The roles of the others remain to be explored. The second molecular feature of the red module

is the presence of six 14-3-3 family proteins (Ywhab, Ywhae, Ywhag, Wnt inhibitor Ywhah, Ywhaq, Ywhaz), with Ywhae as a top hub protein (Figure 6A). Impressively, “14-3-3-Mediated Signaling” in the Red Module is the most significantly enriched IPA Canonical Pathway for all modules in the fl-Htt interactome network (Table S13). The 14-3-3 pathway has been implicated in the pathogenesis of a variety of neurodegenerative disorders (Chen et al., 2003), and four 14-3-3 members have been shown to physically or genetically interact with Htt N-terminal fragments (Kaltenbach et al., 2007 and Omi et al., 2008) (Table S3). Since 14-3-3 proteins are phosphoserine/phosphothreonine

binding proteins (Morrison, 2009), and Htt phosphorylation at several serine residues has been shown to modify HD pathogenesis (Humbert et al., 2002, Gu et al., 2009 and Thompson et al., 2009), it could be a promising direction to investigate whether 14-3-3 proteins in the red module could directly interact with relevant phospho-Htt species to affect the disease process. The third molecular pathway enriched in the red module is “Intracellular Protein Transport” (Dynactin, Dynein, Vcp, and Ran) consistent with the convergent evidence supporting the role of Htt function in the microtubule-based transport process (Caviston and Holzbaur, 2009) and the disruption selleckchem of such function in HD (Gauthier et al., 2004). Although the red module appears to be enriched with proteins from divergent molecular processes, several lines of evidence suggest these proteins indeed have close biological connectivity. First, 26 out of the 61 red module proteins are included in the same IPA network, which is constructed based on the archived IPA Knowledge Base derived from published studies. This network has the highest IPA network score among all of the networks constructed

from Htt interactome modules (Table S13), suggesting that the proteins in red module already have a close functional link based on existing knowledge. Second, the red Endonuclease module has a marked enrichment for proteins implicated in other neurological and genetic disorders. Using another IPA core analysis (IPA Function), the red module has dramatically higher enrichment for proteins in the categories of Neurological Disorders and Genetic Disorders compared to the other modules (Figures S3A and S3B), which cannot be accounted for by enrichment of the HD Signaling Pathway alone (Figure 4C). Furthermore, 16 red module proteins (Figures S3C and S3D) are mutated in neurological disorders ranging from Frontotemporal dementia (Vcp) to Parkinson’s disease (Vps35).

Myc-NGL-2 and myc-NGL2∗ were both subcloned into the pEF-BOS

(Mizushima and Nagata, 1990) vector downstream of the elongation factor promoter. shNGL-2 was subcloned into the pSUPER/Neo vector (Oligoengine) downstream of the H1 promoter. The H1 promoter and shNGL-2 were then subloned into the PacI site of FCK(0.4)GW (a gift from Dr. Pavel Osten, Cold Spring Harbor Laboratory) lentiviral backbone upstream of the CamKII promoter, which contains a 0.4 kb fragment of mouse CamKII protomoter-driving MDV3100 mouse EGFP (Dittgen et al., 2004). FCK(0.4)GW was used as a control. NGL-2 deletion constructs were as follows: NGL2∗ΔLRR (aa 79–287 deleted from full-length mouse NGL2∗) and NGL2∗ΔPDZ (aa 1–648 of full-length mouse NGL2∗). NGL-2-GFP fusion was generated Selleck OSI744 by sequentially subcloning NGL-2 cDNA obtained from

