, 2005, Andretic et al., 2008 and Crocker and Sehgal, 2010). Perhaps the greatest potential of Drosophila for understanding the regulation and function of sleep, however, resides KU-55933 molecular weight in employing forward genetic screens to identify genes that regulate sleep and wakefulness. Previous screens have led to the isolation of mutations

in the voltage-gated potassium channel encoded by Shaker ( Cirelli et al., 2005), and in sleepless ( Koh et al., 2008), which encodes an extracellular membrane-linked peptide that physically associates with the Shaker channel and regulates its abundance and activity ( Koh et al., 2008 and Wu et al., 2010). Hyperkinetic, which encodes the cytoplasmic beta-subunit of the Shaker channel, has also been shown to regulate sleep ( Bushey et al., 2007). In addition to sharply reducing sleep, loss-of-function mutations in each of these genes are associated with reduced longevity, suggesting a link between decreased sleep and lifespan ( Cirelli et al., 2005, Koh et al., 2008 and Bushey et al., 2010). Here, we describe the molecular cloning and characterization of insomniac, a mutant isolated in a forward genetic screen for altered sleep-wake behavior. insomniac animals exhibit severely reduced sleep, shortened sleep bouts, and decreased sleep consolidation. insomniac expression

does not oscillate in a circadian manner, and the circadian clock is intact in insomniac animals, suggesting a function in pathways distinct from the circadian clock. Neuronally restricted depletion http://www.selleckchem.com/products/MLN8237.html of insomniac mimics the phenotype of insomniac mutants, indicating that insomniac is required in the nervous system for the proper regulation of sleep and wakefulness. Conversely,

restoration of insomniac expression to the brains of insomniac animals is largely sufficient to rescue normal sleep-wake behavior. insomniac encodes a protein of the BTB/POZ superfamily. Closely related members of this superfamily function as adaptors for the Cullin-3 (Cul3) ubiquitin ligase complex and thus contribute to protein degradation pathways. Consistent with the hypothesis that Insomniac may function as a Cul3 adaptor, we show that Insomniac can physically interact with Cul3, and that neuronal RNAi directed against Cul3 recapitulates the insomniac phenotype. To identify mutations altering sleep, else we selected a Canton-S (CS) strain exhibiting well consolidated sleep and subjected it to chemical mutagenesis with ethyl methanesulfonate. The screening regimen we employed, in which F2 males are screened, enriches for X chromosome mutations. Over 20,800 animals, representing 3,550 lines, were screened in alternating 12 hr light-12 hr dark (LD) cycles using an automated locomotor activity monitoring system. Three mutant lines exhibiting severe X-linked sleep defects were characterized further. Two of the lines shake under ether anesthesia and fail to complement the sleep defect of Shaker mutants ( Cirelli et al.

Thus it would appear that the extracellular

domains of these neuroligins largely LY294002 ic50 account for the subtype differences in phenotype, while the intracellular domains are exchangeable. To narrow in on the specific region within the extracellular domain that might account for the unique properties of NLGN1, we constructed six additional chimeras with increasingly more of the NLGN3 extracellular domain and less of NLGN1. We found that chimeras containing at least 326 amino acids from the extreme N terminus of NLGN1 possessed the typical NLGN1 NMDAR enhancement, whereas chimeras that contained less than 254 amino acids of the NLGN1 N terminus instead displayed NLGN3 type NMDAR enhancement (Figures 3A and 3E). The difference between NLGN1 and NLGN3 in the region between amino acids 326 and 254 includes an alternatively spliced insertion in NLGN1 previously termed the site B (Ichtchenko et al., 1995; Figure 3B). Interestingly, inclusion of this B site has been shown to determine the specificity with which NLGN1 binds to specific splice variants of neurexin (Boucard et al., VX-809 concentration 2005). We tested an additional mutant of NLGN1 with a deletion of eight amino acids in

the B site and found that it indeed possessed a NLGN3-type NMDAR enhancement phenotype (Figure S3). We have demonstrated that NLGN1, but not NLGN3, is required for LTP in the adult dentate gyrus, but not adult CA1, and that at least some aspects of the phenotypic difference between expression of NLGN1 and NLGN3 are due to the B site insertion in the extracellular domain of NLGN1. What remains is to determine why NLGN1 is required for LTP in dentate gyrus and not CA1 and whether until the B site

