Hazzard for figure assistance.

J.A. was supported by National Eye Institute (NEI)/National Institutes of Health (NIH) grants R01EY018350, R01EY018836, R01EY020672, R01EY022238, R21EY019778, RC1EY020442, Doris Duke Ibrutinib in vivo Distinguished Clinical Scientist Award, Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, Dr. E. Vernon Smith and Eloise C. Smith Macular Degeneration Endowed Chair, and B.J.F. was supported by by NIH T32HL091812 and UL1RR033173. J.A. is named as an inventor on patent applications about age-related macular degeneration filed by the University of Kentucky and is a founder of iVeena Pharmaceuticals, which is commercializing these technologies. “
“Huntington’s disease (HD) is one of the most common dominantly inherited neurodegenerative disorders clinically characterized by a triad of movement disorder, cognitive dysfunction, and psychiatric impairment (Bates et al., 2002). HD neuropathology is characterized by selective and massive degeneration of the striatal medium spiny neurons (MSNs) and, to a lesser extent, the deep layer cortical pyramidal neurons (Vonsattel and DiFiglia, 1998). The disease is caused by a CAG repeat expansion resulting in an elongated polyglutamine (polyQ) stretch near the N terminus of Huntingtin (Htt) (The Huntington’s

Disease Collaborative Research Group, 1993). HD is one of nine polyQ disorders with shared molecular genetic features, such as an inverse relationship between the expanded repeat length and the age of disease onset, and evidence for toxic gain-of-function as a result of PI3K targets the polyQ expansion (Orr and Zoghbi, 2007). However, each of the polyQ disorders appears to target a distinct subset of neurons

in the brain see more leading to disease-specific symptoms. Hence, it is postulated that molecular determinants beyond the polyQ repeat itself may be critical to disease pathogenesis (Orr and Zoghbi, 2007). Protein-interacting cis-domains ( Lim et al., 2008) and posttranslational modifications (PTMs) of polyQ proteins ( Emamian et al., 2003 and Gu et al., 2009) can significantly modify disease pathogenesis in vivo. Thus, studying the proteins that interact with domains beyond the polyQ region may provide important clues to disease mechanisms. In the case of HD, several hundred putative Htt interactors have been discovered using ex vivo methods, such as yeast two-hybrid (Y2H) or in vitro affinity pull-down assays, utilizing only small N-terminal fragments of Htt ( Goehler et al., 2004 and Kaltenbach et al., 2007). Such studies have provided insight into Htt’s normal function as a scaffolding protein involved in vesicular and axonal transport and nuclear transcription ( Caviston and Holzbaur, 2009 and Li and Li, 2006). The caveats of the prior Htt interactome studies include the exclusive use of small Htt N-terminal fragments as baits and the isolation of interactors ex vivo.

Recordings from dissociated LNvs expressing GCaMP1.6 were carried out as in Dahdal et al. (2010). Briefly, 30–60 larval brains were dissociated by treatment with 2 units/ml Dispase II and manual trituration. GCaMP fluorescence from individual neurons was imaged on an inverted epifluorescence microscope (TE2000U, Nikon) via a standard GFP filter set. Cells were continuously superfused at 2 ml/min

with standard saline (128 mM NaCl, 2 mM KCl, 4 mM MgCl2, 1.8 mM CaCl2, 36 mM sucrose, and 5 mM HEPES [pH 7.1]), to which compounds were added as indicated. For low-chloride experiments, standard saline was modified to reduce Cl− Gemcitabine order to 13.6 mM by replacement of NaCl with sodium gluconate. For locomotor activity experiments, adults were entrained to 12:12 LD cycles at 25°C for at least 3 days before transfer to DD. Locomotor activity was recorded