Open biosystems (Thermo Fisher Scientific) in frame with GFP into the pEF-BOS vector downstream of the elongation factor promoter. All constructs were sequenced to verify integrity. NGL1(NGL2LRR) (originally termed pCA NGL1r123-mVenus) was a gift from Elena Seiradake and Alexandru Radu Aricescu. In situ hybridizations were performed as described (Pasterkamp et al., 1999), using 20 μm horizontal P7 and P14 rat brain cryosections. P28 mice were deeply anesthetized with isofluorane, decapitated, and brains were harvested, flash frozen, and stored at −80°C. Crude membranes were isolated by homogenizing each brain in 5 mL homogenization buffer (0.32 mM sucrose, 4 mM HEPES [pH 7.5], and protease inhibitors) using a Dounce homogenizer. Homogenate was spun at 3,000 rpm for 10 min at 4°C. Supernatant (S1) was collected and spun at 10,000 × g for 15 min at 4°C. Each pellet (P2) was resuspended in homogenization buffer and spun at 10,000 × g for 15 min at 4°C. Pellets (P2′) were lysed in RIPA buffer (150 mM NaCl, 20 mM Tris-HCl [pH 7.5], 1% Triton-X, 0.5 M EDTA, protease inhibitors) and rocked for 30 min at 4°C. Samples were centrifuged at 10,000 × g for 20 min at 4°C, and supernatant was removed and mixed with sample buffer

for analysis by western blot. Western blots were probed with mouse anti-NGL2 (Clone N50/35, NeuroMab) and Adenosine rabbit anti-βIII tubulin (Abcam). P14 WT and KO littermate mice were given a lethal dose of sodium pentobarbital and perfused with PBS, followed by 4% paraformalydehyde (PFA) in PBS. We cut 100 μm coronal sections with a vibrating microtome (Vibratome), then blocked them in PBS containing 3% bovine serum albumin and 0.2% Triton X-100 (Sigma) for 1 hr at room temperature, and then immunostained them using standard procedures. See Supplemental Experimental Procedures for more information. P7 mice were given a lethal dose of sodium pentobarbital and perfused with PBS, followed by 4% PFA in PBS. DiI crystals (Invitrogen) were placed in CA3 or EC of fixed brains.

To make sure that the addressed associations were specific for regular use, rather than for substance use in general, analyses were repeated comparing regular compound screening assay users to experimental or less regular users. At age 15–18, regular alcohol and cannabis use were reported by, respectively, 12.2% and 6.3% of the adolescents. Boys were more likely than girls to be regular users of alcohol (χ2 (2 df, N = 1192) = 16.16, p < .01) and

cannabis (χ2 (2 df, N = 1192) = 23.82, p < .001). Mean scores or percentages of the variables used are shown in Table 1. For descriptive purposes, we presented the mean of the unstandardized scores. Genotype frequencies of DRD2 and DRD4 are depicted in Table 2. Allele frequencies were calculated UMI-77 and analyzed for deviations from Hardy–Weinberg equilibrium (HWE) using χ2-tests. No deviations from HWE were detected (p = 0.31 for DRD2 and p = 0.94

for DRD4). Because of the very small number of regular alcohol and cannabis users with two copies of the genetic risk markers DRD2 A1 and DRD4 7R, subsequent analyses were performed comparing the individuals carrying at least one genetic risk factor with individuals carrying no genetic risk factor. This has also been done in many previous studies ( Conner et al., 2010, Conner et al., 2005, Sakai et al., 2007 and van der Zwaluw et al., 2009). The univariate analyses (not depicted in a Table) showed that the A1 allele of the DRD2 TaqIA polymorphism had no direct effect on regular alcohol (OR = 0.98, 95%CI = 0.57–1.70,

p = 0.95) or cannabis use (OR = 0.91, 95%CI = 0.52–1.61, p = 0.75). Similarly, L-DRD4 was not significantly related to regular alcohol (OR = 0.65, 95%CI = 0.37–1.11, Calpain p = 0.11) or cannabis use (OR = 0.79, 95%CI = 0.44–1.41, p = 0.43). DRD2 by parenting measure interactions did not yield any significant associations, indicating that rejection, overprotection, and emotional warmth did not modify the effect of the A1 allele of the DRD2 TaqIA polymorphism on regular alcohol or cannabis use (see Table 3). DRD4 by parenting measure interactions resulted in a significant interaction between DRD4 and emotional warmth. Regression analyses separate for S-DRD4 and L-DRD4 individuals indicated that a higher level of emotional warmth was associated with regular alcohol (versus irregular) consumption in carriers of the L-DRD4 (OR = 1.62, 95%CI = 1.12–2.33, p = 0.01). In S-DRD4 individuals, our findings pointed in the direction of an inverse association between emotional warmth and regular alcohol consumption, though this was not significant at p < 0.05 (OR = 0.84, 95%CI = 0.68–1.03, p = 0.09). Because adjusting for parental substance use might have ruled out part of the variance explained by genetic factors, analyses were repeated without adjusting for parental substance use. These analyses yielded comparable results.

