The F2 progeny were scored to identify animals that lacked GFP ex

The F2 progeny were scored to identify animals that lacked GFP expression under normoxic conditions. To map n5500, the polymorphic Hawaiian CB4856 strain was crossed with n5500; nIs470 animals to obtain F2 progeny for SNP mapping ( Davis et al., 2005). To map the n5500 suppressor n5515 using genetic markers, n5500 II; nIs470 IV; n5515 males were crossed with n5500 II; nIs470 IV; dpy-6(e14) egl-15(n484) X hermaphrodites. Seven out of fifteen Compound Library ic50 Egl non-Dpy F2 progeny segregated GFP-negative n5500-suppressed animals. Refined mapping using SNP analysis further positioned n5515 between dpy-6 and egl-15 in an interval between the SNPs pk6127 and pk6138. To map the dominant suppressor

n5535, n5500; nIs470; n5535 hermaphrodites were crossed with n5500; nIs470 males, and GFP-positive F2 animals were isolated for SNP mapping. Locomotive responses to step changes of O2 were measured using a custom-built multiworm tracker and a gas-flow system controlled in real-time by MATLAB (see Figure S1). The gas flow consisted of pre-mixed 20%, 10%, selleck compound 5%, or 0% O2 balanced by N2. Well-fed young adult hermaphrodites (50–100 per assay) were transferred to a Petri plate freshly seeded with the bacterium OP50 and allowed to stabilize for 1 hr before the assay at 20°C. Worm-tracking

videos were analyzed later using MATLAB to calculate instantaneous locomotion speeds and other behavioral parameters. A hypoxia chamber (Coy Laboratory) that contained 0.5% O2 balanced by N2 was used for experiments involving hypoxia experience. After 24 hr of hypoxia exposure, animals

were allowed to recover in room air at 20°C for 2 hr preceding the acute behavioral assay. For experiments involving H2S exposure, 1 μl of 0.1M NaHS, an established H2S donor that releases 3-mercaptopyruvate sulfurtransferase H2S from solution, was dropped on the edge of agar-containing Petri plates and immediately sealed with tape to ensure airtight conditions. To obtain optimal effects, 24 hr duration of H2S exposure was used for behavioral experiments; 12 hr duration was used for GFP induction; and 1 hr duration was used for biochemical interaction experiments and quantitative real-time PCR. We thank Jo Anne Powell-Coffman, Yuichi Iino, Rene Garcia, Michael Hengartner, Mark Roth, Andy Fire, and Erik Jorgensen for reagents; the Caenorhabditis Genetics Center for strains; Na An, Rita Droste, and Tove Ljungars for technical assistance; Ales Hnizda, Jakub Krijt, and Milan Kodicek for help with characterization of purified CYSL-1; Viktor Kozich for helpful discussion; and Shunji Nakano, Takashi Hirose, Nick Paquin, Howard Chang, and Shuo Luo for comments. This work was supported by grant GM24663 to H.R.H from the NIH. R.V. was supported by grant No. 21709 from Grant Agency of Charles University, Prague, Czech Republic and by the Research Project of Charles University No. MSM0021620806. H.R.H.

, 2000) The human and iPSC data in the current study suggest tha

, 2000). The human and iPSC data in the current study suggest that ADARB2 could play a role in human C9ORF72 ALS by interacting with the C9ORF72 RNA foci. One hypothesis Protease Inhibitor Library is that the interaction of this RBP with the GGGGCCexp RNA might result in its loss of function. Additional studies to investigate the downstream effects of the loss of ADARB2 on biological processes such as RNA editing would be important to further characterize the roles of this protein in CNS cytotoxicity. While we have only thus far investigated

one RBP interactor identified in the present screen (Table S4), further studies are required to determine whether any of the other RBPs might interact with the GGGGCCexp RNA in vivo. Moreover, a GC-rich scrambled RNA sequence was used, which probably forms a G-quadruplex similar to the GGGGCC × 6.5 RNA (Fratta et al., 2012 and Reddy et al., 2013), thereby enriching for structure and sequence-specific protein interactors. However, the stringency of this analysis might have inadvertently excluded RBPs whose binding to the GGGGCC RNA was not apparent since it also showed an affinity to the GC rich scrambled