by using the DAM system (TriKinetics). We used χ2 analysis in ClockLab (Actimetrics) to derive a power and significance for each rhythm over 10 days in DD. We subtracted the significance score from the power to calculate the strength of each rhythm (presented as “power” in Results). Using this analysis, we found that control lines have average powers ranging from ∼270–580 (“rhythmic,” see Table 1), whereas classical clock mutants (per01, ClkJrk, and Clkar) have powers from 10–40 (“arrhythmic”). Pdf > dORKΔC flies, previously described as ∼70% arrhythmic / 30% find more weakly rhythmic ( Nitabach et al., 2002 and Wu et al., 2008), have an average power of 91, establishing a baseline for the effect of manipulations of electrical excitability. All statistical comparisons were many made by ANOVA. We are very grateful to the following for their generous gifts of antibodies and flies: Ravi Allada, Patrick Emery, Paul Hardin, Rob Jackson, Michael Nitabach, Jae Park, Marie-Laure Parmentier, Michael Rosbash, Amita Sehgal, and Mike Young. Additional fly stocks were obtained from the Vienna

Drosophila RNAi and Bloomington Stock centers, and PDF antisera were obtained from the DSHB. We also thank Afroditi Petsakou for advice on qPCR and Ravi Allada, Matthieu Cavey, Claude Desplan, Bambos Kyriacou, and Afroditi Petsakou for helpful comments on the manuscript. B.C. was supported by The Robert Leet and Clara Guthrie Patterson Trust Postdoctoral Fellowship, Bank of America, Trustee. E.A.K. was supported by a Dean’s Undergraduate Research Fellowship from NYU. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant Number C06 RR-15518-01 from the National Center for Research Resources, National Institutes of Health (NIH). Confocal microscopy was performed in the NYU Center for Genomics and Systems Biology Core Facility. This work was supported by NIH grants NS030808 (M.H.A.) and GM063911 (J.B.).

This would argue that CSPα-Hsc70 complex directly participates in oligomerization of dynamin 1 by switching its conformation to one that facilitates self-assembly. It also suggests that CSPα is probably not functioning to protect dynamin 1 from degradation, and in fact we do not find that dynamin 1

is ubiquitinated (data not shown). The apparent decrease in dynamin 1 levels in CSPα KO synapses may be accounted for by a selective PD0332991 in vitro loss of dynamin 1 from the membrane synaptic fractions (Figures S2A and S2B), again consistent with a deficit in membrane-associated oligomerization. We also ruled out that dynamin 1 is aggregated in the CSPα KO (Figure S5D). To investigate whether deletion of CSPα leads to a partial loss of dynamin 1 function, we explored if the CSPα KO phenocopies any aspects of the dynamin 1 KO. Similar to the CSPα KO, deletion of dynamin 1 in mice leads to activity-dependent synaptic find more dysfunction and perinatal lethality after 2 weeks. At an ultrastructural level, CSPα KO synapses have fewer

synaptic vesicles (Figures 5G and 5H) like the dynamin 1 KO (Ferguson et al., 2007 and Hayashi et al., 2008). A decrement in synaptic vesicle number is found in many other endocytic mutants (Dickman et al., 2005) and is consistent with the hypothesis that the CSPα KO has endocytic deficits. In an accompanying paper in this issue of Neuron, Rozas et al. (2012) have directly measured synaptic vesicle recycling in CSPα KO motor neurons with synaptopHluorin imaging and show deficits in dynamin-dependent synaptic Org 27569 vesicle endocytosis, consistent with a loss of dynamin 1 function in the CSPα KO. The distinct interactions of the Hsc70-CSPα chaperone complex with SNAP-25 and dynamin 1 reveal that this chaperone has a dual mode of action and is a testament to the versatility of this chaperone complex. Synapse loss is a cardinal

feature of neurodegenerative diseases such as AD (Selkoe, 2002). Therefore, it was intriguing to determine if a decrement of CSPα-dependent synapse maintenance mechanism plays a role in neurodegenerative diseases. Such an involvement was hinted at by the interaction of CSPα with huntingtin (Miller et al., 2003) (Figure 1D). Hence, we tested the levels of CSPα and Hsc70 in age-matched human control and AD brains. Interestingly, protein levels of CSPα and Hsc70 were both decreased by approximately 40% in the frontal cortex of AD brains (Figure S6), suggesting a possible role in synaptic degeneration. Consistent with previously published results, synaptophysin levels were also decreased in AD brains compared to age-matched controls, and served as a positive control in this cohort (Masliah et al., 2001). In this study, we sought to understand presynaptic mechanisms of synapse maintenance.