, 2000, Takebayashi et al., 2000 and Zhou et al., 2000). OLIG2 knockout results in loss of the pMN domain and consequently complete absence of spinal MNs Dabrafenib supplier (Lu et al., 2002, Takebayashi et al., 2002,

Zhou and Anderson, 2002 and Park et al., 2002). All spinal OL lineage cells are lost as well because OLIG2 is required for OLP development regardless of whether they are generated within or outside of pMN (Lu et al., 2002, Takebayashi et al., 2002, Zhou and Anderson, 2002 and Park et al., 2002). In contrast, OLIG1 has a relatively mild impact on normal development (Lu et al., 2002; J.P.d.F., N. Kessaris, W.D.R., and H.L., unpublished data; but see Xin et al., 2005). However, OLIG1 is believed to be crucial for OL regeneration in demyelinating diseases such as multiple sclerosis (Arnett et al., 2004). The OLIG gene products are members of a large family of helix-loop-helix (HLH) transcription factors, which also includes proneural proteins Neurogenin1/2 (NGN1/2) and MASH1/ASCL1 as well selleck chemicals as cell

lineage regulators MYOD and NEUROD. OLIG2 interacts with different protein partners to regulate specific developmental processes. It can form heterodimers with NGN2 to control MN differentiation, and it can bind NKX2.2 to promote OLP generation and/or differentiation (Novitch et al., 2001, Zhou et al., 2001, Qi et al., 2001, Sun et al., 2003 and Lee et al., 2005). It can also complex with SOX10 or ZFP488 to regulate OLP differentiation and enhance myelin gene expression (Wang et al., 2006, Wissmuller et al., 2006 and Li et al., 2007). Given the central role of OLIG2 in both MN and OL development, we were keen to discover how this one transcription factor can specify two quite different

PAK6 cell types and especially how it participates in the MN-OLP temporal fate switch. We present evidence that OLIG2 controls the switch by reversible phosphorylation on Serine 147 (S147), a predicted protein kinase A (PKA) target; phosphorylation at this site is required for patterning of the ventral neuroepithelium and MN specification, whereas dephosphorylation favors OLP specification. S147 phosphorylation also causes OLIG2 to switch its preferred dimerization partner from OLIG2 (or OLIG1) to NGN2. We propose that this regulated exchange of cofactors is required for and triggers the MN-OLP fate switch. OLIG2 is rich in serine and threonine residues (50 serines and 14 threonines out of a total of 323 amino acids; see Table S1 available online), suggesting that it might possess multiple serine/threonine phosphorylation sites. To test this, we transfected a Myc epitope-tagged OLIG2 expression vector into Cos-7 cells, labeled the cells with [33P]phosphate, and analyzed radiolabeled OLIG2 proteins by immunoprecipitation (IP) with anti-Myc followed by polyacrylamide gel electrophoresis (PAGE). Two major radioactive OLIG2 bands were visible (Figure 1A).

