1 μg/ml) recovered the incremental effect on spine density to a level comparable to that in cells transfected with 1.0 μg/μl of HA-NLG1. Thus, we reasoned that ectodomain shedding negatively regulates the spinogenic effect

of NLG1 in hippocampal granule cells. Next, we analyzed the effects of fragment forms of NLG1 corresponding to its proteolytic products (i.e., NLG1-ΔE and NLG1-ICD) on the spine density (Figure 8A). Unexpectedly, NLG1-ΔE increased the spine density at a similar level to NLG1-FL, suggesting that the NLG1-CTF lacking the ectodomain retains the spinogenic effect. However, NLG1-ICD failed to increase the spine density. Thus, the function of membrane-tethered form of NLG1-ICD (aka, NLG1-ΔE or

NLG1-CTF) was abolished by liberation from the membrane by the γ-secretase cleavage and subsequent degradation. Finally, to directly test whether NLG1 shedding modulates GS-7340 price the spinogenic function, we analyzed selleck the dendritic spines of transfected rat hippocampal primary neurons obtained from E18 pups (Figure 8C). We transfected wild-type or PKQQ/AAAA mutant NLG1 together with green fluorescent protein (GFP) into primary neurons at DIV6 and fixed them at DIV20. The numbers of spines in neurons expressing wild-type NLG1 showed an increased trend compared to those in mock-transfected neurons, but not with a statistical significance. However, the spine density was significantly increased in neurons transfected with the Thalidomide mutant NLG1 (Figure 8D), suggesting that cleavage-deficient mutation enhanced the NLG1 function in primary neurons. Taken

together, our results indicate that the sequential processing of NLG1 negatively regulates the spinogenic activity. To date, all known γ-secretase substrates are shown to be first shed at the extracellular domain to generate a soluble ectodomain as well as a membrane-tethered CTF. ADAM10 is a well-characterized physiological sheddase for a number of γ-secretase substrates (e.g., APP, cadherin, and Notch) (Reiss et al., 2005; Jorissen et al., 2010; Kuhn et al., 2010). Both γ-secretase and ADAM10 have been implicated in the regulation of neural stem cell number by modulation of Notch signaling in the developing CNS (Jorissen et al., 2010). Recently, it was shown that metalloprotease and γ-secretase-mediated cleavage in mature neurons regulates the synaptic function (Rivera et al., 2010; Restituito et al., 2011). Here we systematically analyzed the processing of NLG1 by pharmacological and genetic approaches. Using specific inhibitors and Cre-mediated gene excision, we found that ADAM10 is responsible for NLG1 shedding and that C-terminal stub of NLG1 is subsequently cleaved by γ-secretase (Figure 1F). Notably, significant reduction in the sNLG1 production was similarly observed in two distinct lines of Adam10flox/flox mice (i.e.

, 1980, Jack et al., 1981, Magee, 1999, Magee and Cook, 2000 and Stricker et al., selleckchem 1996). Furthermore, the local integration of synaptic inputs also appears to depend on dendritic region. For example, synaptic inputs to the distal apical dendrites of layer 5 pyramidal cells (Schiller et al.,

1997 and Yuste et al., 1994) or CA1 pyramidal cells (Golding and Spruston, 1998) can trigger local dendritic spikes, and the gating (Larkum et al., 1999) and boosting (Stuart and Häusser, 2001) effects of backpropagating spikes on neighboring synaptic input (Jarsky et al., 2005) can also be region specific. Finally, plasticity mechanisms also appear to depend on dendritic location (Gordon et al., 2006, Letzkus et al., 2006 and Sjöström and Häusser, 2006). These region-specific differences in dendritic properties may also be reflected in the preferential targeting of different types of inhibitory inputs (Somogyi,