A NMDA dose-response curve for both GluN2B2A(CTR)/2A(CTR) and GluN2B+/+ neurons revealed no difference in their EC-50 s ( Figure S2J). Based on these NMDA dose-responses, we predicted that an application of 17 and 21 μM NMDA to GluN2B+/+ neurons would induce the same current as an application of 30 and 50 μM, respectively, to GluN2B2A(CTR)/2A(CTR) neurons ( Figure 2E). This was Enzalutamide then confirmed experimentally; application

of 17 and 30 μM NMDA (hereafter NMDAC1) applied to GluN2B+/+ neurons and GluN2B2A(CTR)/2A(CTR) neurons, respectively, induced equivalent currents ( Figure 2F), as did application of the higher pair of NMDA concentrations: 21 and 50 μM NMDA (hereafter NMDAC2) applied to GluN2B+/+ neurons and GDC-0449 nmr GluN2B2A(CTR)/2A(CTR), respectively ( Figure 2F). Given that NMDAR-dependent excitotoxicity is predominantly Ca2+-dependent, we next studied the intracellular Ca2+ elevation triggered by NMDAC1 and

NMDAC2. Treatment with NMDAC1 caused similar Ca2+ loads in GluN2B2A(CTR)/2A(CTR) and GluN2B+/+ neurons, as did NMDAC2 ( Figure 2G). Satisfied that these doses of NMDA elicit equivalent NMDAR-dependent currents and Ca2+ loads, we next studied their effects on neuronal viability. Strikingly, we found that NMDAC1 and NMDAC2 both promoted more death in GluN2B+/+ neurons than in GluN2B2A(CTR)/2A(CTR) ( Figures 2H and 2I). Thus, swapping the GluN2B CTD for that of GluN2A in the mouse genome reduces the toxicity of NMDAR-dependent Ca2+ influx. This is in agreement with our studies based on the overexpression of GluN2A/2B-based wild-type and chimeric subunits ( Figure 1), thus confirming the importance of the CTD subtype by two independent approaches. We also performed a similar set of experiments in DIV18 neurons.

Because there remained a difference in whole-cell currents (around 25%), Sitaxentan we again generated NMDAR current dose-response curves to allow us to pick pairs of NMDA concentrations (15 and 20 μM; 30 and 40 μM) which would trigger equivalent currents ( Figure S2K). Consistent with our observations at DIV10, we once again saw increased NMDA-induced death in GluN2B+/+ neurons compared to GluN2B2A(CTR)/2A(CTR) neurons experiencing equivalent levels of NMDAR activity ( Figure S2L). We next wanted to determine whether maximal levels of neuronal death could be achieved in neuronal populations devoid of CTD2B if NMDAR activity were high enough. We treated GluN2B2A(CTR)/2A(CTR) neurons with a high dose (100 μM) of NMDA and found that this triggered near-100% neuronal death, as it also did in GluN2B+/+ neurons ( Figures 2H and 2I). Thus, the influence of excitotoxicity on the GluN2 CTD subtype is abolished when insults are very strong. In the adult mouse forebrain, GluN2B and GluN2A are the major GluN2 NMDAR subunits (Rauner and Köhr, 2011 and Sheng et al.

This is consistent with the absence of β-neurexins from the invertebrate genomes of Drosophila and C. elegans ( Tabuchi and Südhof, 2002). We next determined the subcellular localization of endogenous ApNRX by immunocytochemical analysis by using an affinity-purified polyclonal antibody generated against the cytoplasmic tail region of ApNRX (Figure S1).