In contrast, initial exploratory evaluation of a certain brain area frequently requires larger regions of inactivation, particularly when the relation of the area to behavior is tenuous. Small changes in behavior in such cases may go undetected, so a larger inactivation would probably be necessary to reveal the contribution of the structure to behavior. The limited size of the inactivation might well have contributed to the lack of any behavioral effect of channel rhodopsin injections made in monkey motor cortex (Diester et al., 2011). Optogenetic inactivation differs from chemical inactivation in having greater precision and added flexibility. With chemical inactivation, the effect on behavior is

dependent on the overlap of the area of neurons active in

generating the behavior (in our case a saccade) and the area of neurons inactivated by the chemical. With the optogenetic approach, however, there is http://www.selleckchem.com/products/ve-822.html a third gradient: the illumination from the optrode on targeted neurons. Essentially, the outcomes and interpretations of each optogenetic experiment are governed by the interaction of these three gradients. The effect of these gradients on behavior was interesting: size of the effect depended on the saccade’s distance from the optrode, and we would expect this factor to govern behavioral effects in any brain area. In addition, we found a systematic change in the direction of this shift that depended on the location of the injection. Gamma-secretase inhibitor 3-mercaptopyruvate sulfurtransferase Saccade related neurons are mapped on the intermediate layers of the SC as vectors pointing to different regions of the visual field. Activity during a saccade is the result of a large population of such neurons (Munoz and Wurtz, 1995), the average of whose vectors determines the generated saccade (Lee et al., 1988). In fact, the precision of the optogenetic method provides the most convincing evidence so far (Figure 3A) that shifts in saccade endpoints can be predicted if one knows the shift in the vector average resulting from inactivation. In our experiments, the shifts are most easily interpreted as the action of the injection gradient

and the light gradient acting on the SC neuronal vector gradient, as indicated by the analysis in Figures 3C–3E. Although chemical inactivation might well have a place in studying the brain, optogenetic techniques allow a new set of strategies with remarkable temporal and spatial precision, some of the principles of which we have illustrated here. Three adult male monkeys (OZ, OM, RO; Macaca mulatta) provided data for different aspects of these experiments. Monkeys weighed between 8 and 11 kg and had implanted scleral search coils for measuring eye position, had recording cylinders for accessing SC neurons, and had posts for immobilizing the head during experiments as described previously ( Sommer and Wurtz, 2000).

Unc5Ig1 binds to FLRT2LRR burying a total of ∼1,280 Å2 protein surface, which is highly sequence conserved on both sides (Figures 2C and 2D). Superposition of Unc5AIg12T with Unc5DIg1 as found in complex with FLRT2LRR generates a model in which the domains downstream of Unc5 Ig1 extend away from the interface with FLRTLRR, suggesting that the Ig1 domain is the only interacting domain (Figure 2E). Based on this model alone, we cannot rule out that the extracellular FLRT regions downstream of the LRR domain also interact with Unc5. However, in SPR experiments we measured similar Unc5-binding affinities for FLRTecto and

FLRTLRR constructs (data not shown), suggesting that there is no major second Unc5-binding site on FLRT. We provide further support for this conclusion using a mutagenesis AZD9291 clinical trial approach (see

next section). The core of the FLRT2-Unc5D-binding interface contains predominantly hydrophobic and positively charged residues (Figures 2F and 2G). The conserved FLRT2 histidine H170 forms a central anchor point that reaches deep into a hydrophobic pocket formed by Unc5D F82, K84, W89, V135, W137, Dasatinib cost and K144 and likely provides a hydrogen bond to Unc5D W137 (Figure 2G). FLRT2 R191 and L215 may stabilize this arrangement by providing additional contacts to Unc5D F82 and W137. The main residues forming the hydrophobic FLRT2-binding surface of Unc5D are fully conserved in Unc5B (Figure 2H), with the exception of F82, which is replaced by a tyrosine (Y78). The high the degree of sequence conservation at the FLRT-Unc5-binding interface is in agreement with the observed binding promiscuity. Subtle differences in binding affinities

