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Both the neuregulin 1 (Nrg1) and α7 nicotinic acetylcholine receptor (α7*nAChRs) genes have been linked to schizophrenia and associated sensory–motor gating deficits. The prominence of nicotine addiction in schizophrenic patients is reflected in the normalization of gating deficits by nicotine self-administration. To assess the role of presynaptic type III Nrg1 at hippocampal–accumbens synapses, an important relay in sensory–motor gating, we developed a specialized preparation of chimeric circuits in vitro. Synaptic relays from Nrg1tm1Lwr heterozygote ventral hippocampal slices to wild-type (WT) nucleus accumbens neurons (1) lack a sustained, α7*nAChRs-mediated phase of synaptic potentiation seen in comparable WT/WT circuits and (2) are deficient in targeting α7*nAChRs to presynaptic sites. Thus, selective alteration of the level of presynaptic type III Nrg1 dramatically affects the modulation of glutamatergic transmission at ventral hippocampal to nucleus accumbens synapses.
Neuregulin 1 (Nrg1)–ErbB signaling regulates synapse formation, synaptic plasticity, and the maintenance of synaptic connections, in part by regulating the levels of functional neurotransmitter receptors (Yang et al., 1998; Huang et al., 2000; Wolpowitz et al., 2000; Liu et al., 2001; Kawai et al., 2002; Falls, 2003; Okada and Corfas, 2004; Gu et al., 2005; Kwon et al., 2005; Chang and Fischbach, 2006; Bjarnadottir et al., 2007; Li et al., 2007). The implication of Nrg1 as a schizophrenia susceptibility gene underscores the importance of understanding the relationship between Nrg1 signaling and circuits affected in schizophrenia (Stefansson et al., 2004; Harrison and Weinberger, 2005).
The majority of patients with schizophrenia are heavy smokers, consistent with proposed roles of nicotine as a form of self-medication (Batel, 2000; Kumari and Postma, 2005; Strand and Nybaäck, 2005). Nrg1–ErbB signaling has been implicated in the regulation of neuronal nicotinic acetylcholine receptors (nAChR), in particular the α7*nAChRs (Yang et al., 1998; Liu et al., 2001; Kawai et al., 2002; Chang and Fischbach, 2006; Mathew et al., 2007; Hancock et al., 2008), renowned for their role in nicotine-induced plasticity of corticolimbic and mesolimbic circuits (McGehee et al., 1995; Dajas-Bailador and Wonnacott, 2004; Jo et al., 2005; Mansvelder et al., 2006; Couey et al., 2007). Because genetic studies have linked both the Nrg1 and α7 subunit genes to major endophenotypes of schizophrenia (Leonard et al., 1998; Harrison and Weinberger, 2005; Mathew et al., 2007), we tested whether reduced expression of type III Nrg1 alters nicotine responsiveness in the ventral striatum, specifically in the nucleus accumbens shell (nAcc), in which convergent inputs from pre-frontal cortex, ventral hippocampus/subiculum (vHipp), ventral tegmental area, and amygdala are integrated to produce context-informed volitional behaviors (Lisman and Grace, 2005; Ronesi and Lovinger, 2005). We demonstrate that presynaptic type III Nrg1 determines normal levels of presynaptic targeting of α7*nAChRs along axons of ventral hippocampal neurons.
Animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The region of ventral CA1 and subiculum of hippocampi from single wild-type (WT) animals or animals heterozygous for an isoform-specific disruption of type III Nrg1 (Nrg1tm1Lw +/−) (Wolpowitz et al., 2000) were sliced into 150 × 150 μm pieces and plated in minimal volume of culture media (50 μl). Dispersed WT nAcc neurons (embryonic day 16 to postnatal day 1) were added after the vHipp explants had attached. Additional details of the specialized technique developed for these studies can be found in the legend of Figure 1 and in supplemental data (available at www.jneurosci.org as supplemental material).