RNA structure. Finally, it is possible that multiple RBPs UMI-77 cost bind to the expansion, leading to either additive or synergistic effects, implicating various downstream pathways. RAN protein has been reported in C9ORF72 tissue and hypothesized to contribute to disease toxicity. Our studies document that C9ORF72 iPSC neurons have at least

one RAN protein and thus recapitulate the pathology seen in human C9ORF72 postmortem brain. However, despite the mitigation of excitotoxicity, large reductions in RNA foci and RBP aggregation and the normalization of various genes by the ASO therapy, RAN peptide can still be detected after ASO treatment. This would suggest that the detected accumulated cytoplasmic peptide was not a major contributor to neurotoxicity in our culture model. Although it is important to note that it is possible that our ASO treatment could rescue many the formation of newly synthesized RAN peptides and that longer ASO treatment would eventually show a reduction of RAN products. Furthermore, there could be other RAN peptides such as, e.g., antisense RAN peptides, contributing to the observed phenotypes, but that were not detected with the present antibodies, since our antibody preferentially detects the poly-(Gly-Pro) RAN product. While C9ORF72 protein exhibits structural similarity to the DENN protein family, the function of the C9ORF72 protein remains unclear (Levine et al., 2013). DENN proteins are GTP-GDP exchange factors (GEFs) for Rab GTPases, thus implicating the C9ORF72 protein in vesicular trafficking (Zhang et al., 2012). C9ORF72 RNA levels are reduced in patients that contain the expanded allele and a recent study has suggested that C9ORF72 haploinsufficiency causes neurodegeneration in a zebrafish model (Ciura et al.

The vaccine has been previously described [24] and was shown in p

The vaccine has been previously described [24] and was shown in pre-clinical studies to protect mice and ferrets from influenza infection and to induce both protective antibodies and, unlike conventional influenza vaccines, potent T-cell responses [25]. Importantly, this vaccine showed excellent cross-protection against heavily drifted strains in mice [24]. This is the first clinical trial with a VLP-based influenza HA vaccine that is produced entirely

in bacteria. Qbeta-VLPs Saracatinib chemical structure can be stockpiled and only the antigen needs to be produced and conjugated to the carrier. Hence, this vaccine could address the shortcomings of current approved vaccines, particularly in cases of an emerging pandemic. The clinical assessment of safety and immunogenicity of gH1-Qbeta is thus an important step toward a proof of concept and here we present its assessment in healthy adult volunteers

of Asian origin. The antigen sequence was derived from hemagglutinin of the influenza A virus strain A/California/07/2009 (H1N1), GenBank accession number: ACP41953.1 (amino acids 49-325) and C-terminally extended with a linker sequence (GGGCG) to a total of 281 amino acids. Purification and refolding of gH proteins has been described [24]. The cGMP manufacture of recombinant gH1 was performed in a 100 L fermenter at Biomeva GmbH (Germany) and was formulated to contain a final concentration of 10% glycerol at 1.9 mg/mL, stored at ≤−65 °C. The cGMP production of the recombinant Akt inhibitor VLP in E. coli RB791 was performed in an 800 L glycerol fed batch at Lonza AG (Switzerland) [26]. Purified Qbeta was stored at 3 mg/mL between −60 °C and −90 °C. To manufacture the drug substance gH1 was cross-linked