In isolated sensory neurons, ApNRX immunostaining was distributed along neurites in a punctate pattern (Figure 2C). The punctate appearance of endogenous ApNRX cluster is similar to the distribution reported for endogenous neurexin in mammals (Dean et al., 2003). Interestingly, clusters of ApNRX also appear to be distributed along the neurites of the motor neuron in sensory-to-motor cocultures (Figure 2D). When GFP was expressed in sensory neurons as a whole-cell marker, it became

3-Methyladenine manufacturer clear that some ApNRX clusters do not colocalize with the neurites of the sensory neuron outlined by GFP fluorescence; thus they presumably are located on the motor neuron. These ApNRX clusters in the motor neuron may represent a Selleckchem SAHA HDAC pool of ApNRX in postsynaptic compartments since there is a significant amount of neurexin in postsynaptic compartments in mammals (Taniguchi et al., 2007). Moreover, it became clear that some ApNRX clusters overlap with some presynaptic sensory neuron varicosities in contact with the major receptive surface of the postsynaptic motor neuron where the majority of functionally competent synaptic connections are found in culture (Figure 2D, Kim et al., 2003). These ApNRX clusters overlapping with presynaptic varicosities may represent enrichment of ApNRX in presynaptic sensory neuron varicosities, clustering

of ApNRX in postsynaptic motor neuron in apposition to presynaptic sensory neuron varicosities, or perhaps both. Since many known functions of neurexin and neuroligin require their binding to each other across the synaptic cleft, we set out to find whether ApNRX and ApNLG also bind to each other. To address this point, we generated Ig fusion constructs that contain the extracellular domain of either HA-tagged found ApNLG or VSV-G-tagged ApNRX with the Ig Fc at their C-termini (ApNLG-Fc and ApNRX-Fc). We then incubated soluble ApNRX-Fc with cell lysates prepared from GFP-ApNLG transfected HEK293 cells and soluble ApNLG-Fc with cell lysates prepared from GFP-ApNRX transfected HEK293 cells, respectively. We found that ApNRX-Fc specifically binds to GFP-ApNLG, but not to GFP control (Figure 3A), and conversely, ApNLG-Fc binds to GFP-ApNRX, but not to GFP control (Figure 3B). These results provide evidence that recombinant ApNRX and ApNLG bind to each other, as is the case for their vertebrate counterparts. Next, we wondered whether endogenous ApNRX and ApNLG colocalize at the synapse. Thus, we immunostained sensory-to-motor neuron cocultures with both ApNRX and ApNLG antibodies.

Furthermore, our study opens the possibility that changes in cell migration may more generally participate in the evolution of brain connectivity. Consistently, LY294002 supplier it was shown that the formation of the corpus callosum, a mammalian-specific tract, relies on the migration of guidepost neurons (Shu et al., 2003a and Niquille

et al., 2009). Indeed, the mammalian telencephalon is characterized by a complex choreography of tangential neuronal migrations originating in both dorsal and ventral regions, which is essential for its functioning because cell migration defects have been involved in the etiology of several pathologies (McManus and Golden, 2005). Thus, our study raises the intriguing possibility that tangential neuronal migrations may have promoted the evolution of the telencephalon at the expense of its developmental robustness. Wild-type and GFP-expressing transgenic mice (Hadjantonakis et al., 1998), maintained in Swiss OF1 background, were used for expression analysis and tissue culture. Slit1−/−, Slit2−/−, and Slit1−/−;Slit2−/− mutant embryos were obtained by crossing Slit1+/−, Slit2+/−, and Slit1−/−;Slit2+/− parents ( Plump et al., 2002) maintained in B6D2 background.