has ramifications for LTP as well as the baseline synaptogenic phenotype of NLGN1. It has been shown that the dentate gyrus, one of two sites in the brain that incorporates substantial adult born neurons throughout life, remains more plastic into adulthood, perhaps accounting for the susceptibility to loss of a synaptogenic molecule (reviewed in Deng et al., 2010). Indeed, previous reports indicate that halting adult neurogenesis reduces the expression of LTP in the dentate gyrus (Massa et al., 2011; Singer et al., 2011). Perhaps then CA1 neurons would be susceptible to a knockdown of NLGN1 at an earlier developmental time point when the initial connections are still forming. To test this hypothesis we switched to in utero electroporations. By introducing the NLGN1 miR construct in utero we can check the basal state of synaptic currents and LTP in cells lacking NLGN1 at a very young age (Figure 4A).

It also includes the morphogenetic check details processes that change myelinating cells to process bearing cells forming regeneration tracks,

and the conversion of Schwann cells to cells equipped to rapidly clear myelin from injured nerves (Stoll et al., 2002; Chen et al., 2007; Vargas and Barres, 2007; Wang et al., 2008; Gordon et al., 2009; Höke and Brushart, 2010; Angeloni et al., 2011). The exceptional repair potential of peripheral nerves is likely due to the coordinated functions of the repair program. Yet individual factors can also be presumed to play a prominent role, as exemplified by the enhanced regeneration seen when GDNF and artemin levels are increased in c-Jun mutant facial nerves (Fontana et al., 2012). c-Jun is absent from Schwann cell precursors, expressed in immature cells in vivo and in cultured Schwann cells, suppressed by Krox-20 on myelination, but rapidly re-expressed at high levels in Schwann cells of injured nerves (Parkinson et al., 2004, 2008; D.K.W., unpublished). Among potential intracellular activators of c-Jun is the AP-1 transcription complex, of which c-Jun is a key component. AP-1 activity, in turn, is controlled by numerous signals, including the major MAPK pathways Erk1/2, JNK, and p38. These are all activated in injured nerves and therefore potential upstream regulators of c-Jun (Sheu et al.,

2000; Myers et al., 2003; Harrisingh et al., FG-4592 chemical structure 2004; Jessen and Mirsky, 2008: Parkinson et al., 2008; Napoli et al., 2012; Yang et al., 2012). Genetically, the transcription factor Sox2 is not downstream of c-Jun, since

Sox2 remains normally upregulated in injured c-Jun mutant nerves (Figure S4). We described previously that c-Jun shows cross-inhibitory interactions with the pro-myelin transcription factor Krox20 (Parkinson et al., 2008). Mirroring the function of c-Jun in denervated cells, Krox20 is involved in the regulation of 100–200 genes in myelinating Schwann cells (P. Topilko, personal communication) and is required for the normal activation of the myelin program. We therefore suggest PAK6 that Krox20/c-Jun are central components of a cross-inhibitory switch that regulates cell fate in injured and regenerating nerves. The long term persistence of Schwann cell lipid droplets and large multivacuolated (foamy) macrophages in transected mutant nerves suggests problems with lipid clearance and macrophage activation and exit. Recent evidence indicates that failure of lipid breakdown may delay regeneration (Winzeler et al., 2011). The reduced macrophage numbers in the mutant early after injury is unlikely to contribute substantially to the regeneration problems, a conclusion supported by the microfluidic chamber experiments, where axon growth fails in the presence of mutant Schwann cells, even in the absence of macrophages.

Within approximately 20 min, the resting membrane potential of most neurons returned to their initial values (81%, 17/21). Near the site of high-intensity laser exposure, a gap of ∼5 μm became visible, and distal parts of the axon showed typical beading and degeneration (Figures 4A and S2). Axotomy proximal

to the node (P) was made either in the internode (P, 90–140 μm, n = 5, Figure 4A, right) or within the AIS (15–50 μm, selleck kinase inhibitor n = 7). To isolate the impact of axotomizing the first branchpoint from nonspecific changes (asymmetric current flow at the sealed end, heat-related swelling, phototoxicity, etc.), a control group was included in which the axon was cut distal from the identified first node (D, 120–160 μm, n = 5, Figure 4A, PLX4032 middle). Figures 4A and 4B show an example in which the same axon was axotomized at two different locations with an interval of

