attenuated responding for ethanol when infused 3 h before the beginning of the self-administration session. Although the decrease in ethanol intake was similar in the DLS and DMS, BDNF infused in the DLS, but not in the DMS, induced an early termination of the drinking episode. Furthermore, the action of BDNF in the DLS was specific for ethanol, as infusion of the neurotrophic factor in the DMS, but not DLS, resulted in a reduction of sucrose intake. Together, these findings demonstrate that the BDNF pathway within the DLS controls the level of ethanol self-administration. Importantly, our results suggest that an endogenous signaling pathway within the same brain region that mediates drug-taking behavior also plays a critical role in gating the level of ethanol intake.

Scalable method for micro-CT analysis enables large scale quantitative characterization of brain lesions and implants.

Anatomic evaluation is an important aspect of many studies in neuroscience; however, it often lacks information about the three-dimensional structure of the brain. Micro-CT imaging provides an excellent, nondestructive, method for the evaluation of brain structure, but current applications to neurophysiological or lesion studies require removal of the skull as well as hazardous chemicals, dehydration, or embedding, limiting their scalability and utility. Here we present a protocol using eosin in combination with bone decalcification to enhance contrast in the tissue and then employ monochromatic and propagation phase-contrast micro-CT imaging to enable the imaging of brain structure with the preservation of the surrounding skull. Instead of relying on descriptive, time-consuming, or subjective methods, we develop simple quantitative analyses to map the locations of recording electrodes and to characterize the presence and extent of hippocampal brain lesions.

Protein kinase Cdelta regulates ethanol intoxication and enhancement of GABA-stimulated tonic current.

Ethanol alters the distribution and abundance of PKCdelta in neural cell lines. Here we investigated whether PKCdelta also regulates behavioral responses to ethanol. PKCdelta(-/-) mice showed reduced intoxication when administered ethanol and reduced ataxia when administered the nonselective GABA(A) receptor agonists pentobarbital and pregnanolone. However, their response to flunitrazepam was not altered, suggesting that PKCdelta regulates benzodiazepine-insensitive GABA(A) receptors, most of which contain delta subunits and mediate tonic inhibitory currents in neurons. Indeed, the distribution of PKCdelta overlapped with GABA(A) delta subunits in thalamus and hippocampus, and ethanol failed to enhance tonic GABA currents in PKCdelta(-/-) thalamic and hippocampal neurons. Moreover, using an ATP analog-sensitive PKCdelta mutant in mouse L(tk(-)) fibroblasts that express alpha4beta3delta GABA(A) receptors, we found that ethanol enhancement of GABA currents was PKCdelta-dependent. Thus, PKCdelta enhances ethanol intoxication partly through regulation of GABA(A) receptors that contain delta subunits and mediate tonic inhibitory currents. These findings indicate that PKCdelta contributes to a high level of behavioral response to ethanol, which is negatively associated with risk of developing an alcohol use disorder in humans.

A Practical Method for Creating Targeted Focal Ischemic Stroke in the Cortex of Nonhuman Primates.

Ischemic stroke is a major cause of disability among adults worldwide. Despite its prevalence, few effective treatment options exist to alleviate sensory and motor dysfunctions that result from stroke. In the past, rodent models of stroke have been the primary experimental models used to develop stroke therapies. However, positive results in these studies have failed to replicate in human clinical trials, highlighting the importance of nonhuman primate (NHP) models as a preclinical step. Although there are a few NHP models of stroke, the extent of tissue damage is highly variable and dependent on surgical skill. In this study, we employed the photothrombotic stroke model in NHPs to generate controlled, reproducible ischemic lesions. Originally developed in rodents, the photothrombotic technique consists of intravenous injection of a photosensitive dye such as Rose Bengal followed by illumination of an area of interest to induce endothelial damage resulting in the formation of thrombi in the illuminated vasculature. We developed a quantitative model to predict the extent of tissue damage based on the light scattering profile of light in the cortex of NHPs. We then employed this technique in the sensorimotor cortex of two adult male Rhesus Macaques. In vivo optical coherence tomography imaging of the cortical microvasculature and subsequent histology confirmed the formation of focal cortical infarcts and demonstrated its reproducibility and ability to control the sizes and locations of light-induced ischemic lesions in the cortex of NHPs. This model has the potential to enhance our understanding of perilesional neural dynamics and can be used to develop reliable neurorehabilitative therapeutic strategies to treat stroke.

Ligand-induced down-regulation of the cannabinoid 1 receptor is mediated by the G-protein-coupled receptor-associated sorting protein GASP1.