Robo1−/−, Robo2−/−, and Robo1−/−;Robo2−/− were obtained by crossing Robo1+/−, Robo2+/−, and Robo1+/−;Robo2+/− BI-6727 parents ( Grieshammer et al., 2004, Long et al., 2004 and Ma and Tessier-Lavigne, 2007), which were maintained in CD1, C57BL/6, and mixed CD1–C57BL/6 backgrounds, respectively. Animals were kept under French and EU regulations. Chinese soft-shelled turtle embryos (Nagashima et al., 2009) and corn-snake embryos (Gomez et al., 2008) were fixed

in 4% paraformaldehyde (PFA) for 24–48 hr, kept in methanol, rehydrated, and cut into 100 μm thick sections on a vibratome. Sheep embryo was obtained by permission from a slaughterhouse in Cartagena (Spain), perfused with 4% PFA, Non-specific serine/threonine protein kinase postfixed overnight, embedded in paraffin, and cut into 8 μm sections. Human embryos were obtained from legal abortions (procedure approved by the French National Ethical Committee CCNESVS), staged, fixed in 4% PFA, cryoprotected, and cut into 12 μm thick sections as described previously (Verney et al., 2001). For in situ hybridization, mouse or chicken brains were fixed overnight in 4% PFA in PBS. Twenty micrometer frozen sections or 80 μm free-floating vibratome sections were hybridized with digoxigenin-labeled probes as described before (Lopez-Bendito et al., 2006). For immunohistochemistry, cultured slices/explants and mouse or chicken embryos were fixed in 4% PFA at 4°C for 30 min and for 2–3 hr, respectively. Immunohistochemistry was performed on culture slices, Matrigel pads, and 100 μm free-floating vibratome sections as previously described (Lopez-Bendito et al.

, 2008). In other words, there are signal-sequence-independent mechanisms that direct mRNA localizations to the ER. Since the ER permeates the entire neuron including the axonal processes, some mRNAs could be simply carried by the ER into axons (Figure 1B). Accumulating evidence has shown

that axons of nonmammalian neurons and embryonic mammalian neurons have the capacity to synthesize proteins, and in vivo in the adult mouse, ribosomes could be transferred Autophagy Compound Library from Schwann cells to the injured distal axons of the peripheral nerve (Twiss and Fainzilber, 2009, and the references therein). Nevertheless, ribosomes were rarely observed in axons of mature central nervous system neurons in mammals, although they could be found in the axon hillock (Steward, 1997). The cellular mechanisms that prevent ribosomes from entering into the axons of mature neurons remain unclear, although it is conceivable that in immature growing neurons, ribosomes may move into axons as part of the vectorial flow of cytoplasm (Bradke and Dotti, 1997). Perhaps as the neurons mature and become polarized, their axon initial segment (AIS) is established and the AIS serves as a selective cytoplasmic “filter” (Song et al., 2009) that excludes ribosomes from getting into axons. This possibility could, in principle, be examined in mature neurons in which the AIS is acutely disrupted Pazopanib by conditional deletion of the specific ankyrin

G isoform (Grubb and Burrone, 2010). Finally, it will also be interesting to examine whether this mechanism of integrating two major types of target-derived signals, i.e., neurotrophic factors stimulating the axonal synthesis only of SMADs and TGFβ-superfamily factors forming retrograde

signaling endosomes, is used elsewhere in the nervous system for retrograde specification of neuronal subtype identities. “
“The function of the nervous system relies on billions of neurons and their synapses. Loss of neurons and synapses is a feature of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases (Lin and Koleske, 2010). This feature can be replicated in mice lacking cysteine string protein α (CSPα) (Chandra et al., 2005 and Fernández-Chacón et al., 2004), a presynaptic vesicle protein that has been implicated in the pathogenesis of neurodegenerative diseases (Nosková et al., 2011). Knockout of CSPα causes activity-dependent synapse loss, progressive defects in neurotransmission, neurodegeneration, and early lethality in mice (Chandra et al., 2005 and Fernández-Chacón et al., 2004). CSPα KO is therefore a useful tool to study mechanisms underlying synapse loss and neurodegeneration. A thorough understanding on how CSPα works at synapses is a prerequisite to understand the mechanisms underlying synapse loss in CSPα KO mice. In this issue of Neuron, Zhang et al. (2012) and Rozas et al. (2012) found a new role of CSPα—regulation of synaptic vesicle endocytosis via interaction with the vesicle fission protein dynamin 1 ( Figure 1).

