Acetylcholine Acts through Nicotinic Receptors to Enhance the Firing Rate of a Subset of Hypocretin Neurons in the Mouse Hypothalamus through Distinct Presynaptic and Postsynaptic Mechanisms,

Neurons expressing the neuropeptide hypocretin regulate many behavioral functions, including sleep, motivation, and behaviors related to addiction. The ability of nicotine to stimulate nicotinic acetylcholine receptors (nAChRs) is essential for its addictive properties, but little is known about whether, and how, nicotine and the endogenous neurotransmitter acetylcholine affect hypocretin neurons.

Nicotine addiction is mediated by nicotinic acetylcholine receptors (nAChRs). Histochemical studies have shown that nAChRs are also expressed in hypothalamus (Avissar et al., 1981;Wada et al., 1989;O'Hara et al., 1998). Specifically, Hcrtϩ neurons receive appreciable cholinergic innervation arising from basal forebrain (Sakurai et al., 2005;Henny and Jones, 2006), which indicates that the activity of Hcrtϩ neurons might be modulated by nAChRs. nAChRs are pentameric, non-selective cation channels. Activation of nAChRs results in a physiological net flow of inward current, which directly depolarizes the cell and generally affects neuronal input and/or output, influencing subsequent behaviors. nAChRs may be located both presynaptically and postsynaptically. Stimulation of these receptors is known to increase neurotransmitter release, and can also depolarize the postsynaptic neuron (Gioanni et al., 1999;Mansvelder et al., 2002;Lambe et al., 2003). Previous studies have shown that nicotine can alter the firing of Hcrtϩ neurons in rat as measured by c-fos immunoreactivity (Pasumarthi et al., 2006;Pasumarthi and Fadel, 2008), but it is not known whether nAChRs are expressed on these neurons, whether the effects on c-fos immunoreactivity were due to postsynaptic signaling, presynaptic signaling, or network changes due to changes in other neuronal subtypes. Since the ability of nicotine to alter hypocretin signaling may be important for its addiction liability (Hollander et al., 2008), it is important to understand the cellular mechanisms underlying these physiological effects.
To investigate the effect of nAChR stimulation on function of the hypocretin system, we first investigated how acetylcholine (ACh) in the presence of atropine affects spontaneous action potential firing, which is a measure of nicotinic influence on the output of Hcrtϩ neurons. Next, we separated presynaptic and postsynaptic modulation by ACh and nicotine using synaptic blockers, and used pressure application (puff) or fast pipetting of drugs to identify electrophysiological changes following the stimulation of nAChRs. A transient puff of ACh or nicotine at high concentration was used to mimic phasic transmission to determine the postsynaptic response (Alexander et al., 2009). nAChRs can increase release of glutamate from presynaptic terminals in several brain areas, including the ventral tegmental area (Mansvelder et al., 2002) and the prefrontal cortex (Gioanni et al., 1999;Lambe et al., 2003). To assess the effects of cholinergic transmission on the basal level of presynaptic glutamatergic transmission, spontaneous miniature EPSCs (mEPSCs) were recorded following a puff of ACh as well as in response to bath-applied nicotine at a concentration that mimics brain levels during smoking. We show that the activity of both Hcrtϩ neurons and the presynaptic glutamatergic terminals projecting to these cells is modulated by nAChR signaling in a manner that appears to enhance the output of a subset of Hcrtϩ neurons during phasic ACh signaling.

Animals
Male and female adult Hcrt-GFP mice  (2-5 months old, backcrossed onto the C57BL6/J genetic background for at least 10 generations) were group housed and maintained on a 12-12 h light-dark cycle with food and water available ad libitum. Use of animals was in strict accordance with NIH Care and Use of Laboratory Animals Guidelines.

Significance Statement
Neurons expressing the neuropeptide hypocretin regulate many behavioral functions, including sleep, motivation, and behaviors related to addiction. The ability of nicotine to stimulate nicotinic acetylcholine receptors (nAChRs) is essential for its addictive properties, but little is known about whether, and how, nicotine and the endogenous neurotransmitter acetylcholine affect hypocretin neurons. This study suggests that phasic acetylcholine release can enhance the firing of a subset of hypocretin neurons through postsynaptic nAChRs, while disrupting tonic activation of presynaptic nAChRs necessary for maintaining functional glutamatergic inputs to hypocretin neurons. We propose that this mechanism could enhance the signal-to-noise ratio of the electrical response to nicotine or acetylcholine in the nAChR-positive subset of neurons. cording to the method described in http://www.brainslicemethods.com).