The cannabinoid 1 receptor (CB1R) is one of the most abundant seven transmembrane (7TM) spanning/G-protein-coupled receptors in the central nervous system and plays an important role in pain transmission, feeding, and the rewarding effects of cannabis. Tolerance to cannabinoids has been widely observed after long-term use, with concomitant receptor desensitization and/or down-regulation depending on the brain region studied. Several CB1R agonists promote receptor internalization after activation, but the postendocytic sorting of the receptor has not been studied in detail. Utilizing human embryonic kidney (HEK293) cells stably expressing the CB1R and primary cultured neurons expressing endogenous CB1R, we show that treatment with cannabinoid agonists results in CB1R degradation after endocytosis and that the G-protein-coupled receptor-associated sorting protein GASP1 plays a major role in the postendocytic sorting process. Thus, these results may identify a molecular mechanism underlying tolerance and receptor down-regulation after long-term use of cannabinoids.

Targeted cortical reorganization using optogenetics in non-human primates.

Brain stimulation modulates the excitability of neural circuits and drives neuroplasticity. While the local effects of stimulation have been an active area of investigation, the effects on large-scale networks remain largely unexplored. We studied stimulation-induced changes in network dynamics in two macaques. A large-scale optogenetic interface enabled simultaneous stimulation of excitatory neurons and electrocorticographic recording across primary somatosensory (S1) and motor (M1) cortex (Yazdan-Shahmorad et al., 2016). We tracked two measures of network connectivity, the network response to focal stimulation and the baseline coherence between pairs of electrodes; these were strongly correlated before stimulation. Within minutes, stimulation in S1 or M1 significantly strengthened the gross functional connectivity between these areas. At a finer scale, stimulation led to heterogeneous connectivity changes across the network. These changes reflected the correlations introduced by stimulation-evoked activity, consistent with Hebbian plasticity models. This work extends Hebbian plasticity models to large-scale circuits, with significant implications for stimulation-based neurorehabilitation.

Widespread optogenetic expression in macaque cortex obtained with MR-guided, convection enhanced delivery (CED) of AAV vector to the thalamus.

BACKGROUND: In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Previous work has shown that parenchymal delivery of adeno-associated virus (AAV) in the thalamus by convection-enhanced delivery (CED) can lead to large-scale transduction via axonal transport in distal areas including cortex. We used this approach to obtain widespread cortical expression of light-sensitive ion channels. NEW METHOD: AAV vectors co-expressing channelrhodopsin-2 (ChR2) and yellow fluorescent protein (YFP) genes were infused into thalamus of three rhesus macaques under MR-guided CED. After six to twelve weeks recovery, in vivo optical stimulation and single cell recording in the cortex was carried out using an optrode in anesthetized animals. Post-mortem immunostaining against YFP was used to estimate the distribution and level of expression of ChR2 in thalamus and cortex. RESULTS: Histological analysis revealed high levels of transduction in cortical layers. The patterns of expression were consistent with known thalamo-cortico-thalamic circuits. Dense expression was seen in thalamocortiocal axonal fibers in layers III, IV and VI and in pyramidal neurons in layers V and VI, presumably corticothalamic neurons. In addition we obtained reliable in vivo light-evoked responses in cortical areas with high levels of expression. COMPARISON WITH EXISTING METHODS: Thalamic CED is very efficient in achieving large expressing areas in comparison to convectional techniques both in minimizing infusion time and in minimizing damage to the brain. CONCLUSION: MR-guided CED infusion into thalamus provides a simplified approach to transduce large cortical areas by thalamo-cortico-thalamic projections in primate brain.

Neutrophil protein kinase Cdelta as a mediator of stroke-reperfusion injury.

Thrombolysis is widely used to intervene in acute ischemic stroke, but reestablishment of circulation may paradoxically initiate a reperfusion injury. Here we describe studies with mice lacking protein kinase Cdelta (PKCdelta) showing that absence of this enzyme markedly reduces reperfusion injury following transient ischemia. This was associated with reduced infiltration of peripheral blood neutrophils into infarcted tissue and with impaired neutrophil adhesion, migration, respiratory burst, and degranulation in vitro. Total body irradiation followed by transplantation with bone marrow from PKCdelta-null mice donors reduced infarct size and improved neurological outcome in WT mice, whereas marrow transplantation from WT donors increased infarction and worsened neurological scores in PKCdelta-null mice. These results indicate an important role for neutrophil PKCdelta in reperfusion injury and strongly suggest that PKCdelta inhibitors could prove useful in the treatment of stroke.

A Large-Scale Interface for Optogenetic Stimulation and Recording in Nonhuman Primates.

While optogenetics offers great potential for linking brain function and behavior in nonhuman primates, taking full advantage of that potential will require stable access for optical stimulation and concurrent monitoring of neural activity. Here we present a practical, stable interface for stimulation and recording of large-scale cortical circuits. To obtain optogenetic expression across a broad region, here spanning primary somatosensory (S1) and motor (M1) cortices, we used convection-enhanced delivery of the viral vector, with online guidance from MRI. To record neural activity across this region, we used a custom micro-electrocorticographic (µECoG) array designed to minimally attenuate optical stimuli. Lastly, we demonstrated the use of this interface to measure spatiotemporal responses to optical stimulation across M1 and S1. This interface offers a powerful tool for studying circuit dynamics and connectivity across cortical areas, for long-term studies of neuromodulation and targeted cortical plasticity, and for linking these to behavior.