, 2007, Nassi and Callaway, 2007 and Wickersham et al., 2007a), that EnvA-pseudotyped SADΔG-GFP rabies viruses can be used to selectively label the direct inputs to a targeted neuronal population or even a single neuron (Haubensak et al., 2010, Marshel et al., 2010, Miyamichi et al., 2011, CP 673451 Rancz et al., 2011, Stepien et al., 2010, Wall et al., 2010, Wickersham et al., 2007b and Yonehara et al., 2011), and that a combination

of EnvB-pseudotyped rabies viruses and a bridge protein with TVB can selectively target infection to specific neuron types that bind to the bridge protein (Choi et al., 2010). While ΔG rabies viruses have already proven to be a powerful tool for revealing neural circuit structure, understanding how neural circuits develop and function requires direct links to be made between neural circuits, activity monitoring, and manipulation of activity or gene expression. We therefore aimed to extend the utility of a recombinant rabies virus by incorporating the potential to exploit other novel genetic technologies that have recently been pioneered. For example,

much progress has been made at the interface of optical and genetic technologies (Luo et al., 2008 and Scanziani and Häusser, 2009). In vivo this website two-photon imaging of calcium transients in neurons labeled with indicator dyes allows monitoring of activity from many neurons simultaneously (Kerr et al., 2005, Ohki et al., 2005, Runyan et al., 2010 and Svoboda and Yasuda, 2006), and the incorporation of genetically-encoded calcium indicators allows the monitoring of genetically-targeted neurons (Miyawaki,

2005 and Tian et al., 2009). Genetic strategies for activating or inactivating selected neurons have also opened up new possibilities for understanding circuitry and behavior. In particular, optical stimulation, or optogenetics, has allowed for manipulation of the activity of genetically-defined Mannose-binding protein-associated serine protease neurons with high temporal and spatial resolution (Boyden et al., 2005, Cardin et al., 2009 and Sohal et al., 2009). Finally, the last decade has seen the development of a large number of floxed, fretted, or tTA-dependent mouse lines (Branda and Dymecki, 2004) and viral vectors (Kuhlman and Huang, 2008 and Luo et al., 2008) to allow selective and inducible knockout of genes of interest, allowing investigations of the roles of particular genes in the development, plasticity, or function of defined components of the nervous system. By incorporating each of these classes of genetic tools into the ΔG rabies viruses, it is possible to combine their power with the ability to target connectionally-defined neuronal networks.

Manipulations of the SP/NK1R system have been shown to influence several addiction-related RAD001 behaviors. For example, NK1R knockout mice do not display morphine-CPP and self-administer morphine at lower rates. Morphine-induced locomotor activation

and psychomotor sensitization are also blunted in these mice (Murtra et al., 2000; Ripley et al., 2002). Lesions of NK1R-containing neurons in the AMG, but not NAC, suppressed morphine-induced CPP, a finding suggesting that NK1Rs in the AMG contribute to rewarding properties of morphine (Gadd et al., 2003). Reduced opioid reward after NK1R blockade was recently also supported by observations that this treatment attenuates the ability of morphine to lower intracranial self-stimulation thresholds (Robinson et al., 2012). Coadministration of SP and morphine prevents the internalization and acute desensitization of the mu opioid receptor typically induced by morphine, which may account for the involvement of the NK1R in opioid reward (Yu et al., 2009). These data collectively support a role of NK1R activation in rewarding properties of opioids and suggest the possibility that NK1R antagonists may be useful for the treatment of opioid addiction through blockade of opioid reward. Surprisingly,