1977 and Somogyi et al., 1998) and excitatory inputs (Markram et al., 1997, Thomson and Bannister, 1998, Petreanu et al., 2009 and Richardson et al., 2009) to specific dendritic domains. While these functional differences in macroscopic regions of the dendritic tree are now well established, it remains unclear whether the rules for synaptic integration are also heterogeneous on a smaller scale, and in particular at the level of single dendritic branches. This is especially important given the recent emphasis on the role of single dendritic Selleckchem DAPT branches as fundamental functional compartments for synaptic integration and plasticity (Larkum and Nevian, 2008, Losonczy and Magee, 2006, Losonczy et al., 2008, Major et al., 2008, Poirazi et al., 2003 and Branco and Häusser, 2010). Do synaptic inputs along a given dendrite behave approximately

equally in terms of their integrative properties, or are there systematic functional differences even along a single dendrite? To address this question we have taken advantage of the precise spatial and temporal control why of synaptic activation possible with two-photon glutamate uncaging, and probed the thin basal and apical oblique branches of layer 2/3 and layer 5 pyramidal cells, which receive the majority of the synaptic input to these neurons (Larkman, 1991 and Lübke and Feldmeyer, 2007). While strong EPSP attenuation occurs along individual branches of pyramidal cell basal dendrites (Nevian et al., 2007), it is not known if inputs at different distances along a branch are integrated similarly. We show that single cortical pyramidal cell dendrites exhibit a gradient of temporal summation and input gain that increases from proximal to distal locations. This suggests a progressive shift of computational strategies for synaptic inputs along single dendrites.

AAV-CaMKIIa-eNpHR3.0-eYFP or AAV-CaMKIIa-eYFP (from Gene Therapy Center at University of North Carolina at Chapel Hill, courtesy of Dr. Karl Deisseroth) was injected bilaterally in OFC under stereotaxic guidance at AP −3.0 mm, ML ± 3.2 mm, and DV 4.4 and 4.5 mm from the brain surface. A total 1–1.2 μl of virus (titer ∼1012) per hemisphere was

delivered at the rate of ∼0.1 μl/min by Picosptrizer microinjection system (Parker, Hollins, NH). Two rats that received eNpHR3.0 transgene were saved for later slice work; the remaining rats designated for behavioral testing had optic fibers (200 μm in core diameter; Thorlab, Newton, NJ) implanted bilaterally at AP −3.0 mm, ML ± 3.2 mm, and DV 4.2 mm. At the end of the study, these rats were perfused with phosphate buffer saline and then 4% PFA. The brains were then immersed in 30% sucrose/PFA for at least 24 hr. The brains were sliced at 40 μm with a microtome. The http://www.selleckchem.com/products/BMS-777607.html brain slices were then stained with DAPI (through Vectashield-DAPI, Vector Lab, Burlingame, CA) or NeuroTrace (Invitrogen, Carsbad, CA) and mounted to slides with Vectashield (in the case of staining with NeuroTrace)

mounting media. The location of the fiber tip and NpHR-eYFP or eYFP expression was verified using an Olympus confocal microscope. The Z-stack images were merged and processed in Image J (National Institutes of Health). Approximately 2 months after surgery, two rats that had received AAV-CaMKIIa-eNpHR3.0-eYFP injection were anesthetized with isoflurane and perfused selleck kinase inhibitor transcardially with ∼40 ml ice-cold NMDG-based artificial CSF (aCSF) solution containing (in millimoles) 92 NMDG, 20 HEPES, 2.5 KCl, 1.2 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 30 NaHCO3, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, and 12 N-acetyl-L-cysteine (300–310 mOsm, pH 7.3∼7.4). After perfusion, the brain was immediately removed and Megestrol Acetate 300 μm coronal brain slices containing the OFC were made using a Vibratome (Leica, Nussloch, Germany). The brain slices were recovered for less than 15 min at 32°C in NMDG-based aCSF and then transferred and stored for at least 1 hr in HEPES-based aCSF containing

(in mM) 92 NaCl, 20 HEPES, 2.5 KCl, 1.2 NaH2PO4, 1 MgSO4, 2 CaCl2, 30 NaHCO3, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, and 12 N-acetyl-L-cysteine (300–310 mOsm, pH 7.3∼7.4, room temperature). During the recording, the brain slices were superfused with standard aCSF constituted (in millimoles) of 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2.4 CaCl2, 26 NaHCO3, 11 glucose, 0.1 picrotoxin, and 2 kynurenic acid, and was saturated with 95% O2, and 5% CO2 at 32°C–34°C. Glass pipette (pipette resistance 2.8–4.0 MΩ, King Precision Glass, Claremont, CA) with K+-based internal solution (in millimoles: 140 KMeSO4, 5 KCl, 0.05 EGTA, 2 MgCl2, 2 Na2ATP, 0.4 NaGTP, 10 HEPES, and 0.05 Alexa Fluor 594 [Invitrogen, Carlsbad, CA], pH 7.3, 290 mOsm) was used throughout the experiment.