25 min. The intrinsically burst firing neuron (236 Hz in control) continued firing at high frequency (240 Hz) when axotomized distal to the branchpoint, but switched to RS mode after a second cut proximal to the branchpoint (9.6 Hz). Data from multiple recordings showed that axotomy of the first branchpoint in IB neurons (230 ± 3 Hz) led to a RS mode (10.2 ± 0.4 Hz, n = 6; unpaired t test p < 0.001; Figure 4C). In contrast, in RS cells the firing rates at steady current injections remained similar to control (control, 7.7 Hz versus cut, 5.6 Hz, paired t test p > 0.09, Figure 5C). Axotomy proximal to the branchpoint did not affect the input resistance at resting potential (1.9 ± 1.0 MΩ increase, paired t test p > 007, n = 12, Figure S2). Similar to axons cut in the slice preparation, axotomy proximal to the first node significantly increased the AP threshold during steady current injections (+6.8 ± 1.1 mV, p < 0.01, Figure 4D) and reduced the ADP amplitude (−3.8 ± 0.6 mV, n = 5, p < 0.05,

Figure 4E). However, single APs were not affected when the axotomy was made distal of the first node; the AP amplitude (+0.3 ± 1.7 mV change), ADP (−0.5 ± 0.5 mV change), and voltage threshold (+1.6 ± 0.5 mV change) remained similar to control values (for all, paired t test p > found 0.3, n = 5). The only specific impact of cutting within the AIS (on average 35 ± 5.4 μm, n = 7) was a significantly larger reduction in the ADP (−12.0 ± 2.7 mV, compared to internodal axotomy p < 0.05, n = 7, Figure 4E). Interestingly, large negative ADP amplitudes observed after laser axotomy in the AIS were quantitatively similar to the ADP amplitudes associated with axons that were cut in the AIS during the slice-cutting procedure (acute axotomy: −12.0 mV for a 35 μm axon versus slice cut: −10.5 mV for a 32 μm axon), suggesting that the functional consequences of acute transections (30 min) are comparable to the lasting impact by slice cutting (2–8 hr).

, 1995 and Bruno and Sakmann, 2006). During early postnatal development, receptive fields in layer 4 emerge in a process that is driven

by whisker experience (Feldman and Brecht, 2005). The rapid development of receptive fields (Stern et al., 2001) and the experience-dependent synaptic plasticity of many cortical pathways also occur at this time (Bender et al., 2006, Allen et al., 2003 and Cheetham et al., 2007). Nevertheless, it is not clear how synaptic and anatomical changes at the critical level of individual connections interact to produce network architecture that is capable of processing sensory information. To understand Selleckchem SNS032 the development and organization of local circuits it is necessary to

investigate connectivity and the synaptic properties of connections between individual identified neurons in sufficient number to allow a quantitative description of the circuit. Mammalian neocortex is composed of heterogeneous, sparsely connected neuronal populations. This presents a major obstacle for the analysis of circuit connectivity because of the difficulty of identifying and stimulating individual neurons. Simultaneous electrophysiological recordings have been used to analyze multiple neurons (e.g., Thomson et al., 2002), but this is very time consuming, limiting the practicality of a detailed analysis of circuit development. Optical stimulation methods have been used (Nikolenko et al., 2007, Fulvestrant datasheet Matsuzaki et al., 2008, Dantzker and Callaway, 2000 and Petreanu et al., 2007), but, so far, such approaches have not been shown to be suitable for probing local circuit connections with single-cell resolution (but see Fino

and Yuste, 2011). This is largely due to relatively low spatial resolution of the excitation illumination profile leading science to stimulation of multiple and/or off-target cells. We now describe the development of a high-resoution 2P glutamate uncaging technique that reliably and selectively activates single, identified neurons in intact tissue. Combined with patch-clamp electrophysiology and 2P imaging of dendritic structure, we used this technique to analyze the developmental and experience-dependent changes in the layer 4 excitatory stellate cell network in barrel cortex. For glutamate uncaging to be useful in identifying synaptic connections with single-cell resolution, the photostimulation must fulfill six key criteria: (1) repeatable trial-by-trial activation of the targeted neuron, (2) reliable activation of the targeted neuron, (3) single-cell spatial resolution of uncaging, (4) specificity of activation to the visually identified targeted neuron and not neighbors or dendrites of passage, (5) unambiguous detection of evoked synaptic events, and (6) selective activation of monosynaptic connections.