however, an initial human laboratory study found that a single administration of the NK1R antagonist aprepitant potentiated, rather than inhibited, subjective as well as physiologic responses to an opioid challenge in prescription opioid abusers (Walsh et al., 2012). A direct assessment of opioid self-administration after NK1R blockade is therefore critical but has to date not been obtained in laboratory animals or humans. Furthermore, the selleck screening library role of the NK1R in opioid-related behaviors influenced by stress, for example, stress-induced reinstatement of opioid seeking after extinction, has not been explored. In contrast to its role in opioid-related behaviors, disruption of NK1R signaling does not affect cocaine CPP, self-administration, or locomotor sensitization (Gadd et al., 2003; Murtra et al., 2000; Ripley et al., 2002). However, there is some evidence that NK1R antagonists these can suppress

cocaine-induced locomotion (Kraft et al., 2001) and that relapse to cocaine seeking after extinction can be triggered by ICV infusion of a specific NK1R agonist (Placenza et al., 2005) or intra-VTA infusion of an SP analog (Placenza et al., 2004). However, an NK1R specific antagonist was unable to prevent reinstatement of cocaine seeking induced by cocaine priming (Placenza et al., 2005). One possibility is therefore that exogenous SP is able to activate pathways involved in reinstatement of cocaine seeking, but that this does not reflect actions of endogenous SP. Alternatively, cocaine-induced reinstatement may be mediated by an NK receptor other than NK1R, such as NK3R. Finally, it is possible that the NK1R is involved in reinstatement of cocaine seeking triggered by some stimuli, but not that induced by drug priming.

, 2001, Sato

and Schall, 2003 and Thompson et al., 1996) and these selection signals do not depend on the generation of a saccade (Thompson et al., 1997). Moreover, when the saccade is directed to a stimulus outside the RF, FEF neurons are activated by distracters similar to the target (Bichot and Schall, 1999) confirming that visual Wnt drug selection signals are independent of saccade production signals in the FEF. Finally, electrical microstimulation of the FEF in an antisaccade task demonstrated that covert attention is independent of the actual saccade preparation (Juan et al., 2004). Although the evidence listed above argues against a causal role of saccadic activity in attentional processes, a direct test should include a comparison of the responses of all classes of FEF neurons (Bruce and Goldberg, 1985) in both covert attention and saccade tasks, as well as a comparison of their roles in top-down attentional feedback to visual cortex. Our study now does that. We employed an endogenous attention task and a manual response, to preclude any preparation for a saccade. An earlier study also examined the source of attentional signals among FEF neurons (Thompson et al., 2005). Using a pop-out visual search task that required no saccadic response, the authors showed that only cells with visual responses in the FEF (visual

and visuomovement) modulated their activity with the locus of attention. Saccade-related movement neurons were suppressed in the attention task and this suppression was not spatially selective. Our data on firing rates 3-Methyladenine are in large agreement with Thompson et al. and extend their results in two ways. First, during sustained attention, we found that only purely visual neurons increased their activity with attention to the RF and at this time the activity of movement neurons decreased when attention was directed toward their movement

field. The suppression of saccade-related movement neurons with attention may be the result of local processing within the not FEF so that saccades are inhibited downstream based on behavioral context. Indeed, SC, which lies closer to the brainstem saccade generator, receives projections mainly from the infragranular layers of the FEF where most movement neurons lie (Fries, 1984, Pouget et al., 2009 and Segraves and Goldberg, 1987). Second, while Thompson et al. used a task characterized by exogenous shifts of attention (pop-out), we used a task that required endogenous shifts of attention. It has been previously suggested that endogenous, rather than exogenous, shifts of attention are mediated by oculomotor processes related to the preparation for a saccade (Awh et al., 2006, Klein, 1980 and Rizzolatti et al., 1994). The two studies together, therefore, demonstrate that neither in exogenous nor in endogenous attention do FEF saccade-related movement neurons contribute to shifts of attention.