solium and Trichinella spp. In the SE Asian nations where T. solium is endemic, there exists a window of opportunity for a concerted effort to identify hot-spots of endemicity and pin-point control programs designed in consultation with effected communities. However, for this to be achieved, improved diagnostic methods for use in a multi-Taenia species environment are urgently required, a thorough understanding of pork supply networks and the

political will to deliver services to poor marginalised communities selleck chemical will also be important ( Conlan et al., 2009 and Willingham et al., 2010). In an environment where people have a preference for consuming uncooked or partially cooked meat from domestic or wild pigs, the control of trichinellosis will remain problematic. T. spiralis incidence will most likely

continue to decline in the more developed countries such as Thailand, but sporadic cases and outbreaks will continue to occur in Laos and Vietnam. Sensitisation of health officials to trichinellosis is required so that cases and outbreaks can be more thoroughly investigated and documented and the Trichinella worms circulating and causing disease in a population can be accurately identified ( Odermatt et al., 2010). The relative rarity of vector-borne protozoan diseases in SE Asia make it difficult to predict what impact environmental and socio-cultural changes will have on the distribution and incidence of P. knowlesi Thymidine kinase and Leishmania spp. infection. Like trichinellosis, health Selleckchem MK0683 officials will need to be sensitised to these parasitic zoonoses in the differential diagnosis for patients. In addition, vector competence studies will be important to effectively monitor the emergence of these medically important parasites. It is apparent that A. ceylanicum is highly endemic in the dog population of some SE Asian nations

with spill over into the human population. At this stage we do not have a good understanding of the clinical significance of A. ceylanicum, but historical data indicates that it does cause clinical disease. In areas where this zoonotic hookworm is prevalent in humans and dogs, the wide spread use of anthelmintics such as mebendazole in the human population will have limited impact on A. ceylanicum distribution and it may even provide a niche environment for A. ceylanicum to thrive ( Thompson and Conlan, in press). Therefore, the use of anthelmintics to control hookworm in dogs will need careful consideration to avoid clearing T. hydatigena from dogs and consequently altering the infection pressure on pigs, potentially creating a pig population with greater susceptibility to T. solium infection ( Thompson and Conlan, in press). The ecological changes currently taking place in SE Asia present new risks for the emergence or re-emergence of clinically important parasitic zoonoses while at the same time presenting new opportunities for disease control.

This work was supported by grants from the EU (FP7-ICT-270212, ERC-2010-AdG-269716), the DFG (SFB 936/A1/A2/A3/B2/C1), and the BMBF (031A130). We thank Tobias Donner, Peter König, Friedhelm Hummel, and Christian Moll for helpful comments on the manuscript. “
“Social dysfunction is one of the core diagnostic criteria for autism spectrum disorders (ASD) and is also the most consistent finding from cognitive neuroscience studies (Chevallier et al., 2012, Gotts et al., 2012, Losh et al., 2009 and Philip et al., 2012). Although there is evidence for

global dysfunction at the level of the whole brain in ASD (Amaral et al., 2008, Anderson et al., 2010, Dinstein et al., 2012, Geschwind and Levitt, 2007 and Piven et al., 1995), DAPT ic50 several studies emphasize abnormalities in the amygdala both morphometrically see more (Ecker et al., 2012) and in terms of functional connectivity (Gotts et al., 2012). Yet all functional data thus far come from studies that have used neuroimaging or electroencephalography, leaving important questions about their precise source and neuronal underpinnings. We capitalized on the comorbidity between epilepsy and ASD (Sansa et al., 2011) with the ability to record from clinically implanted depth electrodes in patients with epilepsy who are candidates for neurosurgical temporal lobectomy.

