The dorsal raphe nucleus (DRN) contains large populations of serotonergic (5-HT) neurons. This nucleus receives GABAergic inhibitory afferents from many brain areas and from DRN interneurons. Both GABAergic and 5-HT DRN neurons express functional nicotinic acetylcholine receptors (nAChRs). Previous studies have demonstrated that nicotine increases 5-HT release and 5-HT DRN neuron discharge rate by stimulating postsynaptic nAChRs and by increasing glutamate and norepinephrine release inside DRN. However, the influence of nicotine on the GABAergic input to 5-HT DRN neurons was poorly investigated. Therefore, the aim of this work was to determine the effect of nicotine on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of 5-HT DRN neurons and the subtype of nAChR(s) involved in this response. Experiments were performed in coronal slices obtained from young Wistar rats. GABAergic sIPSCs were recorded from post hoc-identified 5-HT DRN neurons with the whole cell voltage patch-clamp technique. Administration of nicotine (1 μM) increased sIPSC frequency in 72% of identified 5-HT DRN neurons. This effect was not reproduced by the α4β2 nAChR agonist RJR-2403 and was not influenced by TTX (1 μM). It was mimicked by the selective agonist for α7 nAChR, PNU-282987, and exacerbated by the positive allosteric modulator of the same receptor, PNU-120596. The nicotine-induced increase in sIPSC frequency was independent on voltage-gated calcium channels and dependent on Ca 2+ -induced Ca 2+ release (CICR). These results demonstrate that nicotine increases the GABAergic input to most 5-HT DRN neurons, by activating α7 nAChRs and producing CICR in DRN GABAergic terminals.

the dorsal raphe nucleus (DRN) is located in the brain stem and contains the largest population of serotonergic (5-HT) neurons in the brain (Dahlström and Fuxe 1964). This nucleus provides 5-HT innervation to several targets including the forebrain and limbic structures (see Michelsen et al. 2008 for review) and is involved in several behavioral functions such as sleep-wake states, feeding, nociception, neuroendocrine regulation, learning and memory, and stress-induced responses (Hale et al. 2012; Jacobs and Azmitia 1992; Meneses 2013). In addition, dysregulation of this nucleus has been associated with psychiatric disorders such as depression and anxiety (Lowry et al. 2008; Sharp and Cowen 2011). Clinical studies have demonstrated that transdermal nicotine improves mood in patients with major depression (Salín-Pascual and Drucker-Colín 1998) and reduces anxiety in both smokers and nonsmokers (Gilbert 1979; Kassel and Unrod 2000). Likewise, in rodents, nicotine reduces stress and anxiety induced by restraint behavior (Hsu et al. 2007).

Experimental studies have shown that nicotine increases the firing rate of the majority of DRN neurons (Li et al. 1998; Mihailescu et al. 2002) as well as 5-HT release in several brain areas such as prefrontal cortex (Ribeiro et al. 1993) and DRN itself (Mihailescu et al. 1998). Immunocytochemical studies have demonstrated the presence of α7 and α4β2 nicotinic acetylcholine receptor (nAChR) subtypes in 5-HT DRN neurons (Bitner et al. 2000; Commons 2008). Other studies have indicated that nicotine increases the firing activity of 5-HT neurons through direct stimulation of their somatodendritic α7 and α4β2 nAChRs (Galindo-Charles et al. 2008) and through presynaptic release of glutamate (Garduño et al. 2012) and norepinephrine (Li et al. 1998).

GABAergic inhibitory afferents to DRN originate in several brain areas such as lateral and posterior hypothalamus, lateral preoptic area, ventral pontine periaqueductal gray, substantia nigra, and ventral tegmental area (VTA), as well as interneurons from the DRN itself (Gervasoni et al. 2000). Serotonergic neurons are tonically inhibited by GABA through both GABA A and GABA B receptors (Bowery et al. 1987; Gervasoni et al. 2000). Supporting this idea, the administration of GABA A antagonists, such as picrotoxin or bicuculline, increases action potential frequency in 5-HT DRN neurons (Gallager 1978; Gallager and Aghajanian 1976). It was suggested that the GABAergic input to DRN neurons increases during rapid eye movement (REM) sleep (Nitz and Siegel 1997), which explains the low activity of 5-HT DRN neurons registered during this period. However, there are few electrophysiological studies concerning the influence of nicotine on the GABAergic input to 5-HT DRN neurons, as well as the nicotinic receptor involved in this response. This is an interesting topic considering the above-mentioned stimulatory effect of nicotine on the firing rate of 5-HT DRN neurons. Thus the aim of this work was to determine the effect of nicotine on the GABAergic input to 5-HT DRN neurons and to identify the nAChR subtype involved in this effect.