For α-bungarotoxin (αBgTx) labeling, the coverslips were incubated with αBgTx conjugated to Alexa 594 (1:1000; Invitrogen) for 30 min at 37°C before fixation. For standard immunodetection, coverslips were fixed in 4% paraformaldehyde/4% sucrose/PBS (20 min, 4°C), treated with 0.25% Triton X-100/PBS (5 min at room temperature) and 10% preimmune donkey serum in PBS (30 min at room temperature), and then incubated in primary antibody for 2 h and secondary antibody for 1 h at 37°C. Antibodies used included the following: anti-vesicular glutamate transporter 1 (vGluT1) 1:250; Synaptic Systems), anti-GAD65 (1:50; Developmental Studies Hybrid-oma Bank), and FITC- and rhodamineconjugated Ig (1:150 to 1:200; Jackson ImmunoResearch). αBgTx clusters (defined as six contiguous pixels at 50% of maximal intensity) were quantified using a custom algorithm with MetaMorph software (version 7.1; Molecular Devices).
Macroscopic and synaptic currents were recorded by whole-cell configuration of the patch-clamp technique, with cells held at −60 mV. Preparations were continuously perfused with extracellular solution containing the following (in mm): 145 NaCl, 3 KCl, 2.5 CaCl2, 10 HEPES, and 10 glucose, pH 7.4. The intracellular solution included the following (in mm): 3 NaCl, 150 KCl, 1 MgCl2, 1 EGTA, 10 HEPES, 5 MgATP, and 0.3 NaGTP, pH 7.2. Voltage-clamp recordings were performed with a List EPC-7 Patch Clamp Amplifier (Medical Systems). CNQX, AP-5, bicuculline (Tocris Cookson), αBgTx, and TTX (Sigma) were included in the perfusate as noted. (−)-Nicotine (hydrogen tartrate salt) and glutamate were applied via local pressure ejection (Picospritzer; General Valve).
Macroscopic and synaptic currents were filtered at 10 kHz with a eight-pole Bessel filter (direct current amplifier/filter; Warner Instruments) before acquisition and digitization through a DigiData 1200B analog-to-digital interface with pClamp8 (Molecular Devices). Peaks of macroscopic currents were determined by pClamp8 (Fetchan), and decay time constants were calculated with Origin; Microcal Software). Spontaneous synaptic currents, amplitude, rise time, half-decay time, and frequency of miniature EPSCs (mEPSCs) were measured with MiniAnalysis (Synaptosoft). Normally distributed data were assessed for statistical significance by ANOVA with a post hoc test for multiple comparisons and group means with unequal sample size. Non-normally distributed data were analyzed using nonparametric methods (Kolmogorov–Smirnov test).
We developed a specialized preparation of hippocampal–striatal circuits in vitro to study the effects of genetic manipulation of presynaptic neurons in mouse CNS synapses (Fig. 1). vHipp and subicular regions were extirpated from WT or Nrg1tm1Lwr +/− mice (Fig. 1A). Microexplants were plated in minimal volume and allowed to spread [WT (Fig. 1C, c1), +/− (Fig. 1D, d1)] before the addition of dispersed target neurons from the nucleus accumbens shell (Fig. 1B). We focused our analysis on the role of type III Nrg1 in the presynaptic vHipp projections in regulating plasticity at hippocampal–striatal synapses by keeping the nAcc genotype (WT) constant and varying the genotype of the vHipp slices.
The general features of chimeric Nrg1tm1Lwr +/− preparations were indistinguishable from those of sibling cocultures from WT mice. The overall profile of hippocampal glutamatergic fiber outgrowth (vGluT+ fibers), the number of vGluT+ puncta along vHipp axons, the survival of nAcc neurons (GAD65+), and the percentage of nAcc neurons that received synaptic input within 1 week were found to be independent of the presynaptic genotype (Fig. 1E–G).
Patch-clamp recording from contacted WT nAcc neurons after 4−7 d in vitro revealed ongoing glutamatergic (microslice-derived) and GABAergic (nAcc to nAcc) synaptic activity, whether the ventral hippocampal slice was derived from WT or from Nrg1tm1Lwr +/− mice (see Figs. 1G, ,2,2, ,3).3). Glutamatergic miniature postsynaptic currents (Glu mEPSCs) were recorded in the presence of bicuculline (20 μm) and TTX (2 μm), and Glu mEPSCs were blocked by application of CNQX (10 μm) and APV (50 μm).