Correct placement of the cannulae and fibers was verified by injections of fluorescent beads and post hoc analysis. Based on incorrect positioning, three rats (in which BL, as a consequence, did not decrease freezing responses) were excluded from further analysis. (Figure 5A, see also Experimental Procedures). To ensure basic activation of the amygdala during the behavioral Entinostat ic50 experiments, we trained all rats in a 2-day contextual fear-conditioning

protocol (see Figure 5B and Experimental Procedures) that resulted in similar freezing in the majority of animals (n = 25) after 2 days of conditioning (Figures 5C1 and 5D1). One animal was excluded from the experiment due to unusually low freezing levels. Hormonal cycle did not appear to affect these freezing levels (Figure S5). To assess acute effects of BL on freezing behavior, we placed rats on day 3 in the fear-conditioning box after optic fibers had been inserted through the guide cannulae to target the CeL. All rats exhibited maximal freezing upon and throughout exposure

to the context (Figure 5C1). After 10 min, 10 ms, 30 Hz BL pulses were given for either 20 or 120 s. As expected from the central role of the CeM in freezing behavior (Ciocchi et al., 2010 and Haubensak et al., 2010) and the inhibitory effects of BL on the CeM in vitro (Figure 4), BL efficiently decreased freezing (from 57.5 ± 0.9 to 32.1 ± 5.6 s/min, n = 6; one-way ANOVA, p < 0.05; Figure 5C1). The onset of see more this decrease (Figure 5C2; see also Movie S1) started in two rats as

fast as 2 s after BL onset and on average with a delay time of 21.5 ± 9.7 s across all animals (n = 6). Freezing returned after 70 ± 21 s upon termination of the 20 s BL stimulation and 108 ± 20 s after the 120 s BL exposure (n = 3 per group). The inhibiting effects of BL appeared specific to the fear-induced freezing response, because BL exposure in the same animals in a non-fear-conditioning context did not affect basic locomotor activity (Figure 5C3). To confirm involvement of endogenous OT release in these BL responses, we injected OTA on day 3 through the same guide cannulae through which the optic fibers were subsequently inserted and applied BL immediately very for 120 s before the rats were re-exposed (after removal of the optic fibers) to the fear-conditioning context. We thus measured the remaining block on the effects of BL by OTA, while at the same time providing more freedom of movement to the rats (now unobstructed by any attached optic fibers). We compared freezing behavior between four groups of rats, namely “Ctrl” (no BL, but optic fibers inserted prior to testing), “OTA” (OTA injected + optic fibers without BL), “BL” (BL application prior to exposure to context) and “OTA + BL” (injection of OTA followed by BL application prior to exposure to context). Ctrl or OTA-injected rats exhibited high freezing levels (Figure 5D2) comparable to those measured previously (Figure 5C1).

Here we overcome this challenge by using chronic two-photon calcium imaging with the genetically encoded calcium indicator GCaMP3. By selectively imaging the activity of ensembles of mitral cells and inhibitory granule cells, we show that the transition from the awake to anesthetized brain state modulates olfactory bulb circuits and odor coding. Furthermore, we monitored the dynamics Proteasome inhibitor of odor responses of the same populations of mitral cells over months in awake mice to test how odor experience affects olfactory bulb odor representations. These approaches revealed a surprisingly dynamic nature of odor representations, which is sensitive to brain state and experience. We expressed

GCaMP3 (Tian et al., 2009) specifically in olfactory bulb principal cells (mitral and tufted cells) by injecting a Cre recombinase-dependent viral vector (Atasoy et al., 2008) into the olfactory bulbs of protocadherin-21 (PCdh21)-Cre mice, which express Cre exclusively in olfactory bulb principal cells (Nagai et al., 2005). Several weeks after injection, virtually all glomeruli had GCaMP3-expressing dendrites, and immunostaining with a mitral/tufted cell-specific antibody (Tbx21) (Yoshihara et al., 2005) showed that the majority of Tbx21-positive cells (67%, n = 3 mice, 644 cells) express GCaMP3