for different homologs are likely due to sequence variations at the periphery of the binding interface (Figure 2I). Histidine residues have a side chain pKa(His) of ∼6, below which they are protonated. We predicted that the protonated FLRT2 H170 would be incompatible with binding to the hydrophobic binding pocket on Unc5D. Indeed, at pH ∼5.7, Unc5Decto does not interact with FLRT2ecto (Figure S2A). Based on the crystal structures, we designed mutations in the FLRT2-Unc5D interface to disrupt binding. In FLRT2 H170E and H170N, we replaced the central histidine with a negative-charged residue or an N-linked glycosylation site, respectively. Neither of these mutants binds Unc5D in our assays, confirming the binding site we describe is essential for the interaction (Figure 3A). Also, the Unc5D mutants E88A+W89A+H91A and W89N+H91T show poor binding to FLRT2 (Figure 3A). Binding was unaffected by FLRT2 and Unc5D mutations at sites involved in minor interactions in the crystal (FLRT2 D248N+P250T, Unc5D L101N+E103T), suggesting that these sites are not physiologically relevant (Figure 3A). For subsequent functional analysis we chose the non-Unc5-binding FLRT2 mutant H170N and the non-FLRT2-binding Unc5D mutant W89N+H91T.

, 2006, Howarth and Attwell, 2006 and Paulson and Newman, 1987). However, the current generated by glutamate transport is small compared to that generated by astrocytic K+ uptake at the synaptic cleft in olfactory glomeruli (De Saint Jan and Westbrook, 2005), and it seems unlikely that the blockade of a comparatively small current would reduce functional hyperemia as much as observed. Second, CBF may increase as a result of metabolic activation induced by glutamate uptake. Sodium/glutamate

cotransport consumes energy for the restoration of the ionic gradient by the Na+-/K+-ATPase, and for the conversion of glutamate to glutamine. While the contribution of these processes to the brain’s energy

budget is small (Attwell and Laughlin, 2001), glutamate Vorinostat research buy uptake into astrocytes also directly initiates astrocytic nonoxidative glycolysis and lactate release (Pellerin, 2005). Lactate itself may initiate vasodilation (Gordon et al., 2008), but it is also possible that sodium ions cotransported into astrocytes with glutamate may trigger a vasoactive click here pathway. Sodium ions shuttled into astrocytes by this cotransport propagate as interastrocytic sodium waves in cell cultures (Bernardinelli et al., 2004), and they have also been shown to couple synaptic activity and astrocytic nonoxidative glucose consumption (Voutsinos-Porche et al., 2003). This stimulation of nonoxidative glycolysis in astrocytes is thought to underlie the disproportionate rise of CBF and glucose compared to a smaller increase in oxygen consumption—the mismatch that forms the basis of functional brain imaging (Magistretti and Pellerin, 1999). Therefore, glutamate transport into astrocytes may simultaneously activate functional hyperemia and nonoxidative glycolysis in astrocytes, and may contribute to the high temporal and spatial correlation of

CBF increase and glucose consumption observed in functional brain imaging (Raichle and Mintun, 2006). Advances in imaging and cellular manipulation may be harnessed to overcome the oxyclozanide uncertainties regarding the role of astrocytic molecular agents in functional hyperemia. Optical imaging during physiological activity can, in principle, be extended to any small molecule for which there is an appropriate fluorescent indicator (Zhang et al., 2002). Genetic manipulations may also be valuable, particularly if the perturbations can be performed in a cell-type-specific and temporally precise manner (Kennedy et al., 2010). Methods to stimulate or downregulate the expression of genes, such as those for glutamate transporters, specifically in mature astrocytes are increasingly becoming available (Colin et al., 2009).

99 ± 0.05; Spine 2, 0.96 ± 0.11; dendrite, 1.05 ± 0.04; Figures 4E, 4H, and 4L). To address how NLG1 cleavage affects synaptic function, we developed a system to acutely and selectively cleave NLG1 on demand (Figure 5A; Movie S1). For this, we inserted

the thrombin (Thr) proteolytic Ku-0059436 in vivo recognition sequence LVPRGS into the stalk domain of NLG1 downstream of the dimerization domain, replacing the endogenous sequence TTTKVP. In these experiments, the NLG1ΔA splice variant lacking splice site A was chosen, due to its stringent partitioning into excitatory synapses (Chih et al., 2006). This Thr-cleavable mutant (GFP-Thr-NLG1) localized to synapses in a manner indistinguishable from wild-type GFP-NLG1 in DIV21 hippocampal neurons (Figures 5A–5D). Incubation with 5 U/ml Thr for 30 min resulted in rapid and extensive reduction of GFP-Thr-NLG1 synaptic fluorescence (fractional fluorescence