Data analysis
Electrical recording data of nAChR currents were analyzed using Clampfit 10 (Molecular Devices). Traces were filtered with Gaussian low-pass with 50 Hz cutoff before current amplitude measurements. Other electrical data were analyzed using Axograph X 1.5.5 (Axograph Scientific). To analyze miniature EPSCs, we took 150 Ϫ 200 events for baseline and washout from each cell, during a time period ranging from 60 to 120 s. For Ach, we took all events from within 90 s after application of ACh. Traces were filtered with Gaussian low-pass with 500 Hz cutoff before event searching. A template EPSC was defined with amplitude Ϫ20 pA, rise 0.5 ms, and decay 3 ms, and only those EPSCs with amplitude ՆϪ10 pA were counted. Following programmed event detection, all events were examined by eye to be counted as EPSCs. All statistics were done on raw data. Unpaired Student's t tests and chi square ( 2 ) test were used for determining statistically difference. Significance was set as p Ͻ 0.05, and high significance as p Ͻ 0.01. Values are presented as mean Ϯ SEM.

Nicotinic stimulation boosts spontaneous action potential firing in hypocretin neurons
To determine whether phasic nicotinic stimulation modulates the electrical output of Hcrtϩ neurons, we patched the cells with an ATP-rich (5 mM, vs 3 mM in other experiments) intracellular solution and investigated the effect of pressure-applied ACh on spontaneous action potential firing. An adequate level of intracellular ATP is required for normal Hcrtϩ neuron function (Liu et al., 2011), potentially because Hcrtϩ neurons are involved in the regulation of energy status (Girault et al., 2012;Gao and Horvath, 2014). In the presence of atropine, Hcrtϩ neurons regularly fired action potentials. Mechanical interference as a result of puffing bath solution induced a temporary reduction in firing frequency (Ͻ50% of baseline) in 7 out of 11 cells (Fig. 1B1,C), despite inducing no, or minimal, outward current (Ͻ10 pA, data not shown). Firing in 4 out of 11 cells was not altered by puffing bath solution (50% Ͻ change Ͻ 200% at 0 Ϫ 10 s). In contrast, in 7 out of 20 cells, a puff of ACh (1 mM) increased the firing rate (Ͼ 200% of baseline) with a significant depolarization of the membrane potential ( Fig. 1B2,D). We note that this experimental design is not suitable to detect decreases in firing due to presynaptic mechanisms. Only blocking all mEPSCs using CNQXϩAP-5 induces even a small change in firing rate of a hypocretin neuron (ϳ2 Hz to ϳ1.6 Hz; Li et al, 2002), which is not distinguishable using the current criteria (50% Ͻ change Ͻ 200%). In nine cells, an ACh puff decreased the firing rate, and in four cells the ACh puff had no effect. Thus, stimulation of nAChRs depolarizes approximately one-third of Hcrtϩ neurons and boosts the action potential firing of these cells.
To identify the receptor type(s) that mediate this depolarization and increase in firing rate, we applied an ACh puff in the presence of the heteromeric (␣4␤2) nAChR antagonist, dihydro-␤-erythroidine (DH␤E), or the homomeric (␣ 7) nAChR antagonist, methyllycaconitine (MLA). When applied alone, DH␤E significantly decreased the firing rate by 62.9% (p ϭ 0.00027) at 10 M, whereas MLA did not change the firing rate of Hcrtϩ neurons at a concentration of 10 nM (Fig. 2, top; 20 Ϫ 30 s after puff vs 10 Ϫ 20 s before puff: p ϭ 0.58). However, after addition of either reagent, most cells recovered to a new stable firing state that lasted for several minutes with attachment of the recording pipette (Fig. 2, bottom). DH␤E (10 M) significantly reduced the ability of ACh to increase firing in 11 of 11 Hcrtϩ neurons ( 2 test, p ϭ 0.0069) and increased the likelihood that an ACh puff could decrease the firing rate (p ϭ 6.8E-6, 10 Ϫ 20 s after puff; Fig. 1E). In contrast, although MLA (10 nM) antagonized the effects of an ACh puff in the majority of Hcrtϩ neurons, ACh still increased the firing rate in 2 of 16 cells in the presence of the homomeric nAChR antagonist ( 2 test, p ϭ 0.00039; Fig.  1F). In this data set, some cells increased their firing rate during the ACh puff and returned to normal right after puff cessation, whereas 10 of 17 cells responded with a prolonged period of firing increase. These prolonged periods of firing may be due to increased activity of Hcrt neurons triggering action potentials in a recurrent circuit.
Muscarinic acetylcholine receptors (mAChRs) are expressed by ϳ30% of hypocretin neurons (Sakurai et al., 2005), and may contribute significantly to cholinergic modulation of these cells. To determine the overall response of hypocretin neurons to cholinergic inputs, we tested the effect of ACh in the absence of atropine to engage both nAChRs and mAChRs. ACh induced depolarization and increased firing rate in 57% (8 out of 14) of hypocretin neurons (Fig. 1G), a significant increase compared to the presence of atropine ( 2 test, p ϭ 0.0040). The remaining cells showed no effect (1 out of 14 cells) or responded to ACh with decreased firing (5 out of 14 cells).