to Qbeta using succinimyl 6-[(maleimidopropionamido)-hexanoate] and formulated in PBS at a concentration of 1.9 mg/mL containing 0.01% Tween-20. Purity and integrity of the VLP were confirmed by SDS-PAGE and size-exclusion HPLC respectively, during for details see Supplemental Material and Methods. For clinical use gH1-Qbeta (batch 12036) was formulated in 20 mM sodium phosphate, 150 mM sodium chloride, 1.5% (v/v) glycerol, 0.01% (v/v) Tween-20 and water for injection (pH 7.2) and filled and finished by Symbiosis Pharmaceutical Services Ltd. (Scotland, UK). It was supplied in 2 mL single-use vials, filled with 350 μL at a concentration of 0.4 mg/mL (determined by protein content) and stored at ≤−65 °C. The purity and the integrity of the VLP were assessed by scanning densitometry after SDS-PAGE and SE-HPLC, respectively. The coupling density of gH1-Qbeta was determined by SDS-PAGE as 31% and endotoxin levels (according to Ph. Eur.2.9.19) were <0.6 EU/mg protein. Other components of the vaccine (adjuvant, diluent) were provided in the same 2 mL single use vials.

Only the CDK inhibitor, roscovitine, led to net dephosphorylation

Only the CDK inhibitor, roscovitine, led to net dephosphorylation of SAD-A as indicated by a shift toward the 76 kDa form (Figure 7A and data not shown). We also treated cultured DRG neurons with roscovitine and found a similar although less pronounced shift in SAD-A mobility (Figure 7A). These results suggest that selleck chemicals llc CDKs are physiological regulators of CTD phosphorylation. To test this idea directly, we coexpressed either wild-type or catalytically inactive CDK5 with the p35 coactivator and SAD-AWT or SAD-A18A. Expression of active but not inactive CDK5 caused SAD-AWT protein to migrate exclusively at 85 kDa, while migration of SAD-A18A

was only slightly affected (Figure 7B). Moreover, whereas expression of active CDK5 completely

eliminated ALT phosphorylation of SAD-AWT, ALT phosphorylation of SAD-A18A was largely resistant to CDK5-mediated inhibition (Figure 7B). The fact that the SAD-A18A mutant is not completely refractory to the inhibitory effects of CDK5 suggests that there may be other check details residues involved in mediating SAD-A inhibition. Thus, CDK5 can phosphorylate SAD-A in the CTD, preventing activating phosphorylation at the ALT. These results reveal a mechanism in which activation of SAD kinase by canonical activation loop phosphorylation is inhibited by phosphorylation of distal sites in the CTD. To ask which phosphorylation sites in the SAD-A CTDs are important for inhibition of SAD activation, we divided them into two groups and mutated each separately: the 13 sites in the PXX[S/T]P motifs N-terminal to the D box (aa 428–468, mutant called SAD-A13A) or the 5 sites (4 of which are [S/T]P) C-terminal to the D box (aa 490–513, Fossariinae mutant called SAD-A5A; Figure S6A), We expressed the mutants in 293T cells with CDK5 and examined the effects on SAD ALT phosphorylation. CDK5 activation suppressed SAD ALT phosphorylation of both the SAD-A13A and SAD-A5A mutants (Figure S6B). We conclude that no single phosphorylation event in the CTD regulates SAD activity, but rather phosphorylation of residues throughout

the SAD CTD is sufficient to block SAD ALT phosphorylation. What signaling pathway does NT-3 use to regulate phosphorylation of the SAD CTD? We analyzed SAD-A immunoprecipitates from untreated and NT-3 treated cells using the anti-p[S/T]P antibody. Consistent with results presented above (Figure 6C), SAD-A protein from untreated cells was strongly phosphorylated at [S/T]P sites, whereas SAD-A protein from NT-3 treated cells lacked [S/T]P phosphorylation (Figure 7C). Thus, NT-3/TrkC signaling induces net SAD-A CTD dephosphorylation. Inhibitors of MEK1/2 or PLCγ decreased NT-3 dependent SAD-A CTD dephosphorylation in TrkC+ HeLa cells (Figure 7D), indicating that both of these pathways are capable of regulating SAD-A CTD dephsophorylation in response to NT-3.