METHODS Slice preparation. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee of the Universidad Nacional Autónoma de México. Experiments were performed in young (postnatal day 18–21) Wistar rats that were deeply anesthetized with isoflurane and then decapitated. Their brains were quickly removed and placed into ice-cold (4°C) artificial cerebrospinal fluid (ACSF) consisting of (in mM) 125 NaCl, 3 KCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 1 MgCl 2 , 1.2 CaCl 2 , and 25 glucose, 300 mosM, pH = 7.3 by bubbling with 95% O 2 -5% CO 2 . Coronal slices (350 μm thick) containing the DRN were obtained with a Vibratome 1500 (Vibratome, St. Louis, MO) and allowed to rest in carbogen-bubbled ACSF at room temperature for at least 1 h before recording. Electrophysiological recordings. Individual slices were transferred into a custom-made Plexiglas recording chamber and perfused with ACSF at a rate of 4–5 ml/min at 33°C maintained by an in-line solution heater (TC-324; Warner Instruments). DRN neurons were visualized with a videomicroscopy system (Olympus BX51WI) fitted with a ×60 water-immersion objective, differential interference contrast and infrared filter. The image from the microscope was enhanced with a CCD camera and displayed on a monitor. Whole cell current- and voltage-clamp recordings were performed with a Multiclamp 700A amplifier (Axon Instruments, Union City, CA) and monitored with a PC running Clampex 8 software (Axon Instruments). Signals were digitized by a Digidata 1320 series analog-to-digital converter at 10 kHz and stored online with pCLAMP8 software (Molecular Devices). Only one cell was recorded per brain slice. Micropipettes used for recordings were pulled from borosilicate glass tubes (WPI, Sarasota, FL) with a Flaming-Brown puller (Sutter Instrument, Novato, CA) (resistance 4–7 MΩ). The internal solution consisted of (in mM) 70 K-gluconate, 70 KCl, 5 NaCl, 1 MgCl 2 , 0.02 EGTA, 10 HEPES, 2 Mg 2 ATP, and 0.5 Na 2 GTP, with biocytin 0.1%, pH = 7.3 with Trizma base, 280–300 mosM. Spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded at a holding potential of −70 mV, and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX; 10 μM) and dl-2-amino-5-phosponovaleric acid (APV; 50 μM) were used to block glutamatergic currents. Miniature inhibitory postsynaptic currents (mIPSCs) were equally recorded according to the same protocol, but with tetrodotoxin (TTX; 1 μM) also added to the perfusion solution. Series resistance was monitored throughout the experiment; if it was unstable or exceeded four times the electrode resistance, the cell was discarded. Drug administration. Several protocols of drug administration were used, as imposed by the purpose of the experiments. Recordings of at least 10 min were allowed for stabilization of sIPSC frequency, in which blockers of glutamate receptors (CNQX and APV) were present in the perfusion solution. Afterwards, bicuculline, nicotine, or nAChR agonists were added to the perfusion fluid and their effects were recorded during the administration (8–10 min) and during the period of washout (15 min or more). In the experiments using drugs susceptible to alter nicotine effects [nAChR antagonists, PNU-120596, TTX, blockers of calcium-induced calcium release (CICR) and of voltage-gated calcium channels (VGCCs)], one of these drugs was administered before nicotine and its effects on sIPSC frequency and amplitude were followed for at least 10 min. Nicotine was then added to the perfusion fluid for 10 min, and its effects on sIPSC frequency were recorded during the administration as well as 15 min after the washout of drugs. Immunohistochemistry. Neurons were filled with biocytin present in the internal solution during recordings. To identify whether recorded cells were 5-HT, we used an anti-5-HT antibody. After electrophysiological recording, slices were fixed in 4% paraformaldehyde and 1% picric acid in 0.1 M PBS (pH 7.4) until