Postsynaptic nAChR currents can be induced in one third of hypocretin neurons
Although mRNAs encoding nAChR subunits are expressed in lateral hypothalamus (Clarke et al., 1985;Wada et al., 1989) where Hcrtϩ neurons reside, it is not yet clear on which cell types and in what subcellular structure nAChRs are functional, and whether Hcrtϩ neurons express nAChRs. To dissect the mechanisms underlying nicotinic stimulation of Hcrtϩ neurons, we evaluated postsynaptic nAChRs on Hcrtϩ neurons selectively by using synaptic blockers to rule out contributions from glutamate-and GABA-activated currents. We recorded from Hcrtϩ neurons in voltage clamp mode in the presence of TTX (0.5 M), PTX (100 M), CNQX (10 M), AP-5 (30 M), and atropine (4 M), while puffing 1 mM ACh onto the cell body of the recorded cell. A brief puff of ACh evoked an inward current in about one-third of Hcrtϩ neurons (32 out of 92 cells). The peak amplitudes of the inward currents ranged from Ϫ10 to Ϫ850 pA, with an average size of Ϫ71.1 Ϯ 30.9 pA (n ϭ 32). We also tested the nAChR agonist nicotine (100 M). A nicotine puff produced a response similar to that induced by ACh in Hcrtϩ neurons. Inward currents were recorded in 9 out of 29 cells, ranging from Ϫ10 to Ϫ1500 pA, with an average size of Ϫ413 Ϯ 178 pA (n ϭ 9; Fig. 3). We also wished to determine whether the small currents might be sufficient to alter the function of the Hcrtϩ neurons. Injection of a small inward current (Ϫ10 pA) markedly increased the spontaneous action potential firing rate to 324% Ϯ 101% (Student's t test, p ϭ 1.0E-5, n ϭ 3) of the baseline level (Fig. 1B4), suggesting that nicotinic currents are likely to have a significant effect on the output of these cells. nAChRs are known to be easily desensitized by agonists (Giniatullin et al., 2005). Consistent with these observations, we observed that postsynaptic nAChR-mediated currents on Hcrtϩ neurons were desensitized following 5 s exposure to 1 mM ACh or 100 M nicotine. Previous studies have shown that most nAChR subtypes, including ␣4␤2, ␣3␤4 and ␣7, recover from the desensitization induced by brief (1 Ϫ 5 s) exposure to 1 mM ACh in 60 s or less (Reitstetter et al., 1999;Meyer et al., 2001;Paradiso and Steinbach, 2003;McCormack et al., 2010). While the majority of currents did not recover rapidly following ACh stimulation, we observed a rare subset of Hcrtϩ neurons (3 out of 121 cells) that responded to repeated ACh or nicotine puffs every 60 s (Fig. 3A2), which happened only when the current size was greater than 500 pA.

