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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(.)

Zhou WL, Gao XB, Picciotto MR - eNeuro (2015 Jan-Feb)

Bottom Line: Stimulation of postsynaptic nAChRs with ACh or nicotine resulted in a highly variable inward current in approximately one-third of hypocretin neurons.In contrast, ACh or nicotine (1 μM) reliably decreased the frequency of miniature EPSCs (mEPSCs).Finally, blocking internal calcium release reduced the frequency of mEPSCs, occluding the suppressive effect of presynaptic ACh.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Psychiatry and Comparative Medicine, Kavli Insitute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06508.

ABSTRACT

Hypocretin/orexin neurons regulate many behavioral functions, including addiction. Nicotine acts through nicotinic acetylcholine receptors (nAChRs) to alter firing rate of neurons throughout the brain, leading to addiction-related behaviors. While nAChRs are expressed in the hypothalamus and cholinergic fibers project to this structure, it is unclear how acetylcholine modulates the activity of hypocretin neurons. In this study, we stimulated hypocretin neurons in mouse brain slices with ACh in the presence of atropine to dissect presynaptic and postsynaptic modulation of these neurons through nAChRs. Approximately one-third of tested hypocretin neurons responded to pressure application of ACh (1 mM) with an increase in firing frequency. Stimulation of postsynaptic nAChRs with ACh or nicotine resulted in a highly variable inward current in approximately one-third of hypocretin neurons. In contrast, ACh or nicotine (1 μM) reliably decreased the frequency of miniature EPSCs (mEPSCs). Antagonism of nAChRs with mecamylamine also suppressed mEPSC frequency, suggesting that an endogenous, tonic activation of presynaptic nAChRs might be required for maintaining functional mEPSC frequency. Antagonism of heteromeric (α4β2) or homomeric (α7) nAChRs alone suppressed mEPSCs to a lesser extent. Finally, blocking internal calcium release reduced the frequency of mEPSCs, occluding the suppressive effect of presynaptic ACh. Taken together, these data provide a mechanism by which phasic ACh release enhances the firing of a subset of hypocretin neurons through postsynaptic nAChRs, but disrupts tonic, presynaptic nAChR-mediated glutamatergic inputs to the overall population of hypocretin neurons, potentially enhancing the signal-to-noise ratio during the response of the nAChR-positive subset of neurons.

No MeSH data available.


Related in: MedlinePlus

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 Hcrt+ 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 Hcrt+ cell did not change action potential firing rate upon ACh puff. D, In the same cell, a puff of ACh decreased mEPSC frequency.
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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 Hcrt+ 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 Hcrt+ cell did not change action potential firing rate upon ACh puff. D, In the same cell, a puff of ACh decreased mEPSC frequency.

Mentions: 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 Hcrt+ 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 α4β2* nAChRs are most critical for postsynaptic responses to ACh and nicotine in Hcrt+ neurons; postsynaptic α7 nAChRs also contribute to Hcrt+ nicotinic responses, but are less prominent. Blockade of α4β2* receptors by DHβE not only decreases mEPSCs by disrupting presynaptic nicotinic signaling, but also prevents the activation of postsynaptic nicotinic receptors, and therefore affects spontaneous firing of Hcrt+ neurons. In contrast, the role of presynaptic nAChRs has not been reported previously. In our experimental paradigm, either pressure application of ACh (1 mM) onto the soma and the proximal processes or bath application of nicotine (1 μM) suppressed the spontaneous mEPSC frequency in Hcrt+ 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 accompanied the reduction in mEPSC frequency (Figs. 4 – 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 – 6). Partial blockade of α4β2 or α7 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 Hcrt+ neurons. These results suggest that both ACh and nicotine likely desensitize nAChRs and interrupt glutamatergic transmission onto Hcrt+ 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 (Li and van den Pol, 2006), that result in negative feedback. These paradoxical presynaptic and postsynaptic effects of ACh may improve the signal-to-noise ratio of selective Hcrt+ 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 Hcrt+ neurons expressing nAChRs, while also reducing activity in the remaining two-thirds of Hcrt+ neurons by blocking synaptic excitatory events supported by tonic activation of presynaptic nAChRs (Fig. 8).


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(.)