This gave us the opportunity to record intracranially from the amygdala in two rare neurosurgical patients who had medically refractory epilepsy,

but who also had a diagnosis of ASD, comparing their data to those obtained from eight control patients who also had medically refractory epilepsy and depth electrodes in the amygdala, but who did not have a diagnosis of ASD (see Tables S1 and S2 available online for characterization of all the patients). Perhaps the best-studied aspect of abnormal social information processing in ASD is face processing. People with ASD show abnormal fixations onto (Kliemann Thymidine kinase et al., 2010, Klin et al., 2002, Neumann et al., 2006, Pelphrey et al., 2002 and Spezio et al., 2007b) and processing of (Spezio et al., 2007a) the features of faces. A recurring pattern across studies is the failure to fixate and to extract information from the eye region of faces in ASD. Instead, at least when high functioning, people with ASD may compensate by making exaggerated use of information from the mouth region of the face (Neumann et al., 2006 and Spezio et al., 2007a), a pattern also seen, albeit less prominently, in their first-degree relatives (Adolphs et al., 2008). Such compensatory strategies may also account for the variable and often subtle impairments that have been reported regarding recognition of emotions from facial expressions in ASD (Harms et al., 2010 and Kennedy and Adolphs, 2012).

Glutamate can be released from astrocytes by several mechanisms, including exocytosis, volume regulated anion channels, as well as hemichannels (Hamilton and Attwell, 2010). Because this field is still at a relatively early stage, there is still discussion over which mechanism predominates under specific conditions and whether there are region-specific variations in release pathway. In the dentate gyrus, there is considerable evidence for an exocytotic mechanism of release of glutamate from astrocytes. For example, astrocyte-to-synapse modulation is attenuated by intracellular glial dialysis of tetanus toxin, and immunoelectron microscopy has revealed the presence

of vesicular glutamate transporter associated with small electron lucent vesicles in astrocytes associated with synapses in the dentate gyrus (Bezzi et al., 2004 and Jourdain et al., 2007). Because BTK phosphorylation it is not feasible with current technology to image exocytosis in brain slices, they examined exocytosis in cultured astrocytes using total internal reflection microscopy (TIRF) to see if it could provide

a clue to a gliotransmission switching mechanism. In TIRF one images the first ∼100 nm adjacent to the plasma membrane allowing the resolution of vesicles that are docked and ready to be released. Santello et al. (2011) transfected astrocytes with two vesicle-targeted constructs, one is pH sensitive and dequenches upon exocytosis, while the other is pH insensitive and allowed for the examination of the location of vesicles in relation to the plasma membrane. With this strategy, they showed that TNFα regulates the number of vesicles resident at the astrocytic plasma membrane. check details TNFα does not change the total number of vesicles but increases two-fold those that are present at the plasma membrane ready for exocytosis. As a consequence the presence of TNFα regulates the rate of exocytosis. In the absence of TNFα, exocytosis is slow and asynchronous with kinetics determined by the rate of

delivery of distant vesicles. However, the ability of TNFα to increase those vesicles already resident at the plasma membrane permits burst-mode exocytosis (Figure 1). How could this change in exocytosis mode gate gliotransmission in situ? Santello et al. (2011) reasoned that glutamate released by a slow asynchronous mechanism would be scavenged by local glutamate transporters almost which would prevent this gliotransmitter from accessing neuronal NMDA receptors that are required to modulate presynaptic release. Under the influence of TNFα, burst-mode release would provide sufficient temporally coincident glutamate to allow this transmitter to escape reuptake transporters. If this is the case, they predicted, and demonstrated, that pharmacological attenuation of glutamate transporters would permit astrocytic glutamate to access neuronal NMDA receptors and induce the consequent increase in mEPSC frequency under conditions of slow asynchronous release (TNFα−/−).

Females with higher-risk genotypes may encounter difficulties at later stages of their lives that manifest as a different diagnostic category, or that reduces fecundity. If true, the disorder would most likely be one with a gender bias opposite that of ASDs, such as anorexia nervosa (Fairburn and Harrison, 2003). Our genetic theory of autism, as discussed above, largely depends on dominant acting genetic variants of variable

penetrance. We think the theory is sufficient to explain most of the genetic basis of autism, both simplex and multiplex, but certainly not all. For example, the role of recessive mutations in individuals from consanguineous marriages has been demonstrated (Morrow et al., 2008). We have observed only a single case of inheritance of a rare homozygous null state. A striking finding of all the studies of de novo mutation

in children with ASDs is the apparent number of distinct target loci. Even discounting 25% of events PI3K Inhibitor Library as incidental (based on a 2% frequency in sibs and 8% in probands), there are a large number of target regions and few recurrences. Only CNVs at 16p11.2 are present in more than 1% of cases (ten out of 858 children). We can make an estimate of the minimum number of target regions by analysis of recurrence. Combining two large studies (ours and that of Pinto et al., 2010), we observe 39 overlaps at 12 recurrent loci in 121 events. Excluding the highly recurrent 16p11.2 locus (with 13 hits in the combined dataset) and Selleck LY2835219 discounting