Acetylcholine and nicotine decrease the frequency of mEPSCs in hypocretin neurons
Next, we tested whether nAChRs are expressed in presynaptic glutamatergic terminals that impinge onto Hcrtϩ neurons, and what physiological changes might occur to synaptic transmission following presynaptic receptor stimulation. To do this, we applied TTX, PTX, and atropine in the bath and recorded spontaneous mEPSCs from the Hcrtϩ neurons. A brief puff of ACh above the tissue surface next to the Hcrtϩ neuron reliably decreased mEPSC occurrence for ϳ1 min, followed by full recovery (Fig 4A,B). The average instantaneous frequencies of mEPSCs at baseline, upon ACh application, and after washout were 3.76 Ϯ 0.19 Hz, 0.64 Ϯ 0.12 Hz, and 3.48 Ϯ 0.31 Hz, respectively. The average peak amplitudes of mEPSCs at baseline, upon ACh application, and washout were 26.4 Ϯ 0.3 pA, 21.8 Ϯ 0.8 pA, and 30.0 Ϯ 0.5 pA, respectively. Analysis shows that phasic stimulation of preterminal nAChRs significantly reduced vesicle release probability by 83.0% (p ϭ 2.06E-39). The average peak size of mEPSCs was also slightly reduced by 17.4% (p ϭ 1.88E-7; Fig. 4C,D).
Nicotine is a strong exogenous agonist of nAChRs, and also a prevalent drug of addiction. During cigarette smok-ing, nicotine concentration increases rapidly in the brain (Berridge et al., 2010;Rose et al., 2010). To mimic this fast onset of brain nicotine concentration, we pipetted 5ϫ nicotine solutions into the recording chamber within 5 s, to a final concentration of 1 M (Henningfield et al., 1993).

Antagonists of nAChRs mimic the effect of agonist application on mEPSC frequency
Under physiological conditions, activation of nAChRs induces excitatory inputs by evoking inward currents. The suppressive effect on mEPSCs might therefore be due to desensitization of nAChRs. To test this possibility, we applied mecamylamine (MEC), a nonselective antagonist of nAChRs. The application of MEC alone caused a substantial suppression of mEPSCs (Fig. 6), with instantaneous frequency decreased to 37.6% (from 6.49 Ϯ 0.32 Hz to 2.44 Ϯ 0.24 Hz, p ϭ 1.82E-23), and amplitude decreased to 80.7% (from 31.6 Ϯ 0.4 pA to 25.5 Ϯ 0.4 pA, Figure 2 The effects of ␣4␤2 receptor antagonist, DH␤E, and ‫ء7␣‬ receptor antagonist, MLA, on the action potential firing of Hcrtϩ neurons. Top, Change of firing rate (action potentials/5 s) before and after bath application of DH␤E (red) and MLA (blue). Bottom, A representative cell reduced in firing rate upon bath application of DH␤E, and partially recovered over time.

Blockade of Ca 2؉ internal stores occludes the effect of ACh on mEPSC frequency
Synaptic vesicle release is a calcium-dependent process (Neher and Sakaba, 2008;Südhof, 2012). Spontaneous vesicle release, unlike action potential-evoked events, is independent of extracellular calcium and voltage-gated calcium channel opening, but is instead driven largely by calcium release from internal stores (Llano et al., 2000;Emptage et al., 2001;Han et al., 2001). To determine whether nAChR stimulation affects calcium release from internal stores, we applied dantrolene (50 M) and (Ϫ)-Xestospongin C (XeC) (5 M) to block ryanodine and IP 3 receptors in the endoplasmic reticulum membrane (Dickinson et al., 2008). Following 30 min incubation with dantrolene and XeC, the mEPSCs instantaneous frequency was reduced to 50.7% of its baseline value (4.90 Ϯ 0.48 Hz to 2.48 Ϯ 0.38 Hz, p ϭ 7.12E-4; Fig. 7), in agreement with previous reports that approximately half of spontaneous vesicle release is due to internal calcium stores (Emptage et al., 2001). The peak amplitude was reduced to 80.6% (21.1 Ϯ 0.7 pA to 17.0 Ϯ 0.5 pA, p ϭ 5.18E-5).
In the continued presence of dantrolene and XeC, we pressure applied ACh onto Hcrtϩ neurons and found that both amplitude (from 17.0 Ϯ 0.5 pA to 18.0 Ϯ 0.5 pA, p ϭ 0.664) and frequency of mEPSCs (from 2.48 Ϯ 0.38 Hz to 2.25 Ϯ 0.37 Hz, p ϭ 0.151) remained unchanged (Fig.  7C,D). Thus, blockade of calcium release from internal store occluded the effect of subsequent nAChR stimulation.