Continuous recording of glutamatergic transmission at +/+ to +/+ synapses during and after a brief exposure to a low concentration of nicotine (1 min, 100−500 nm) revealed a sustained (>30 min) enhancement of transmission (Fig. 2). The frequency of Glu mEPSCs increased 2.8 ± 0.3-fold (from 3−4 to 8−14 Hz; n = 8) when nicotine was applied. The initial increase in Glu mEPSC frequency was followed by a sustained, 2.0 ± 0.1-fold increase above the pre-nicotine Glu mEPSC frequency (Fig. 2A–C). The nicotine-induced enhancement of glutamatergic synaptic transmission was partially blocked by nAChR subtype-selective antagonists and completely blocked by general nicotinic antagonists (e.g., mecamylamine; data not shown). Most notably, pretreatment with the α7*nAChRs-selective antagonist αBgTx eliminated the sustained enhancement of glutamatergic transmission but left the transient enhancement of transmission by nicotine intact (Fig. 2B). Brief application of nicotine also resulted in sustained, focal increases in [Ca2+]i along vHipp axons (Fig. 2D) (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). The sustained, nicotine-induced increases in presynaptic [Ca2+]i were blocked by αBgTx but not by dihydro-β]-erythroidine (DHβE). A non-α7nAChRs agonist (5-Iodo-A-85380) did not elicit a sustained increase in presynaptic [Ca2+]i.
We next examined the effects of nicotine on glutamatergic transmission at synapses between Nrg1tm1Lwr +/− vHipp and +/+ nAcc neurons. The magnitude of the rapid nicotine-induced facilitation detected at chimeric synapses was comparable with that detected at +/+ to +/+ synapses (Fig. 3A–C). However, at chimeric synapses, the nicotine-induced synaptic facilitation was short-lived (Fig. 3A–C), returning to control levels immediately after washout of nicotine (Fig. 3B). The nAChR-mediated enhancement of glutamatergic transmission at chimeric synapses was insensitive to the α7*nAChRs-selective antagonist αBgTx (Fig. 3B).
The brief nature of the nicotine-induced enhancement of glutamatergic transmission at chimeric synapses was paralleled by a transient, rather than a sustained, increase in presynaptic [Ca2+]i (Fig. 3D). Pooled data summarizing the effect of presynaptic, monoallelic deletion of type III Nrg1 on the modulation of hippocampal glutamatergic transmission and on presynaptic [Ca2+]i are presented in Figure 3D and supplemental Figure 2 (available at www.jneurosci.org as supplemental material). In chimeric circuits, the sustained, αBgTx-sensitive component of nicotine-enhanced transmission was abolished, whereas the transient effects on both glutamate release and Ca2+ signaling were preserved. These data are consistent with a selective loss of functional α7*nAChRs at presynaptic sites along Nrg1tm1Lwr +/− vHipp axonal projections, without loss of non-α7*nAChRs that support transient responses to nicotine.
To assess whether the loss of α7nAChRs response at type III Nrg1 chimeric synapses was attributable to decreased axonal α7nAChRs expression, we measured α7nAChRs levels in vHipp explants and along vHipp projections. Analysis of hippocampal axons revealed >70% decrease in the fraction of vGluT+ axons that colabeled with αBgTx in Nrg1tm1Lwr +/− vHipp to WT nAcc compared with +/+ vHipp to +/+ nAcc cocultures (Fig. 4A,B). Total α7 protein levels in vHipp microslices from Nrg1tm1Lwr +/− mice were ~40% lower than levels in WT slices (Fig. 4D). These results indicate that WT levels of type III Nrg1 signaling are required for expression of functional pre-synaptic α7*nAChRs.