remaining; 0.20 ± 0.03; Figures 5A–5G; Movie S1). Control neurons Dabrafenib molecular weight transfected with GFP-NLG1 lacking a Thr recognition sequence exhibited no change in GFP fluorescence upon Thr treatment (fractional fluorescence remaining; 0.99 ± 0.02; Figure 5H; Movie S2 right panel), indicating that the reduced GFP fluorescence was not due to photobleaching. To determine how acute NLG1 shedding affects postsynaptic morphology and integrity, we cotransfected neurons with mCherry (mCh) or PSD95-mCh and compared fluorescence changes after 30 min of Thr incubation. No significant changes in spine volume measured by the mCh cell fill (mCh fluorescence ratio post/prethrombin; 1.04 ± 0.03; Figures 5A and 5F; Movie S1) or Adenosine PSD95-mCh puncta intensity (PSD95-mCh fluorescence ratio post/prethrombin; 0.97 ± 0.02; Figures 5B and 5F) were detected after Thr treatment. To test whether acute cleavage of NLG1 regulates presynaptic NRX1β, we sequentially transfected neuronal cultures with GFP-Thr-NLG1 and NRX1β-mCh lacking splice site 4, obtaining

distinct populations of neurons expressing each construct. This approach generated pre- and postsynaptic pairs labeled with NRX1β-mCh and GFP-Thr-NLG1, respectively. At dually labeled synapses, acute Thr treatment caused a rapid and pronounced decrease in NRX1β-mCh fluorescence (Figures 5E–5G; Movie S2; fractional fluorescence remaining: 0.51 ± 0.04). Further analysis of the kinetics of GFP-Thr-NLG1 and NRX1β-mCh level at a higher sampling rate revealed that loss of both proteins occurs in tandem (Figures S5A–S5D). Importantly, there was no detectable change in presynaptic synaptophysin-mCh signal intensity (fractional fluorescence remaining: 0.99 ± 0.08) under similar conditions (Figures 5D and 5F), indicating that destabilization of NRX1β by NLG1 cleavage is specific and not due to indirect structural changes in presynaptic terminals. This was further confirmed by immunolabeling of the vesicular glutamate transporter VGLUT1, which was unaffected by acute NLG1 cleavage (Figures S5E–S5G).

Some of these cells undergo two waves of migration—one rostrally to form the nucleus

posterior limitans (PLi) and the lateral habenula selleck chemicals (LHa) and the other ventrally into the ventral LGN which is largely populated by Dlx-expressing cells. The expression of Dlx in the vLGN offers a repulsive signal to the invading Sox14 cells, thus ensuring only a few Sox14 cells populate the vLGN. In the absence of Dlx1/2 this repulsive signal is abolished leading to mass migration of Sox14 cells into the vLGN turning it into an IGL phenotype territory ( Figure 1A). The Sox14-deficient mice in which the Sox14 coding sequence is replaced by GFP show no significant changes in the overall organization of the SVS except for the redistribution of cell populations in the IGL/vLGN region. The presumptive Sox14 cells fail to migrate ventrally to partially populate the vLGN and remain in the IGL region (Figure 1A), increasing their density. Sox14 appears to be dispensable for expression

of Gad1 and NPY suggesting the Sox14-deficient cells are still neurotransmitter competent. An increased density of Sox14-GFP cells in the IGL also correlated with increased immunoreactivity for NPY. Taken together, the combined function of Sox14 and Dlx define the spatial distribution of Sox14-expressing cells in the IGL and vLGN region of the SVS. The loss of Sox14 leads to changes in the regional distribution of Sox14 cells and consequently nearly potential changes in the pattern and strength of connectivity of IGL with INCB024360 purchase target regions. To assess the functional consequence of the loss of Sox14 expression, Delogu et al. (2012) used a suite of behavioral tests. Intact pupillary light reflex and