Discussion
Hypocretin/orexin neurons are involved in multiple behaviors, including those related to arousal and addiction (Mahler et al., 2012;de Lecea and Huerta, 2014). Understanding how the electrical activity of Hcrtϩ neurons is modulated by endogenous neurotransmitters and exogenous drugs such as nicotine is critical for understanding the role of these cells in complex behaviors, including nicotine dependence. As a candidate modulator, nAChRs have been shown to be expressed in hypothalamus. Stimulation of these receptors can activate Hcrtϩ neurons (Pasumarthi et al., 2006;Pasumarthi and Fadel, 2008;Morgan et al., 2013); however, the underlying mechanisms by which nAChRs modulate the activity of Hcrtϩ neurons are still unclear.
In the current study, we used pressure application to mimic phasic ACh neurotransmission (Alexander et al., 2009) and investigated the effects of ACh and nicotine acting through presynatpic and postsynaptic nAChRs on glutamatergic transmission and postsynaptic electrical activity in Hcrtϩ neurons. We found that nAChRs are expressed postsynaptically in around one-third of Hcrtϩ neurons, as well as presynaptically on glutamatergic axon terminals to Hcrtϩ neurons. Stimulation of presynaptic or postsynaptic nAChRs produced opposing effects on electrical activity in Hcrtϩ neurons, however. Stimulation of postsynaptic nAChRs evoked an inward current in a fraction (approximately one-third) of Hcrtϩ neurons (Fig.  3). This current, which was as small as 10 pA, ranging to more than 1000 pA, depolarized the membrane potential and increased spontaneous firing of Hcrtϩ neurons (Fig.  1). In contrast, stimulation of presynaptic nAChRs reliably decreased the frequency of mEPSCs in Hcrtϩ neurons (Fig. 4), indicating the efficacy of glutamatergic transmission was decreased. At a concentration consistent with  B 1 , B 2 , and B 3 , respectively. Inset shows the full trace of an example mEPSC event. C, Cumulative probability plot of the mEPSC amplitude at baseline and upon ACh application from six cells. Inset shows the mean value of the mEPSC amplitude at three time periods: baseline, under the influence of ACh, and washout. ‫‪p‬ءء‬ Ͻ 0.01. D, Interevent intervals of mEPSCs at baseline and upon ACh application from six cells (same cells as in C). Inset shows the mean of instantaneous frequency of the mEPSC at three periods of time: baseline, under the influence of ACh, and after washout. cigarette smoking, nicotine suppressed mEPSCs in a majority (ϳ70%) of Hcrtϩ neurons (Fig. 5). The nonselective nAChR antagonist mecamylamine depressed mEPSC frequency to a similar extent (Fig. 6), suggesting that the suppressing effect of ACh on mEPSCs was likely mediated by desensitization of presynaptic nAChRs, and that an endogenous, tonic activation of presynaptic nAChRs might be required for maintaining normal, functional mEPSC frequency. It should be noted that inhibitory inputs impinging on hypocretin neurons are also likely to express nAChRs, and therefore could also be regulated by ACh; however, inhibitory inputs were excluded using picrotoxin in this study.
To explore the mechanism underlying the AChmediated decrease in mEPSC frequency, we determined the role of calcium release from internal stores (Llano et al., 2000;Cheng and Lederer, 2008), since intracellular calcium stores are thought to be required for spontaneous vesicle release (Emptage et al., 2001). In the presence of the ryanodine receptor blocker dantrolene and the IP3 receptor blocker XeC (Dickinson et al., 2008), mEPSC frequencies in Hcrtϩ neurons were significantly decreased. The application of ACh did not further decrease mEPSC frequencies (Fig. 7), indicating that phasic nAChR stimulation (i.e., endogenous ACh release from cholinergic synapses) could desensitize tonically active nAChRs and thereby decrease calcium release from internal stores, preventing Ca 2ϩ -dependent spontaneous vesicle release. Previous studies have shown that nicotine can transiently facilitate neurotransmitter release, and one study in mouse vas deferens indicated that this is mediated by calcium-induced calcium release from a ryanodine-sensitive calcium store in nerve terminals (Brain et al., 2001). Here, we propose that in the mouse central nervous system, at least in glutamatergic synapses impinging onto Hcrtϩ neurons, this mechanism is also present. Tonic activation of presynaptic nAChRs contributes to spontaneous vesicle release via transient opening of ryanodine receptors and/or IP3 receptors in local internal calcium stores. Levels of nicotine delivered through cigarette smoking are sufficient to interfere with this function, and this mechanism could therefore contribute to behaviors related to nicotine dependence.