one-quarter of the remaining 108 events as incidental, we observe 11 recurrent loci in approximately 80 causal events. If we assume a uniform rate of copy-number almost mutation, we estimate the number of target loci at 250–300. However, targets do not have a uniform rate of copy-number mutation, so this figure would be an underestimate of total targets. We derive a similar estimate for target size by a completely different method, based on many assumptions including the rate of new mutations that damage a gene in humans (about one gene per three births), the incidence of ASDs among males (approximately 1 in 100), a genetic model that predicts that about half of ASDs result from new mutations (Zhao et al., 2007), and high penetrance of a select set of single mutational hits. The latter assumption is based on the observation of dominant transmission in multiplex families (Zhao et al., 2007). An organism will be vulnerable to a single mutational hit at only a small subset of its genetic elements. We imagine that vulnerable targets may arise by two distinct cellular mechanisms: insufficient or uncorrectable dosage compensation resulting from (for example) altered stoichiometry of protein complexes; and monoallelic gene expression, which could result in subpopulations of functionally null neurons, perhaps confined to specific subtypes (Gimelbrant et al., 2007 and Gregg et al., 2010).

To determine if the persistent synapsin particles seen in the above experiments were associated with vesicular cargoes moving in fast axonal transport, we cotransfected neurons with GFP:synapsin-I

(labeling all synapsin at steady state) and synaptophysin:monomeric red fluorescent protein (mRFP; transmembrane protein moving in fast transport) and simultaneously imaged both cargoes in thin distal axons by using dual-camera imaging Roxadustat in vitro (DC2 Dual-Cam system, Photometrics, Tucson, AZ). We found that a large fraction of persistent synapsin particles were colocalized with synaptophysin as shown in Figure 4B. Collectively the data show that while a large population of synapsin moves as a slowly migrating wave (likely with intricate particle kinetics), a subpopulation of somatically derived synapsin associates with vesicles conveying other fast synaptic proteins and moves persistently. These data help reconcile the seemingly conflicting observations by live imaging that synapsin particles clearly move rapidly as transport packets (Ahmari et al., 2000) and the fact that radiolabeling studies have established that the majority of synapsin is conveyed in slow axonal transport (Baitinger and Willard, 1987 and Petrucci et al., selleck 1991). The data above support

a model where the majority of the cytosolic protein population organizes into particles that undergo slow transport via intricate kinetics. Particles are invariably seen upon immunostaining of cytosolic proteins in neurons and L-NAME HCl (at least a fraction of them) probably represent the cargo structure moving in slow transport. What is the nature of these punctate structures and how are they being transported? One possibility is that they represent the association of synapsin and CamKIIa molecules with a proteinaceous particle or macromolecular complex that is transported by motors (Lasek et al., 1984). Another possibility is that these puncta represent the association of individual monomers with vesicles. Indeed, association of both synapsin and CamK with synaptic vesicles is well established (Takamori et al., 2006) and it is possible that they have transient vesicular associations in nonsynaptic compartments as well. To address the biochemical nature of synapsin and CamKII cargoes in mouse brains in vivo we first used classical assays to isolate synaptosomal-rich and synaptosomal-depleted fractions (P2 and S2 respectively, see strategy in Figure 5A). We reasoned that the S2 fractions, largely free from the pre- and postsynaptic terminals (Dunkley et al.

AF64α injection into the PPTg also resulted in a ∼10-fold upregulation of Shh transcription in the ipsilateral vMB compared to the contralateral control ( Figure 6I). Thus, the comparative analysis of Shh-nLZC/C/Dat-Cre mice, which are unable

to express functional Shh, and of Shh-nLZC/+/Dat-Cre control animals allows the distinction of Shh dependent and independent regulation of gene expression in the experimentally undisturbed striatum that occurs in response to AF64α injection into the PPTg. Utilizing this experimental paradigm, we found that the expression of ChAT and vAChT in the ipsilateral striatum were downregulated to a similar extent, regardless of Shh expression by DA neurons compared to the contralateral striatum (Figure 6K).