Competitive blockade of group I and group II mGluRs with the LY34

Competitive blockade of group I and group II mGluRs with the LY341495 (100 μM) (Kingston et al., 1998) also failed to prevent bicuculline-induced downregulation of surface GluA1 and GluA2/3 (Figures S1C and S1D). The failure of CP and MCPG or LY341495 to block scaling to bicuculline suggests that mGluR activation is not due to glutamate

released at synapses or glutamate that might accumulate in the medium. To further examine this issue, we added glutamate-pyruvate transaminase (GPT) to the medium during Ibrutinib chronic bicuculline treatment at a concentration reported to prevent local accumulation of glutamate at synapses (Matthews et al., 2000 and Pula et al., 2004). The Kd of GPT for glutamate is ∼8 μM and this is close to the measured level of glutamate in the medium of our

cultures (∼7 μM). GPT did not alter the effect of chronic bicuculline to downregulate surface GluA1 and GluA2/3 (Figures S1E and S1F). Effects of group I mGluR GDC-0941 purchase antagonists on bicuculline-evoked scaling were examined in parallel using electrophysiological recordings. Chronic treatment with Bay and MPEP produced an increase in the miniature excitatory postsynaptic current (mEPSC) amplitudes (control 20.9 ± 1.1 pA; n = 24 cells versus Bay/MPEP 26.7 ± 1.9 pA; n = 21 cells; #p < 0.05), and blocked the effect of bicuculline (bic 14.1 ± 0.2 pA; n = 28 cells versus bic/Bay/MPEP 25.7 ± 2.0 pA; n = 18 cells) (Figures 1E and 1F). By contrast, chronic CP and MCPG did not block the effect of bicuculline (amp: 13.5 ± 0.4 pA, ∗∗p < 0.01; frequency: 22.8 ± 2.6 Hz, n = 22 cells), and did not produce an increase in the basal mEPSC amplitude (21.8 ± 0.9 pA, n = 11 cells, Figures 1E and 1F) or frequency (22.0 ± 2.8 Hz, n = 11 cells). The cumulative histogram below of mEPSCs indicates that Bay 36-7620 and MPEP produced a multiplicative increase of amplitudes, suggesting the antagonists produce a scaling up of relative synaptic weights (Turrigiano and Nelson, 2000). Chronic Bay and MPEP also increased the frequency of mEPSCs (control 23.4 ± 2.6 Hz;

n = 24 cells versus Bay/MPEP 36.4 ± 4.4 Hz; n = 21 cells; #p < 0.05). This is consistent with either a presynaptic action of group I mGluRs or the possibility that Bay and MPEP convert silent synapses to active synapses. To assess if Homer1a can activate group I mGluRs in cortical neurons to downregulate surface AMPARs, we expressed Homer1a transgene by Sindbis virus infection for 24 hr, and assayed surface AMPARs by biotinylation and IHC. We compared effects of Homer1a with that of Arc (Shepherd et al., 2006). Neurons were treated with tetrodotoxin (TTX, 1 μM) to reduce native expression of Homer1a and Arc, and thereby isolate the action of the transgenes. Surface GluA1 and GluA2/3 were reduced in neurons that expressed Homer1a or Arc, compared to GFP (Figures 2A and 2B).

, 1997) This signaling follows presynaptic inhibition of GABA re

, 1997). This signaling follows presynaptic inhibition of GABA release and is dependent on G protein activation and PKC activity (Rodríguez-Moreno SB203580 clinical trial and Lerma, 1998 and Rodríguez-Moreno

et al., 2000). Later, this nonconventional mode of signaling was compellingly established in dorsal root ganglion neurons and was shown to be independent of ion flux (Rozas et al., 2003). Since then, an increasing number of metabotropic actions triggered by KARs have been described in many cell types and in different regions of the CNS, particularly in association with the presynaptic control of neurotransmitter release or the postsynaptic regulation of neuronal excitability (see Rodrigues and Lerma, 2012 for a recent review and Figure 2). However, key aspects of the molecular Dabrafenib mechanisms underlying this noncanonical signaling still remain unclear, including how KARs activate G proteins to trigger these effects and what determines the mode of action