The opening of nAChRs channels leads to the influx of cations, and generally excites the postsynaptic neurons (Léna and Changeux, 1997;Zhou et al., 2001;Mansvelder et al., 2002;Sharma et al., 2008;Huang et al., 2011). In the current study, this direct depolarizing effect of nAChR activation was also demonstrated for mouse Hcrtϩ neurons, likely leading to hypocretin release in downstream neuronal circuits. The pharmacological experiments shown here, along with previous studies (Pasumarthi et al., 2006;Pasumarthi and Fadel, 2008;Morgan et al., 2013), suggest that ‫ء2␤4␣‬ nAChRs are most critical for postsynaptic responses to ACh and nicotine in Hcrtϩ neurons; postsynaptic ␣7 nAChRs also contribute to Hcrtϩ nicotinic responses, but are less prominent. Blockade of ‫ء2␤4␣‬ receptors by DH␤E not only decreases mEPSCs by disrupting presynaptic nicotinic signaling, but also prevents the activation of postsynaptic nicotinic receptors, and therefore affects spontaneous firing of Hcrtϩ neurons. In contrast, the role of presynaptic nAChRs has not been reported previously. In our experimental para-digm, either pressure application of ACh (1 mM) onto the soma and the proximal processes or bath application of nicotine (1 M) suppressed the spontaneous mEPSC frequency in Hcrtϩ neurons, which is consistent with effects reported in the arcuate nucleus of the hypothalamus (Huang et al., 2011). We also observed a consistent, though modest, reduction in mEPSC size that always Figure 8 ACh increased action potential (AP) firing or had no effect. In the same cells, however, ACh decreased mEPSC occurrence despite of its effect on action potential firing. A, A Hcrtϩ cell increased action potential firing rate in response to ACh, in presence of atropine. B, In the same cell, a puff of ACh decreased mEPSC frequency. Yellow bars indicate the time duration of ACh application. C, Another Hcrtϩ cell did not change action potential firing rate upon ACh puff. D, In the same cell, a puff of ACh decreased mEPSC frequency. accompanied the reduction in mEPSC frequency (Figs. 4 Ϫ 7). This could be due to effects of nicotinic signaling on activity of postsynaptic ionotropic glutamate receptors, in particular AMPA receptors. It is also possible that large presynaptic vesicles (Wojcik et al., 2004) or proximal synapses (which produce mEPSCs with larger amplitudes than distal ones; Bekkers and Stevens, 1996) are more significantly recruited by nicotinic signaling. Interestingly, the nicotinic antagonist, MEC, had a similar effect as ACh and nicotine on mEPSC frequency and amplitude (Figs. 4 Ϫ 6). Partial blockade of ␣4␤2 or ␣7 nAChRs by more selective antagonists also reduced mEPSC frequency, indicating that both heteromeric and homomeric nAChRs in brain are present in presynaptic glutamatergic terminals impinging on Hcrtϩ neurons. These results suggest that both ACh and nicotine likely desensitize nAChRs and interrupt glutamatergic transmission onto Hcrtϩ neurons, further suggesting that tonic activation of presynaptic nAChRs might be necessary for the normal function of neurotransmission in glutamatergic synapses impinging onto these neurons (Brain et al., 2001). Phasic cholinergic release may temporarily interrupt or reduce this tonic glutamatergic transmission. It is also possible that stimulation of postsynaptic nAChRs depolarizes membrane potential and releases calcium from internal stores, facilitating release of retrograde signaling molecules, such as endocannabinoids (Huang et al., 2007) or dynorphin , that result in negative feedback. These paradoxical presynaptic and postsynaptic effects of ACh may improve the signal-tonoise ratio of selective Hcrtϩ firing during phasic release, as might occur during exposure to behaviorally relevant stimuli (Dalley et al., 2001;Parikh et al., 2007), or in response to nicotine during smoking or behaviors relevant to drug reinforcement (Hollander et al., 2008). In particular, phasic ACh release would be expected to stimulate the firing of the approximately one-third of Hcrtϩ neurons expressing nAChRs, while also reducing activity in the remaining two-thirds of Hcrtϩ neurons by blocking synaptic excitatory events supported by tonic activation of presynaptic nAChRs (Fig. 8).
In conclusion, these results suggest that ACh modulates the Hcrt system through a multifaceted, nAChRmediated mechanism, which is complementary to the effects of muscarinic modulation of these neurons (Sakurai et al., 2005). More importantly, nAChR-mediated effects of ACh may enhance the output of a selective group of Hcrtϩ neurons expressing nAChRs, and distinguish this subset from the overall hypocretin network.