Alpha4 containing nicotinic receptors are positioned to mediate postsynaptic effects on 5-HT neurons in the rat dorsal raphe nucleus

Abstract

Nicotinic acetylcholine receptors containing the alpha4 and beta2 subunits constitute the most abundant high-affinity binding site of nicotine in the brain and are critical for the addictive qualities of nicotine. Serotonin neurotransmission is thought to be an important contributor to nicotine addiction. Therefore in this study it was examined how alpha4-containing receptors are positioned to modulate the function of serotonin neurons using ultrastructural analysis of immunolabeling for the alpha4 receptor subunit in the dorsal raphe nucleus (DR), a primary source of forebrain serotonin in the rat. Of 150 profiles labeled for the alpha4 subunit, 140 or 93% consisted of either soma or dendrites, these were often small-caliber (distal) dendrites <1.5 um in diameter (63/150 or 42%). The majority (107/150 or 71%) of profiles containing labeling for alpha4 were dually labeled for the synthetic enzyme for serotonin, tryptophan hydroxylase (TPH). Within dendrites immunogold labeling for alpha4 was present on the plasma membrane or near postsynaptic densities. However, labeling for alpha4 was commonly localized to the cytoplasmic compartment often associated with smooth endoplasmic reticulum, plausibly representing receptors in transit to or from the plasma membrane. Previous studies have suggested that nicotine presynaptically regulates activity onto serotonin neurons, however alpha4 immunolabeling was detected in only 10 axons in the DR or 7% of profiles sampled. This finding suggest that alpha4 containing receptors are minor contributors to presynaptic regulation of synaptic activity onto serotonin neurons, but rather alpha4 containing receptors are positioned to influence serotonin neurons directly at postsynaptic sites. Keywordsacetylcholine; nicotine; anxiety; 5-HT-1A; addictionThe dorsal raphe nucleus (DR) is one of the primary sources of serotonin (5-HT) in the forebrain. Cholinergic modulation of the DR likely influences several physiological functions including sleep cycle (Portas et al., 2000), anxiety and pain sensitivity. In addition, the actions of nicotine in the DR may contribute to nicotine addiction. Acute nicotine administration modifies 5-HT release in the forebrain (Toth et al., 1992, Ribeiro et al., 1993, Summers et al., 1996. Chronic nicotine administration produces alterations in 5-HT receptors, transporters and brain metabolism of 5-HT in both humans and animal models (Benwell and Balfour, Correspondence to: Kathryn G. Commons, Ph.D., Children's Hospital, 300 Longwood Ave., Enders 1206e, Boston, MA 02115, Telephone: 617-919-2220, Fax: 617-730-0235, Email: Kathryn.Commons@Childrens.Harvard.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discove...

α4* Nicotinic Acetylcholine Receptors Modulate Experience-Based Cortical Depression in the Adult Mouse Somatosensory Cortex

“…5A). Consistent with previous electron microscopy studies (Nakayama et al, 1995;Arroyo-Jiménez et al, 1999;Commons, 2008), putative ␣4* nAChR expression was found in the endoplasmic reticulum, as well as near the plasma membrane of neuronal cell bodies and their apical dendrites (Fig. 5 B, C, red arrows).…”

“…Our ultrastructural analysis indicated that ␣4* nAChRs were often associated with peri-or extrasynaptic sites on neuronal cell bodies. This finding is supported by electron microscopy studies showing that ␣4* nAChRs are detected near the plasma membrane of neuronal cell bodies and apical dendritic shafts, but not typically at postsynaptic densities (Nakayama et al, 1995;Commons, 2008). Whether ␣4* nAChR are directly apposed by cholinergic terminals is not entirely clear, however our immunofluorescence data suggest a loose spatial relationship between ␣4* nAChRs and cholinergic terminals, similar to that described for ␣7 nAChRs and muscarinic receptors (Jones and Wonnacott, 2004;Yamasaki et al, 2010).…”

“…Furthermore, our thalamic and VGlut2 labeling experiments indicated that only a small fraction of ␣4-YFP puncta colocalized with thalamocortical axons in layer 4 of the barrel cortex. Although this observation may not be generalized to other brain regions such as prefrontal cortex (Vidal and Changeux, 1993;Lambe et al, 2003), we should note that the relatively scant colocalization of ␣4* nAChRs with presynaptic structures in the present work is consistent with ultrastructural examination of these receptors in rat somatosensory cortex and dorsal raphe nucleus (Nakayama et al, 1995;Commons, 2008). Our ultrastructural analysis indicated that ␣4* nAChRs were often associated with peri-or extrasynaptic sites on neuronal cell bodies.…”

Presynaptic α4β2 Nicotinic Acetylcholine Receptors Increase Glutamate Release and Serotonin Neuron Excitability in the Dorsal Raphe Nucleus

“…Also, immunolabeling for the ␣4 receptor subunit has been detected in axon terminals in the DRN (Commons, 2008). Data obtained in our laboratory revealed that postsynaptic nAChRs of 5-HT and non-5-HT DRN neurons are functional (Galindo-Charles et al, 2008) and that nicotine increases the firing frequency of ϳ80% of 5-HT DRN neurons (Mihailescu et al, 1998(Mihailescu et al, , 2002.…”

“…Immunocytochemical studies have demonstrated that both 5-HT and non-5-HT DRN neurons express postsynaptic nAChRs of ␣4␤2 and ␣7 subtypes (Bitner et al, 2000;Bitner and Nikkel, 2002;Commons, 2008). Also, immunolabeling for the ␣4 receptor subunit has been detected in axon terminals in the DRN (Commons, 2008).…”