of KARs (i.e., conventional ionotropic versus noncanonical metabotropic signaling). The evidence for a direct interaction between KARs and G proteins is limited. Prior to describing the metabotropic behavior of KARs, the Pertussis toxin (PTx)-sensitive binding of an agonist to goldfish-purified KARs was demonstrated biochemically, providing a link between KARs and PTx-sensitive proteins (Ziegra et al., 1992). While similar PTx-sensitive KAR agonist-binding was also observed in hippocampal membranes (Cunha et al., 1999), this kind of interaction does not seem to be that related to the functional signal transduced by KAR activation through G protein activity. It is expected that undergoing proteomic analysis of KAR subunits identify partners that could account for the coupling between an ion channel receptor and a G protein. Initially, STK38 it was unclear which subunits might engage this activity and, still, the search to identify the KAR subunit that mediates this noncanonical signaling is not free of controversy. In dorsal root ganglia (DRG) neurons

that exclusively express GluK1 and GluK5 subunits, noncanonical signaling was dependent on GluK1 rather than GluK5 (Rozas et al., 2003). Subsequent studies found that KAR-mediated modulation of IAHP, an action provoked by the noncanonical signaling of KARs (Melyan et al., 2002), was absent in GluK2- (Fisahn et al., 2005) or GluK5- (Ruiz et al., 2005) deficient mice. However, more recent studies reported that noncanonical signaling persisted in GluK5 and GluK4–GluK5 knockout (KO) animals (Fernandes et al., 2009). Indeed, expression of GluK1 in SHSY5 neuroblastoma cells was sufficient to reconstitute metabotropic activity of KARs, as evaluated by the G protein and PKC activation inducing internalization of KARs from the membrane (Rivera et al., 2007). Recent experiments confirmed the involvement of GluK1 in the metabotropic control of glutamate release (Segerstråle et al., 2010 and Salmen et al., 2012).

We thank Violana Nesterova for help with figure preparation Prot

We thank Violana Nesterova for help with figure preparation. Protein expression was done at the Caltech Protein Expression Center. This work was supported by an NIH PARP phosphorylation RO1 grant (NS28182) to K.Z. “
“The Down syndrome cell-adhesion molecule (Dscam) is important for the development of neural circuits in both invertebrates and vertebrates (Fuerst et al., 2008, 2009; Millard and Zipursky, 2008; Schmucker and Chen, 2009; Yamagata and Sanes, 2008). In Drosophila, Dscam undergoes extensive alternative splicing

to generate as many as 38,016 different isoforms ( Schmucker et al., 2000). This diversity is critical for neurite self-recognition ( Hattori et al., 2007, 2008; Zipursky and Sanes, 2010). For example, loss of Dscam function results in a dramatic increase in intraneuronal dendritic crossings in the dendritic arborization (da) neurons ( Hughes et al., 2007; Matthews et al., 2007; Soba et al., 2007) and a failure in sister branch segregation of the axons of mushroom body neurons ( Hattori et al., 2007; Wang et al., 2002). In addition to self-recognition, Drosophila selleck inhibitor Dscam regulates synaptic target selection and axon guidance in several types of neurons ( Chen

et al., 2006; Hummel et al., 2003; Millard et al., 2010; Wang et al., 2002; Zhu et al., 2006). For instance, in mechanosensory neurons of the adult fly, Dscam mutants exhibit profound loss of axon terminal branches as a result of defective branch extension and target selection ( Chen et al., 2006). L-NAME HCl Despite the absence of the remarkable molecular diversity seen in insects, vertebrate Dscam is also essential for neurite self-avoidance and synaptic target selection (Blank et al., 2011; Fuerst et al., 2008, 2009; Yamagata and Sanes, 2008), suggesting that the functions of Dscam in neuron morphogenesis and circuit assembly are evolutionarily conserved (Zipursky and Sanes, 2010). Little is known about how Dscam is regulated, but several observations suggest that its expression must

be tightly controlled. Dscam expression is dynamically regulated in developing brains (Maynard and Stein, 2012; Saito et al., 2000). In mouse, Dscam protein levels peak at postnatal days 7–10 in the cerebral cortex, coinciding with a period of extensive axonal branching (Larsen and Callaway, 2006), and decrease after postnatal day 10 (Maynard and Stein, 2012). Moreover, Dscam expression is elevated in several brain disorders, including Down syndrome (DS) (Saito et al., 2000), intractable epilepsy (Shen et al., 2011), and bipolar disorder (Amano et al., 2008). These findings suggest that appropriate regulation of Dscam expression may be important for development and that inappropriate or dysregulated Dscam expression may lead to developmental abnormalities and diseases.