Type III Nrg1 functions as a bidirectional signaling molecule (Bao et al., 2003; Hancock et al., 2008). To test the possibility that axonal type III Nrg1, acting as a receptor, regulates α7*nAChRs levels along axons, we treated vHipp microslices with the extracellular domain of ErbB4 (B4-ECD, 2 nm) for 1, 6, or 24 h. We visualized α7*nAChRs present on the surface of vHipp axons by staining live preparations with labeled αBgTx (red) before fixation. When vHipp microslices from Nrg1tm1Lwr +/− animals were treated with B4-ECD for 6 or 24 h (but not after 1 h), levels of α7*nAChRs clusters at glutamatergic synapses increased from ~12 per 100 μmto ~40 per 100 μm axon length, a level comparable with that seen in +/+ microslices (treatment of +/+ vHipp microslices with B4-ECD increased the number of α7*nAChRs clusters from ~30 clusters/100 μmto ~40 clusters/100 μm) (Fig. 4B). Microslices from Nrg1tm1Lwr −/− animals did not respond to B4-ECD treatment (supplemental Fig. 3D, available at www.jneurosci.org as supplemental material). To determine whether the B4-ECD-induced increase in α7*nAChRs resulted from recruitment of preexisting intracellular pools, we repeated the B4-ECD treatment of +/+ and +/− microslices in the presence of cycloheximide (CHX) for 6 h. CHX treatment alone did not affect levels of αBgTx staining but eliminated the B4-ECD-induced increase in α7*nAChRs levels (Fig. 4C). B4-ECD treatment of vHipp micro-slices from Nrg1tm1Lwr +/− animals also restored the ability of these neurons to mount a sustained elevation of intracellular calcium in response to brief exposure to nicotine (Fig. 3D). Thus, increased type III Nrg1 back-signaling in Nrg1tm1Lwr +/− vHipp microslices restored functional axonal α7*nAChRs to WT levels.
Using an in vitro microslice preparation that permits examination of CNS synapses comprising genetically distinct presynaptic versus postsynaptic neurons, we demonstrate that type III Nrg1 is required for nicotine-induced sustained potentiation of glutamatergic transmission at hippocampal–accumbens synapses. The persistent phase of glutamatergic facilitation, which lasts up to 1 h after a single, 1 min exposure to 100 nm nicotine, is mediated by presynaptic α7*nAChRs. Decreased expression of presynaptic type III Nrg1 results in an ~80% reduction in functional α7*nAChRs on axonal surfaces, as assessed by αBgTx staining and nicotine-elicited changes in axonal [Ca]i. Incubation of vHipp microslices with recombinant B4-ECD increased the levels of surface α7*nAChRs along glutamatergic projections from WT vHipp microslices and restored levels of surface α7*nAChRs along glutamatergic projections from +/− vHipp microslices. Whether the increase in surface α7*nAChRs reflects a specific effect of ErbB4/Nrg1 signaling on the α7 subunit per se or is secondary to more general response of α7*nAChRs-expressing vHipp projection neurons is not clear at this time. We propose that presynaptic type III Nrg1 is required for the normal levels of expression and axonal targeting of α7*nAChRs.
Expression and somatodendritic trafficking of α7*nAChRs is regulated by Nrg1/ErbB and neurotrophin/Trk signaling (Yang et al., 1998; Liu et al., 2001; Kawai et al., 2002; Chang and Fisch-bach, 2006; Massey et al., 2006; Hancock et al., 2008). Our current results are distinct from previous studies in that the requirement for type III Nrg1 is cell autonomous, i.e., presynaptic type III Nrg1 regulates presynaptic α7*nAChRs. Type III Nrg1 isoforms have the capacity to participate in bidirectional, juxtacrine signaling that involves both transcriptional responses and local signaling in axons (Bao et al., 2003; Hancock et al., 2008). We propose that the α7*nAChRs is a target of both forward signaling downstream of activated ErbB receptors and reverse signaling. Within the hippocampus, Nrg1/ErbB signaling regulates levels of α7*nAChRs on interneurons (Liu et al., 2001; Chang and Fischbach, 2006). We now demonstrate that type III Nrg1 reverse signaling regulates α7*nAChRs expression and targeting to ventral hippocampal axonal projections. In this manner, Nrg1/ErbB signaling affects cholinergic modulation within hippocampal circuits as well as cholinergic modulation of hippocampal output.
The chimeric in vitro preparation from Nrg1tm1Lwr mice described here provides an informative approach for studying the role of Nrg1 signaling in both presynaptic and postsynaptic mechanisms of synaptic plasticity. The modulatory influence of ACh on ventral striatal circuits involves both muscarinic and nicotinic receptors, as well as presynaptic and postsynaptic mechanisms (Ge and Dani, 2005; Wang et al., 2006). Current findings support the proposal that genetic modifications of Nrg1-mediated signaling in presynaptic inputs changes the presynaptic profile of nAChRs and thereby alters the temporal profile of responses to nicotine. Alterations in this temporal profile might lead to deficits in sensory gating by altering glutamatergic transmission in corticostriatal circuits (supplemental Fig. 4, available at www.jneurosci.org as supplemental material). In particular, glutamatergic transmission from vHipp to nAcc is thought to be involved in the regulation of sensory gating or prepulse inhibition (PPI), and PPI deficits are a common endophenotype of schizophrenia. Self-administration of nicotine might represent a means of coping with the altered temporal response to nicotine and might underlie the ameliorating effect of nicotine administration on PPI deficits as proposed previously (Bast and Feldon, 2003; Zornoza et al., 2005).