staining. Slices were infiltrated with 30% sucrose and cut on a vibratome into 40-μm sections. Sections were incubated for 4–6 h in PBS solution containing 0.2 Triton X-100 and streptavidin conjugated to Cy3 (1 mg/ml; Zymed, South San Francisco, CA; diluted 1:100) to label the recorded neuron. Sections were rinsed in PBS and incubated for 18–24 h at 4°C with primary rabbit anti-5-HT antisera (ImmunoStar, Hudson, WI; 1:2,000). After rinsing in PBS, sections were reincubated for 2–4 h with secondary antibodies conjugated to fluorescein (Vector Laboratories, Burlingame, CA; diluted 1:100). The reacted sections were first examined with an appropriate set of filters on an epifluorescence-equipped microscope. Afterwards, sections were mounted in an antiquenching medium (Vectashield, Vector Laboratories) and examined under a confocal microscope (MRC 1024, Bio-Rad, Natford, UK) equipped with a krypton/argon laser. A two-line laser emitting at 550- and 500-nm wavelength was used for exciting Cy3 and fluorescein, respectively. Digitized images were transferred to a personal computer with image-capturing software (Confocal Assistant, T. C. Brelje). Drugs. Drugs were dissolved in ACSF and administered by bath perfusion. The time required for obtaining equilibrated concentrations of the drugs in the recording chamber was ∼3–4 min. TTX, APV, CNQX, (−)bicuculline methiodide, methyllycaconitine (MLA), dihydro-β-erythroidine hydrobromide (DHβE); N-(5-chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596), 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid ethyl (nitrendipine), and biocytin were purchased from Sigma-Aldrich RBI (St. Louis MO). Thapsigargin, ryanodine, (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine oxalate (RJR-2403 oxalate), and N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide (PNU-282987) were purchased from Tocris Bioscience (Ellisville, MO). ω-Agatoxin-TK and ω-conotoxin-GVIA were purchased from Peptide Institute and Alomone Labs, respectively. All reagents were added from freshly prepared stock solution to the bath saline. Data analysis and statistics. Data analysis was performed with Clampfit 8 software (Molecular Devices) and Mini Analysis software (Synaptosoft, Decatur, GA). The Mini Analysis software was used to detect sIPSCs and mIPSCs and to assess their frequency and amplitude. Initially, a noise analysis was conducted for each recorded cell, and detection thresholds were set to exceed noise values. The events were detected automatically and then visually inspected and confirmed. For each recorded cell, cumulative 10-s sIPSC frequency histograms were constructed and the average control frequency of sIPSCs during a period of 10 min was calculated. The average control values of sIPSC frequency obtained for individual cells were fairly heterogeneous. Therefore, the changes in sIPSC frequency produced by drug administration were expressed as a percentage of the control value. The normalized sIPSC frequency histograms obtained from individual cells of a given experimental group were averaged. The Wilcoxon signed-rank test and the Mann-Whitney test on ranks were used for statistical comparisons between samples. Cumulative fractions of amplitude and interevent intervals were compared by Kolmogorov-Smirnoff test. P < 0.05 was taken as significant.

GRANTS This work was supported by Dirección General de Asuntos del Personal Académico (DGAPA)-UNAM Grants IN220112 to S. Mihailescu and IN212313 to S. Hernández-López.

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS Author contributions: F.H.-V., S.H.-L., and S.P.M. conception and design of research; F.H.-V. and K.C. performed experiments; F.H.-V., K.C., J.G., and S.H.-L. analyzed data; F.H.-V., S.H.-L., and S.P.M. interpreted results of experiments; F.H.-V. and J.G. prepared figures; F.H.-V. drafted manuscript; K.C., J.G., S.H.-L., and S.P.M. approved final version of manuscript; S.P.M. edited and revised manuscript.