(Figure 1B). Consistent with the selective expression in mitral/tufted cells, all GCaMP3-expressing cells (n = 2 mice, 323 cells) lacked immunoreactivity for GAD67, a marker for GABAergic interneurons (data not shown). We next performed simultaneous patch-clamp recording and two-photon Metalloexopeptidase imaging in olfactory bulb slices to test the ability of GCaMP3 to report click here action potential firing in mitral cells (GCaMP3-expressing cells in the mitral cell layer). We measured GCaMP3 fluorescence changes in mitral cells in response to spikes elicited at 50–100 Hz via brief depolarizing current steps (1 nA, 3 ms). In agreement with previous findings in cortical pyramidal cells (Tian et al., 2009), we found that increases in GCaMP3 fluorescence

had a relatively linear relationship with the number of action potentials evoked in mitral cells (Figures 1C and 1D). To optically monitor mitral cell activity in vivo, we implanted a cranial window (Holtmaat et al., 2009) over the dorsal olfactory bulb of GCaMP3-expressing mice. Using two-photon imaging selectively in the mitral cell layer allowed us to repeatedly image the same sets of up to 100 mitral cells over months in awake, head-fixed mice (Dombeck et al., 2007; Komiyama et al., 2010) (Figures 1E and 1F). Consistent with previous electrophysiological studies in anesthetized animals (Bathellier et al., 2008; Davison and Katz, 2007; Dhawale et al., 2010; Fantana et al., 2008; Meredith, 1986; Mori et al., 1992; Tan et al., 2010), passive application of structurally diverse odors elicited activity revealed by increases in GCaMP3 fluorescence in overlapping but distinct ensembles of mitral cells (Figure 1G).

This analysis demonstrated that, despite the large number of theoretical response modes that groups of several tens of neurons could generate, local auditory cortex populations generate only a small repertoire of functionally distinct response modes. Interestingly, a similar result was obtained when two second long sounds were presented ( Figure S4). We then sought to determine the spatial organization of the neurons that underlie distinct response modes. We calculated the mean firing rate of neurons in response to the groups of sounds

associated to the different modes, which were identified in the above analysis. Interestingly, pairs of response modes observed in a given population PD-0332991 manufacturer corresponded to the firing of partially overlapping subgroups of neurons (Figures

4A and 4D). To assess the similarity of tuning of neurons associated to the same or different subgroups, we computed their signal correlations. We found that members of the same subgroup had significantly higher signal correlations than neuron pairs across groups (same mode: 0.76 ± 0.07, n = 37; different modes: 0.53 ± 0.11, n = 23 modes, Wilcoxon test p = 2 × 10−4). Furthermore, the centroids of the CDK inhibitor neuronal subgroups corresponding to two distinct response modes were significantly more distant to each other than when the neurons of the local population are spatially randomized (Figure 4E). This indicated an organization of the modes into different spatial domains, which was also visually evident in many examples (Figures 4A and 3C). This observation is consistent with previous estimations of the spatial layout of neurons suggesting a patchy organization of neuronal subgroups in the cortex (Rothschild et al., 2010). The low number of observed response modes suggests that local activity patterns form discrete representations of sounds. A prediction from this scenario would be that for a continuous transition between two stimuli exciting two modes an abrupt change in response patterns would be observed because the population could generate no intermediate response pattern. Alternatively, the low number of response modes could merely

reflect biases or gaps in the set of Phosphatidylinositol diacylglycerol-lyase tested sounds. To determine if abrupt changes in response patterns could be observed, we first identified local populations in anaesthetized mice showing at least two response modes using a broad set of different sounds (Figure 5A). We selected two basis sounds that were falling in either response mode and constructed linear mixtures from them. Next, we retested the same population with the new set of stimuli to map the transition across modes with higher resolution. When the mixture ratio was varied continuously, we observed abrupt transitions in the population activity patterns that are visible in both the raw activity plots and the similarity matrices (Figures 5B, 5C, and S5).

A syp-GFP construct (generated from a mouse complementary DNA clone) was provided by Dr. Niwa (our laboratory). KIF1A and KIF5B expression vectors were generated

by standard molecular methods. Detailed information is provided in the Supplemental Experimental Procedures. Astrocyte cultures were prepared as previously described (Suzuki et al., 2007). The cultures were or were not treated with BDNF (100 ng/ml) for 3 days. Detailed information is provided in the Supplemental Experimental Dinaciclib ic50 Procedures. Neurons were transfected with syp-GFP at 7 DIV and were incubated with or without BDNF (100 ng/ml) for 3 days. At 10 DIV, time-lapse recordings were performed with an LSM710 confocal laser-scanning microscope (Zeiss). We selected the middle part of axons of transfected neurons for live imaging. Images were acquired every 1 s, and syp-GFP containing vesicles moving across the center line of the imaged area were counted. Images were analyzed using ImageJ software. Neurons at 7 DIV were or were not treated with BDNF (100 ng/ml) for 3 days. At 10 DIV, neurons were fixed and immunostained as previously described (Niwa et al., 2008). Cells were fixed with 4% paraformaldehyde in PBS for 10 min, Abiraterone research buy permeabilized

