Nicotinic excitatory postsynaptic potentials in hippocampal CA1 interneurons are predominantly mediated by nicotinic receptors that contain α4 and β2 subunits.

2.Methods 2.1 Animal use A total of 51 animals were used in these studies that were housed in an animal care facility approved by the American Association for the Accreditation of Laboratory Animal Care (AAALAC). Animal experimental procedures followed a protocol approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University (protocol AD20205). This protocol adhered to the ethical guidelines described in the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). All efforts were made to minimize animal suffering and to reduce the number of animals used. Generation of retroviral adeno-associated virus (rAAV) expressing oChIEF in a Cre-dependent manner We obtained the mammalian codon optimized clone of ChIEF (oChIEF) fused to tdTomato( Lin et al., 2009 ; Lin, 2010 )(pCAGGS-I-oChIEF-tdTomato-I-WPRE) (gift of R. Tsien) and ligated it into rAAV-FLEX-rev-ChR2-tdTomato (Addgene, donated by Scott Sternson, ( Atasoy et al., 2008 ))at KpnI and XbaI sites replacing the ChR2-tdTomato sequence. rAAVs (serotype 2/1) were produced by Vector Biolabs (Philadelphia) (1.05 × 10 13 VG/ml). We refer to this virus as rAAV-Flex-rev-oChIEF-tdTomato. Because the sequence coding for oChIEF-tdTomato was reversed and floxed by two incompatible LoxP sites, oChIEF-tdTomato’s expression only occurred in infected cells that also expressed Crerecombinase( Atasoy et al., 2008 ). Stereotaxic injection of rAAV-Flex-rev-oChIEF-tdTomato into the MS/DBB of Chat-Cre mice All Chat-cremice (JAX Stock No. 006410) were initially anesthetized via an intraperitoneal injection of ketamine (100 mg/kg IP) and xylazine (2.5 mg/kg IP). Anesthesia was maintained with O 2 supplemented with 1.0% isoflurane. An incision was made in the skin along the midsagittal suture, and a very small hole was drilled in the skull overlying the septum. Analuminosilicate glass pipette containing undilutedrAAV-Flex-rev-oChIEF-tdTomatowas lowered to the level of the MS/DBB, andtwo midsagittal tracks of viral injections were performed, 1.0 mm and 0.7 mm rostral to Bregma at a rate of 100 nl/min using a motorized nanoinjector (Leica). At each coordinate, 8 × 100 nl injections were made between 3.5-5.0 mm in depth. Two weeks post viral injection, mice were killed for experimentation. Preparation of hippocampal slices Mice (38 to 49 days old) were used for experimentation. Animals deeply anaesthetized with ketamine (200 mg/kg) and xylazine (20 mg/kg), administered by intraperitoneal injection. Once the animals’ heart rate and respiration approached zero and the animals no longer responded to toe pinch, the animals were transcardially perfused with ice cold saline (consisting of (in mM): Sucrose 230, KCl 2.5, CaCl 2, MgCl 2 6, NaHPO 4 1, NaHCO 3 25, glucose 25) and sacrificed by decapitation. The brain was removed, hemi-sected, and horizontal slices containing the mid temporal hippocampus were cut at 350 μm on a vibratome 3000 (Ted Pella Inc, Redding CA). Sections were incubated in a holding chamber kept at 36°C for 30 min and then allowed to return to room temperature. The holding chamber solution consisted of normal saline (in mM): NaCl 125, KCl 3.0, CaCl 1.2, MgCl 2 1.2, NaHPO 4 1.2, NaHCO 3 25, glucose 25 bubbled with 95% O 2 / 5% CO 2 . Light-evoked release of acetylcholine from MS/DBB cholinergic axon terminals Neurons expressing oChIEF-tdTomato were stimulated by blue light transmitted through the epi-illumination light path of an Olympus BX51WI microscope and a 20x water immersion objective (0.95 NA). Blue light flashes (1 ms in duration) were generated from a flash lamp (JML-C2, Rapp Optoelectronic) by passing light through a D455/70x excitation filter focused into a liquid light guide. Blue light exiting the light guide was focused into the epillumination light path of the Olympus BX51WI microscope and back aperture of the 20x water immersion objective (0.95 NA) using a Flashcube 70 (Rapp Optoelectronics) and two dichroic mirrors (515dcxru, Chroma Technology).