Fasudil In contrast, we observed a ∼4-fold downregulation of GDNF expression upon AF64α injection into PPTg of control mice, i.e., mice that produce Shh in DA neurons, but not of Shh-nLZC/C/Dat-Cre mutant animals ( Figure 6K). These results provide genetic evidence that increased Shh signaling specifically originating from mesencephalic DA neurons results in the repression of GDNF transcription in the striatum. The observed upregulation of Shh expression by DA neurons upon neurotoxic insult to the PPTg suggested that expression of Shh by DA neurons is not static but could be regulated by cell extrinsic signals. We therefore explored the dynamic range of Shh expression and whether the striatum in addition to the PPTg might be a source of signals that could contribute to the regulation of Shh expression by DA neurons. We first investigated buy BMN 673 whether the acute interruption of mesostriatal communication by unilateral injection of 6-OHDA into the mFB of C57BL/6 wt and GDNF-LZ-mice alters Shh expression by DA neurons. Thirty hours after toxin injection, and we observed contralateral turning biases in wt and GDNF-LZ animals consistent with reduced DA signaling to

the striatum ( Figures 7A and 7B). In these animals, we found an upregulation of Shh expression in the vMB ipsilateral to the toxin injection. These results suggested that the striatum could be a source of signals that inhibit Shh expression in the vMB ( Figure 7C). Use of the cholinotoxin AF64α afforded us to test next whether signals emanating from striatal ACh neurons could contribute to the repression of Shh transcription in DA neurons. Thirty hours postunilateral striatal injection of AF64α into wt C57B/6 mice, we observed a dose-dependent ipsilateral turning bias consistent with a graded increase in striatal motor output due to a progressive, AF64α induced, inhibition of cholinergic activity (Figures 7A and 7D) (Lester et al., 2010). In the ipsilateral vMB of these animals, we found an AF64α dose-dependent, stepwise, upregulation of Shh transcription correlated with the graded turning bias (R2 = 0.79, p < 0.001; Figure 7E).

, 2001) and slower-frequency synchronization of neural activity representing unattended stimuli (Cohen and Maunsell, 2009; Mitchell et al., 2009). In sum, our results suggest that synchronous oscillations allow dynamic selection of currently relevant neural ensembles. This may be particularly

important in prefrontal cortex, where neurons have highly diverse properties and thus a particular ensemble must be formed from neurons that are also members of other ensembles (Rigotti et al., 2010). JQ1 The dynamic nature of synchronized oscillations may provide a substrate for the ensembles that allows their rapid selection and deselection and, hence, cognitive flexibility. Two macaque monkeys, one male (CC, Macaca fascicularis) and one female (ISA, Macaca mulatta), were trained on a cued task-switching paradigm ( Figure 1A). Neural activity was simultaneously recorded during task performance from two frontal regions: the dorsolateral prefrontal cortex (PFC, area 9/46) and the anterior cingulate cortex (ACC, areas 24c and 32). Only data from the dorsolateral prefrontal cortex are reported here. The recording well targeting PFC was placed in the left hemisphere and was centered approximately 28 mm anterior to the interaural plane and 21 mm lateral from the midline. Stereotaxic positioning

of the well was Epacadostat guided by structural magnetic resonance imaging. Neural activity was recorded during 34 sessions (11 for monkey CC, 23 for monkey ISA). Arrays of

up to sixteen epoxy-coated tungsten electrodes (FHC) were lowered into the PFC during each recording session (median number of electrodes with well-isolated all single neuron activity was 5.5 per session). Electrodes were lowered in pairs by a custom-built microdrive assembly and spaced at least 1 mm apart. Electrodes were lowered acutely each day through an intact dura and allowed to settle before recording. This ensured stable isolation of the single neuron activity. After each recording session, the electrodes were retracted and the microdrive assembly was removed from the well. A Plexon Multichannel Acquisition Processor (MAP; Plexon) was used to perform electrophysiological recordings. The signal from each electrode was filtered by the preamplifier between 154 Hz and 8.8 kHz to isolate spiking activity and between 3.3 and 88 Hz to isolate the local field potential. Both spiking activity and local field potentials were referenced to earth ground (although the same results were observed when rereferencing locally, within PFC). The raw spiking waveforms were digitized at 40 kHz and subsequently sorted into single units offline, based on waveform shape characteristics and principal components analysis (Offline Sorter, Plexon). During recording, electrodes were lowered to maximize the signal-to-noise ratio of spiking activity and were not guided by the task relevance of neural responses.