Zhou WL, Gao XB, Picciotto MR - eNeuro (2015 Jan-Feb)

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 Hcrt+ 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 Hcrt+ cell did not change action potential firing rate upon ACh puff. D, In the same cell, a puff of ACh decreased mEPSC frequency.
© Copyright Policy - open-access
Related In: Results  -  Collection

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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 Hcrt+ 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 Hcrt+ cell did not change action potential firing rate upon ACh puff. D, In the same cell, a puff of ACh decreased mEPSC frequency.
Mentions: 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 Hcrt+ 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 α4β2* nAChRs are most critical for postsynaptic responses to ACh and nicotine in Hcrt+ neurons; postsynaptic α7 nAChRs also contribute to Hcrt+ nicotinic responses, but are less prominent. Blockade of α4β2* receptors by DHβE not only decreases mEPSCs by disrupting presynaptic nicotinic signaling, but also prevents the activation of postsynaptic nicotinic receptors, and therefore affects spontaneous firing of Hcrt+ neurons. In contrast, the role of presynaptic nAChRs has not been reported previously. In our experimental paradigm, either pressure application of ACh (1 mM) onto the soma and the proximal processes or bath application of nicotine (1 μM) suppressed the spontaneous mEPSC frequency in Hcrt+ 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 accompanied the reduction in mEPSC frequency (Figs. 4 – 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 – 6). Partial blockade of α4β2 or α7 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 Hcrt+ neurons. These results suggest that both ACh and nicotine likely desensitize nAChRs and interrupt glutamatergic transmission onto Hcrt+ 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 (Li and van den Pol, 2006), that result in negative feedback. These paradoxical presynaptic and postsynaptic effects of ACh may improve the signal-to-noise ratio of selective Hcrt+ 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 Hcrt+ neurons expressing nAChRs, while also reducing activity in the remaining two-thirds of Hcrt+ neurons by blocking synaptic excitatory events supported by tonic activation of presynaptic nAChRs (Fig. 8).

Bottom Line: Stimulation of postsynaptic nAChRs with ACh or nicotine resulted in a highly variable inward current in approximately one-third of hypocretin neurons.In contrast, ACh or nicotine (1 μM) reliably decreased the frequency of miniature EPSCs (mEPSCs).Finally, blocking internal calcium release reduced the frequency of mEPSCs, occluding the suppressive effect of presynaptic ACh.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Psychiatry and Comparative Medicine, Kavli Insitute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06508.

ABSTRACT

Hypocretin/orexin neurons regulate many behavioral functions, including addiction. Nicotine acts through nicotinic acetylcholine receptors (nAChRs) to alter firing rate of neurons throughout the brain, leading to addiction-related behaviors. While nAChRs are expressed in the hypothalamus and cholinergic fibers project to this structure, it is unclear how acetylcholine modulates the activity of hypocretin neurons. In this study, we stimulated hypocretin neurons in mouse brain slices with ACh in the presence of atropine to dissect presynaptic and postsynaptic modulation of these neurons through nAChRs. Approximately one-third of tested hypocretin neurons responded to pressure application of ACh (1 mM) with an increase in firing frequency. Stimulation of postsynaptic nAChRs with ACh or nicotine resulted in a highly variable inward current in approximately one-third of hypocretin neurons. In contrast, ACh or nicotine (1 μM) reliably decreased the frequency of miniature EPSCs (mEPSCs). Antagonism of nAChRs with mecamylamine also suppressed mEPSC frequency, suggesting that an endogenous, tonic activation of presynaptic nAChRs might be required for maintaining functional mEPSC frequency. Antagonism of heteromeric (α4β2) or homomeric (α7) nAChRs alone suppressed mEPSCs to a lesser extent. Finally, blocking internal calcium release reduced the frequency of mEPSCs, occluding the suppressive effect of presynaptic ACh. Taken together, these data provide a mechanism by which phasic ACh release enhances the firing of a subset of hypocretin neurons through postsynaptic nAChRs, but disrupts tonic, presynaptic nAChR-mediated glutamatergic inputs to the overall population of hypocretin neurons, potentially enhancing the signal-to-noise ratio during the response of the nAChR-positive subset of neurons.

No MeSH data available.


Related in: MedlinePlus