Voltage-sensitive dye imaging

Slices were stained for 30 to 60 min with the voltage-sensitive dye (VSD) NK3630 (0.02 to 0.05 mg/ml) prior to experimentation. Following staining, slices were submerged and continuously perfused in a glass bottom recording chamber with warmed normal saline. The recording chamber was mounted on a fixed stage under an Olympus BX51WI microscope equipped with differential interference contrast (DIC) optics. The image of the slice was collected using transmitted near infrared light (> 775 nm) with a 20x (0.95 NA) water immersion objective. The image was captured (Foresight I-50 frame grabber) with a DAGE-MTI IR1000 CCD camera with contrast enhancement. For voltage-sensitive dye absorbance measurements, slices were illuminated with a tungsten-halogen 100 W lamp passed through a bandpass filter (705 ± 30 nm, Chroma Technology, Rockingham, VT). The transmitted light was collected with a Wutech H-469IV photodiode array that is part of the Redshirtimaging integrated Neuroplex II imaging system, mounted on the front port of the Olympus BX51WI microscope. The data were acquired, displayed and analyzed with Neuroplex software. To evoke synaptic ACh release in hippocampal CA1, 1 - 3 flashes of blue light were delivered at 20 ms intervals. Stimulating blue light was directed to the back aperture of the 20x objective by a 515dcxru dichroic mirror (Chroma Technology) for photostimulation. Transillumintated light responses were collected by the same objective and passed back through the dichroic mirror and a 675 nm long pass filter (Chroma Technology) onto surface of an hexagonal array of fiber optics that collect light for the Wutech photodiode array. Adjacent groups of photodiodes of the array, which were perpendicularly oriented to the SP, were selected to measure the VSD signals in SO, stratum pyramidale (SP), stratum radiatum (SR) and SLM of hippocampal CA1. The signal from one photodiode was spatially averaged with its immediate surrounding photodiodes (nine photodiodes in total) to represent the electrical response in each layer of CA1. Additionally, the VSD signals were an average of approximately 10 measurements. The VSD signals were sampled at 1.6 kHz and low pass filtered at 126 Hz. To eliminate or reduce the artifacts created by the stimulation light of the flash lamp, responses measured in the presence of TTX were subtracted from all measurements.

Electrophysiological Measurements Whole cell patch clamp recordings on hippocampal

CA1 interneurons were performed using patch pipettes (2 to 5 MΩ) pulled from borosilicate glass (8250 1.65/1.0 mm) on a Narishige PP830 pipette puller filled with (in mM): KMeSO4 135, NaCl 8, MgATP 2, NaGTP 0.3, HEPES 10, BAPTAK 4 0.1. Membrane potentials were measured with a Model 2400 patch clamp amplifier (A-M Systems, Port Angeles, WA) and converted into a digital signal by a PCI-6040E A/D board (National instruments, Austin, TX). WCP Strathclyde Software was used to store and analyze membrane potential responses on a PC computer (courtesy of Dr. J Dempster, Strathclyde University, Glasgow, Scotland). Further analysis was performed with Originpro 8.1 (OriginLab Corp., Northampton, MA, USA) and Instat 3.0 (Graphpad Software, La Jolla, CA, USA).

Morphological reconstruction of interneurons displaying nicotinic EPSPs

Slices were fixed in 4% paraformaldehyde in 0.1M phosphate buffer overnight, washed 6 × 10 minutes in phosphate buffered saline (PBS) and incubated for 3 days at 4 degrees in 1:1000 streptavidin Alexa Fluor 633 (Invitrogen) in PBS containing 0.4% Triton X 100 (Fisher). After incubation,theslices were washed 3 × 60 minutes in PBS and mounted in anti-fade mounting media.Processed slices were then reconstructed using a Zeiss LSM 510 META NLO confocal microscope (Carl Zeiss, Jena, Germany). Alex 633 was excited with the 633 nm line of the red HeNe 5W laser and cells were visualized using a 10 x dry lens (0.3 N.A., voxel dimensions 0.57 × 0.57 × 7.8 μm).

Statistics and data analysis

Data were analyzed using WCP software for the electrophysiological measurements and Neuroplex II software for the VSD recordings. Statistics were performed using GraphPadInstat (GraphPad Software, La Jolla, CA). Statistical significances were determined using a one way ANOVA or a repeated measures ANOVA and Bonferroni post hoc tests. Differences were determined to be statistically significant for p values less than 0.05. All data was reported as the mean +/− standard error of the mean (SEM).

Chemicals

All chemicals were purchased from VWR unless otherwise indicated. NK3630 was obtained from Hayashibara Co. (Japan). Dihydro-β-erythroidine (DHβE) and α-ConotoxinPnIA (α-CTX PnIA) were obtained from Tocris Bioscience (Ellisville, Missouri) and 6,7-Dinitroquinoxaline-2,3-dione (DNQX), DL-2-Amino-5-phosphonopentanoic acid (APV), methyllycaconitine (MLA) from Ascent Scientific (Bristol, U.K.).