light induction of c-Fos in the SCN implied normal ontogeny and projection of the ipRGCs in these mice. The circadian activity rhythm under constant darkness also showed normal periodicity, thus indicating no gross defect in the endogenous SCN clock. However, the Sox14 knockout mice showed profound alteration in activity-rest pattern. Several studies have demonstrated the IGL participates in at least three different aspects of daily arousal-rest pattern: overall activity level, entrainment of the circadian clock, and light suppression of activity or masking. Since the loss of Sox14 affects the cellular composition of the IGL and consequently changes the circuitry, the mice show remarkable defects in all three aspects of activity regulation. Increased number of functionally active NPY-positive cells in the IGL and potentially increased NPY-mediated signaling is opposite to IGL lesion resulting in reduced activity (Redlin et al., 1999). Overall, the Sox14 knockout mice appear to exhibit increased basal activity. Light is known to enhance NPY release from the GHT at the SCN, thereby driving early morning NPY release near the SCN (Glass et al., 2010).

We here ask: What is the consequence of HCN1 deletion www.selleckchem.com/products/chir-99021-ct99021-hcl.html for hippocampal place cell firing and place field properties? Do any changes evident in the hippocampus reflect changes in the EC inputs to hippocampus, or are they a result of changes intrinsic to the CA1 or CA3 place cells? We find changes in CA1 and CA3 place cell properties consistent with the observed changes in the EC grid cell

inputs to hippocampus. In addition, we observe a significantly greater change in the response properties of the CA1 neurons consistent with an intrinsic change in CA1 pyramidal cell firing and synaptic plasticity. These results demonstrate how alterations in encoding of the spatial environment by the EC and its transformation in the hippocampus may contribute to changes in long-term spatial memory.

Moreover, these results suggest the interesting possibility that the fine accuracy of spatial encoding and spatial memory storage may be separable. We obtained extracellular recordings using multiple tetrodes and compared firing properties of hippocampal CA1 and CA3 pyramidal neurons in forebrain-restricted HCN1 knockout mice (KO) to those in control littermates (CT) (Nolan et al., 2003 and Nolan et al., 2004). We focused on these two populations of hippocampal neurons because the CA3 neurons express HCN1 weakly KU-55933 ic50 (Santoro et al., 2000) and therefore should not be affected directly by the knockout, whereas the CA1 neurons strongly express HCN1 channels, which normally constrain the ability of the direct layer Florfenicol III EC inputs to excite CA1 neurons. Before examining place cell properties in vivo, we first directly examined the influence of HCN1 in CA3 neurons in acute hippocampal slices, which has not been previously characterized. Whole-cell current clamp recordings indicate that HCN1 plays little direct role in regulating CA3 electrophysiological properties, consistent with previous voltage-clamp results showing that CA3 neurons had little Ih (Santoro et al., 2000). Because of

potential voltage clamp artifacts in slice patch clamp recordings, we assayed Ih in CA3 neurons under current clamp conditions by measuring the voltage sag in response to a hyperpolarizing current injection, a characteristic property of the activation of Ih. In contrast to the large sag in CA1 neurons, the sag in control CA3 neurons was minute, with a sag ratio of only 0.99 (see Experimental Procedures), compared to a typical sag ratio of 0.7 in CA1 (Chevaleyre and Siegelbaum, 2010). The small sag in CA3 neurons was abolished in the KO mice (CT = 0.988 ± 0.001, n = 14; KO = 1.002 ± 0.001, n = 15, p < 0.001). Consistent with a minor role for HCN1 in CA3, HCN1 deletion caused no significant change in CA3 pyramidal neuron resting potential (CT = −73.6 ± 0.8 mV; KO = −73.3 ± 1.4 mV; p = 0.820), input resistance (CT = 133.2 ± 11.3 MΩ; KO = 129.7 ± 9.7 MΩ, p = 0.818), membrane time constant (CT = 21.0 ± 1.1 ms, KO = 21.6 ± 1.8 ms, p = 0.