with 0.1% Triton X-100 in PBS, and blocked with 5% bovine serum albumin in PBS. Cells were incubated with primary antibodies overnight at 4°C, followed by incubation with the appropriate Alexa-labeled secondary antibodies for 1 hr. Images were acquired using an LSM510 confocal laser-scanning microscope (Zeiss). Immunopositive puncta

along MAP2-labeled dendrites and synaptophysin/PSD-95-double-positive puncta were counted. For immunocytochemistry, anti-synaptophysin (mouse monoclonal, Chemicon, 1:1000; rabbit monoclonal, Abcam, 1:2000), anti-PSD-95 (mouse monoclonal, ABR, 1:200), and anti-MAP2 (chicken very polyclonal, Abcam, 1:2000) antibodies were used. Neurons were transfected with syp-GFP alone or cotransfected with syp-GFP and KIF1A or KIF5B at 7 DIV. At 10 DIV, neurons were fixed and immunostained for MAP2 and PSD-95, and images were acquired as described above. Synaptophysin-GFP puncta along MAP2-labeled dendrites and colocalized with PSD-95 were counted. Data were analyzed by the two-tailed t test or one-way ANOVA with a post hoc Dunnett’s test. For analysis of water maze test data, one-way ANOVA and two-tailed t test were used in the probe test, and two-way repeated-measures ANOVA with a post hoc Bonferroni’s test was used to compare differences between groups at several time points. We thank H. Sato, H. Fukuda, N. Onouchi, T. Akamatsu, T. Aizawa, and all other members of the Hirokawa laboratory for technical assistance and discussions. This work was supported by a grant-in-aid for specially promoted research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to N.H.).

, 2010, Goldberg and Reynolds, 2011, Morris et al., 2004, Raz et al., 1996 and Smith et al., 2004) and that show stereotyped burst activity on presentation of salient stimuli (Aosaki

et al., 1994 and Matsumoto et al., 2001). We directly tested the intriguing possibility that activation of intralaminar thalamic glutamate inputs to striatum might also drive DA release via a striatal nAChR-dependent mechanism. Indeed, laser activation of ChR2-eYFP-expressing thalamostriatal axons arising from intralaminar thalamus in CaMKII-Cre mice evoked DA release in coronal striatal slices, and this was prevented by nAChR inhibition and, necessarily, glutamate receptor antagonists but not GABA receptor antagonists (Figure 4; n = 4 animals, TTX-sensitive, Ca2+-dependent). ACh-dependent DA signals can ATR inhibitor therefore be driven by the thalamic inputs that synchronize activity in ChIs in vivo. It is interesting in this regard that the relatively “digital” nature of the stereotyped burst activity in the thalamostriatal network that is associated with salient event detection parallels the lack of simple frequency dependence in the ChI activation of DA release seen here. In any event, these data suggest that DA may be important for conveying

salience- or attention-related signals mediated not through changes in DA neuron firing but through activation check details of DA axons by ChIs and their inputs. Third, we would expect that a ChI-driven DA signal will have key outcomes for DA functions that are encoded by dynamic patterns of activity MRIP in DA neurons themselves. The outcome will depend entirely on the timing of activity in DA neurons relative to ChIs. Pauses in ChIs have been suggested previously to remove a low-pass filter on

DA release during concurrent changes in DA neuron activity (Cragg, 2006). Prior ChI-driven DA release could shunt (limit) the impact of subsequent changes in DA neuron activity, while alternatively, postpause “rebound” facilitation in ChI activity (Aosaki et al., 1995, Apicella, 2007 and Morris et al., 2004), which probably corresponds to increased synchrony in the population, could critically supplement preceding DA signals and promote, for example, the selection of a behavior. In addition, discrete functions for DA could be driven by synchronous activity in ChIs despite an absence of accompanying phasic changes in DA neuron activity, which otherwise would be taken as evidence for functions not requiring phasic DA. Furthermore, what might be the outcome for nicotine action? By desensitizing nAChRs on DA axons, nicotine would be expected to prevent ChI-driven DA release (pilot observations suggest this to be the case, data not shown) and thereby devolve the control of DA release to activity in DA neurons without modulation by ChIs. In this case, DA release might be a more direct reporter of activity in DA neurons than with nAChRs active (Rice and Cragg, 2004).