Genotype-Specific vHipp-nAcc Synaptic Co-cultures
All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80−23, revised 1996). Dispersed nAcc neurons from WT mice (C57BL/6J) were added to vHipp microslices (~150 μm3) plated the prior day onto poly-D-lysine/laminin-coated glass coverslips (BD Sciences, Bedford, MA). The vHipp slices originated from either a single WT or heterozygous Nrg1tm1Lwr animal. The region of ventral CA1 and subiculum were dissected and further sliced into 150×150 μm pieces, and plated in minimal volume of culture media (50 μl) to facilitate attachment. After the microslices settled (1−3 hours at 37°C), 100 μl of media was added. nAcc neurons (ED16 – P1) were dispersed with 0.25% trypsin (GIBCO, Grand Island, NY) for 15 min at 37°C, followed by gentle trituration in plating media (Neurobasal, B-27 (GIBCO, Grand Island, NY) and 20 ng/ml brain-derived neurotrophic factor (R&D Systems, Minneapolis, MN)). Dispersed nAcc neurons were added to the vHipp explants at 0.25 ml/coverslip. Co-cultures were maintained in a humidified 37°C, 5% CO2 incubator.
Ventral hippocampal microslices were loaded with 5 μM Fluo-3 (AM ester, Molecular Probes) in HEPES buffered saline (HBS), 0.02% Pluronic® F-127 (Molecular Probes) for 30 min. at 37°C and 5% CO2. The Fluo-3 solution then was replaced with HBS and the explants were allowed to recover for at least 30 min at 37°C / 5% CO2. The effect of nicotine on wild type and Nrg1tm1Lwr heterozygous vHipp axonal [Ca2+]i, was monitored with a Zeiss LSM 510 NLO multiphoton confocal microscope (excitation wavelength = 488 nm with laser on half power). Culture media was supplemented with 50 μM D-APV, 20 μM CQNX, 10 μM bicuculline, 2 μM TTX to eliminate contribution of other pre and postsynaptic receptors and channels. One minute after acquiring the first image (which served as the resting baseline), 1 μM nicotine was added and images were acquired every 2 minutes over a 30 minute period (using a 63×/0.90 NA water immersion objective, pixel-time = 3.20 μs, 512×512 pixels, the pinhole set on 1 airy unit).
To assess the relative contribution of α7*nAChR vs. non-α7*nAChRs to sustained increases in axonal [Ca2+]i, cultures were maintained under continuous perfusion (0.5 ml/min) in an imaging chamber (Live Imaging Services, Olten Switzerland) mounted on a Olympus IX81 DSU (spinning disk confocal) microscope (Olympus America Inc., Center Valley, PA). Media contained 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES pH7.4, 10 mM glucose with addition of 2 μM TTX (Tocris), 10 μM bicuculline (Tocris), 50 μM D-APV (Tocris) and 20 μM CNQX (Tocris). Z-stack images were collected every 5s for the first 5 min and then at one minute intervals for an additional 30 min. After 1 minute of baseline data collection, 1 μM nicotine was applied by local pressure ejection for 1 minute. The contributions of different nAChRs were assessed by including either 100 nM αBgTx (Molecular Probes) to block α7* nAChRs, 1μM dihydro-β-erythroidine hydrobromide (Dhβe; RBI) to block (αβ)*nAChRs, in perfusion media, or by substituting 10 nM 5-Iodo-A-85380 (Tocris) a (αβ)*nAChRs specific agonist for nicotine.