We performed ex vivo whole-cell voltage and current-clamp recordi

We performed ex vivo whole-cell voltage and current-clamp recordings from VTA DA neurons while optically stimulating VTA GABA neurons. Optical stimulation of VTA GABA neurons led to detectable IPSCs in VTA DA neurons that were abolished by bath application of the GABAA receptor antagonist, gabazine (Figure 4A). To examine the effects of optical activation of VTA GABA neurons on the excitability and activity of VTA DA neurons, the membrane potentials of DA neurons were initially set to −60 mV in current clamp. We then applied SCH 900776 concentration 5 s current injection ramps through the patch pipette to evoke firing in the presence and absence of

5 s light pulses to activate VTA GABA neurons. Activation of VTA GABA neurons reduced excitability of VTA

DA neurons as indicated by a significant increase in the rheobase of the recorded neurons compared to recordings when no stimulation occurred (Figures 4B and 4C). In addition, VTA GABA stimulation reduced the activity of VTA DA neurons as indicated by an increase in the interspike interval and a reduction in total number of spikes evoked by the current injection (Figures 4B, 4D, and 4E). Furthermore, to determine whether activation of VTA GABA neurons could functionally suppress GW-572016 datasheet activity-dependent release of NAc DA in vivo, we performed fast-scan cyclic voltammetry experiments in anesthetized mice. Electrical activation of VTA DA neurons resulted in a stimulation frequency-dependent increase in detected NAc DA release, which was significantly attenuated by coincidental 5 s VTA GABA activation

that started 2.5 s before the electrical stimulation of the VTA (Figure 5). Taken together, these data demonstrate that activation of VTA GABA neurons reduces the excitability and evoked activity of neighboring VTA DA neurons in vitro and in vivo. The activity of VTA neurons and subsequent release of DA, glutamate (Stuber et al., 2010, Tecuapetla et al., 2010 and Yamaguchi et al., 2011), and GABA in forebrain targets, such as the NAc, are important processes that promote crucial aspects of motivated because behavior. Thus, the regulation of DA neuronal activity by both intrinsic and extrinsic mechanisms is required for optimal behavioral performance. Further, the mechanisms that regulate DA neuronal activity in adaptive contexts may underlie the maladaptive actions and responses seen in addiction and neuropsychiatric illnesses. In this study, we show that brief activation of VTA GABA neurons selectively disrupts reward consumption when these neurons are stimulated following reward delivery but not when they are activated during reward-predictive cue presentation.

In bats, as in rodents, grid cells colocalize with head direction

In bats, as in rodents, grid cells colocalize with head direction cells and border cells. More recently, grid cells

have been reported in monkeys, but here the hexagonal firing was determined by where the monkey fixated on a visual image (Killian et al., 2012). The dependence on view location in monkey grid cells is reminiscent of earlier work suggesting that in monkeys, hippocampal and parahippocampal cells fire when the animal looks at certain locations, independently 5-Fluoracil cost of where the animal is located (Rolls and O’Mara, 1995 and Rolls et al., 1997). Collectively, these findings suggest that in primate evolution, grid cells and place cells became responsive not only to changes in the speed and direction of locomotion, but also the velocity of the animal’s eye movements. Whether grid cells of monkeys are driven only by saccades or also by locomotion remains to be determined. The fact that grid cells have been reported in humans performing a virtual reality task (Jacobs et al., 2013) reinforces the view that, in primates, grid activity can be evoked by a spectrum of sensory inputs and that the grid network may be used for multiple purposes. Exploration of the variety of functions Compound Library supplier potentially served by grid cells in primates should certainly have priority. The mammalian space circuit is one of

the first nonsensory cognitive functions to be understood in mechanistic terms. With the presence of grid cells, and with the availability of new tools for selective activation and inactivation of circuit elements, it has become possible to study neural computation at the high end of the cortical hierarchy, far away from sensory inputs and motor outputs. A huge benefit of studying these cells is the close correspondence between the firing pattern and a property of the external world: the animal’s location in the environment. This correspondence provides researchers with easy experimental access to high-end neuronal most coding within the

circuits where the codes are generated. Understanding how space is created in this circuit may provide important clues about general principles for cortical computation that extend well beyond the domain of space, touching on the realms of thinking, planning, reflection, and imagination. We thank the European Research Council (“CIRCUIT” Advanced Investigator Grant, Grant Agreement 232608; “ENSEMBLE” Advanced Investigator Grant, Grant Agreement 268598), the Louis-Jeantet Prize for Medicine, the Kavli Foundation, and the Centre of Excellence scheme and the FRIPRO and NEVRONOR programs of the Research Council of Norway for support. “
“In recent years, there has been an explosion of interest in mapping the brain and its connections systematically across a range of spatial scales and in a number of species. This is embodied in the concept of a connectome as a “comprehensive” map of brain connectivity (Sporns et al., 2005).

All experiments were conducted in accordance with the National In

All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with approval of the National Institute on Drug Abuse animal care and use committee. Microinjection

needles (29G) were connected to a 2 μl Hamilton syringe and filled with purified, concentrated adeno-associated virus (∼1012 infectious units ml−1) encoding EYFP, ChR2-EYFP, or NpHR-EFYP under control of the αCaMKII promoter. Mice were anesthetized with 150 mg kg−1 ketamine and 50 mg kg−1 xylazine and placed in a stereotaxic frame. Microinjection needles were bilaterally placed into the vHipp, basolateral amygdala, prefrontal cortex, or NAc shell and 0.5 μl virus was injected over 5 min. The needles were left in place for an additional CT99021 in vitro 5 min to allow for diffusion of virus particles away from injection site. Mice used for in vivo optogenetic experiments had 200 μm core optical fibers, threaded through 1.25-mm-wide zirconia ferrules, implanted directly above the NAc shell (+1.4 AP, ±1.5 ML, −3.7 DV at an 11° angle). Optical fibers were secured in place Ulixertinib using skull screws and acrylic cement. Wounds of mice destined for confocal imaging or slice electrophysiology were sealed with cyanoacrylate tissue glue. Mice were anesthetized with Euthasol

6–12 weeks after surgery and perfused with ice-cold PBS followed by 4% paraformaldehyde. Brains were removed, postfixed overnight in 4% paraformaldehyde, and sectioned in 100 μm coronal slices on a VT-1200 vibratome (Leica). Sections were mounted using Mowiol with DAPI. Slides were scanned on a confocal microscope (Olympus) with a 10× objective, isolating a single z plane. To enable comparisons, we processed and captured the quantified

images presented in Figures 1B, S3B, and S3C using identical settings. Glass capillary pipettes were pulled to a tip diameter of 30–40 μm and filled with 1% Fluoro-Gold (Fluorochrome) in 100 mM sodium cacodylate (pH 7.5). This micropipette was unilaterally placed in the medial NAc shell of anesthetized mice in a stereotaxic frame. A current of 2 μA was applied in 5 s pulses over 20 min. The micropipette was left in place very for an additional 5 min to prevent flow of tracer back through the needle track. Seven days after surgery, mice were anesthetized and perfused, as described above. Immunohistochemistry and imaging details are available in the Supplemental Experimental Procedures. Starting 4 weeks after surgery, mice in this group either remained in their home cage or were placed in an activity box (38 cm by 30 cm) for 40 min each day over 5 consecutive days. At the same time each day, or 10 min after entering this chamber, mice received intraperitoneal injections of either cocaine (15 mg/kg) or saline (0.9% NaCl). They were prepared for electrophysiological recordings 10–14 days later.