Raw fluorescence images were analyzed in Metamorph. Regions of interest (ROIs) with a diameter of 3 μm were drawn along axons and integrated intensity within these areas was then calculated at each time point. Fluorescence data are displayed as the change in integrated fluorescence intensity divided by the resting fluorescence (i.e. ΔF/F0) resulting in a normalized integrated intensity. Data were analyzed further using Excel and Matlab. Data are plotted in boxplots where the boxes include data points between the twenty-fifth percentile (bottom line) and the seventy-fifth percentile (top line). The middle line indicates the fiftieth percentile. Vertical lines mark the fifth and ninety-fifth percentiles.
vHipp tissue was dispersed in hot SDS sample buffer (2.5% SDS; 0.125 M Tris pH 6.8, 10% 2-mercaptoethanol) with a mini-pestle and then boiled for 5 min. Lysates were separated on 10% SDS-PAGE gels and transferred to nitrocellulose. Filters were blocked in 5% BSA / 0.1% Tween-20 / 10 mM Tris-HCl pH 7.5 / 150 mM NaCl and probed with anti-α7 nicotinic acetylcholine receptor (4 μg/ml; M-220 Sigma RBI), washed and probed with horseradish peroxidase conjugated anti-mouse IgG (1:10,000, Amersham). Immunoreactivity was detected by enhanced chemiluminescence.
Supplemental Figure 1: A: Changes in vHipp axonal [Ca2+]i elicited by nicotine application are indicated on pseudo color scale at different time points following nicotine (1 μM) application in fluo-3-loaded WT vHipp axons. White arrowheads highlight axonal regions affected by nicotine, black arrowheads indicate non-responsive regions.
B: Relative changes in [Ca2+]i at the sites indicated by the white (open) and black (closed) arrows; note that nicotine responsive and nicotine non-responsive regions of axon include areas that are initially at a range of [Ca2+]i. Only the former regions (white arrowheads / open symbols) were included in the analyses shown in Figures 2 and and33.
Supplemental Figure 2: Summary of data pooled from 8 separate experiments examining the αBgTx sensitivity of nicotine enhanced transmission at +/+ vHipp to +/+ nAcc compared with +/− vHipp to +/+ nAcc synapses. The acute effects of nicotine on facilitation of GluR-mediated synaptic currents were comparable in magnitude (open bars) and in αBgTx insensitivity (black bars) for +/+ to +/+ vs. +/− to +/+ synapses. In contrast the sustained, αBgTX-sensitive effect of nicotine that was seen 30 minutes after nicotine treatment at +/+ to +/+ synapses was not detected at +/− to +/+ synapses. Data are expressed relative to control (pre nicotine) levels of Glu mEPSC frequency.
Supplemental Fig. 3: A: Pre-treatment of vHipp cultures with unlabeled αBgTx (100 nM) completely blocked αBgTx-Alexa-594 (red) labeling of surface α7* nAChR along vGluT (green) expressing axons.
B: αBgTx (red) did not stain glutamatergic projections (vGlut, green) from vHipp explants from α7 nAChR −/− mutant mice.
C: vHipp explants from +/+ mice were cultured and were co-labeled with Type III Nrg1 (red) and vGluT (green) by indirect immunofluorescence.
D: vHipp axons from Nrg1tm1Lwr −/− mice were stained with αBgTx (red) and vGluT (green) after a 24 hr treatment with either B2-ECD or B4-ECD. Little αBgTx staining was seen along B2-ECD treated axons and there was no response to B4-ECD treatment.
E. Immunoblot detection of α7*nAChR subunit protein in extracts of whole brain lysates from P0 α7 WT (+/+) vs α7 KO (−/−) mice. No α7 protein was detected in whole brain lysates from α7 (−/−) mice, which confirmed the specificity of the indicated bands in Fig 3.
Supplemental Fig. 4: Model of proposed requirement for higher frequency administration of nicotine to achieve sustained information gating in individuals with one mutant allele of Type III Nrg1. Top: Theoretical response profile of an individual with normal levels of expression of Type III Nrg1 in hippocampus demonstrates sustained enhancement of response (e.g. sensorimotor gating) following nicotine administration. Bottom: Proposed response profile of an individual with reduced levels of expression of Type III Nrg1 in hippocampus illustrates the requirement for repeated nicotine administration to achieve normal level of sustained response enhancement.
This work was funded by Grants NS29071 and DA019941 from National Alliance for Research on Schizophrenia and Depression (Sidney Baer Distinguished Investigator Award to L.W.R.) and the McKnight Foundation (L.W.R.). M.H. was supported by National Institutes of Health Grant T32 DK07328. We thank Drs. S. Siegelbaum, Y. H. Jo, and M. Johnson for suggestions on previous versions of this manuscript.