Timed-pregnant Sprague–Dawley rats (Taconic Farms, USA) were delivered on gestation day 15. All dams were housed in standard animal cages with conventional bedding material and ad libitum
access to food and water in a room with a 12-h light/dark cycle (lights on 06:00 hours). The variable rearing condition models and nomenclature were adopted according to Plotsky et al. (2005)
as follows. The day of birth was considered PND 0. Litters were standardized to 6–8 male pups per dam on PND 2 and were assigned to one of the following rearing conditions from PND 2 to PND 14 inclusive: (1) non-handled [hours of maternal separation (HMS-0)] animals were not exposed to any handling or experimenter manipulation, including cage bedding changes, from PND 2 to PND 14, (2) handling with brief maternal separation for 15 min (HMS-15) from PND 2 to PND 14, (3) handling with longer maternal separation for 180 min (HMS-180) from PND 2 to PND 14 and (4) a control group which consisted of vendor-reared rats (control).
The HMS protocol was initiated in the morning between 09:00 and 11:00 hours. Each dam was removed from the home cage to an adjacent cage, and each litter was removed and placed as a group into a plastic cage (15 × 15 cm) that was transferred to an incubator in an adjacent room. These pup cages were lined with 3 cm of bedding material (wood shavings) and were placed into an incubator set to maintain an ambient temperature of 32 °C. At the conclusion of the separation period, pups were returned to the maternity cage, rolled in the soiled home cage bedding material, and the dam was returned. Rat pups were weaned on PND 21–22. Electrophysiological experiments were performed within the next 2 wk. Care and use of animals was approved by the Institutional Animal Care and Use Committee of the Children's Hospital of Philadelphia.
Rats ranging in age from PND 22 to PND 35 were rapidly decapitated and the head placed in ice-cold oxygenated artificial cerebrospinal fluid (aCSF) in which sucrose was substituted for sodium. The brain was rapidly removed and blocked. Following blocking of the tissue, horizontal 200-μm-thick slices of brainstem containing the LC were cut using a Leica microslicer (Leica, USA) and placed in a holding vial containing aCSF at 34–36 °C bubbled with 95% O2/5% CO2 for 1 h. The composition of the aCSF was (in mm): NaCl 124, KCl 3.0, NaH2PO4 1.25, MgSO4 2.0, CaCl2 2.5, dextrose 10 and NaHCO3 26. After 1 h the slices were kept at room temperature, aCSF bubbled with 95% O2/5% CO2 until transferred to the recording chamber. A single slice was placed in a recording chamber and continuously superfused with oxygenated aCSF at 1.5–2 ml/min at 32 °C maintained by an in-line solution heater (TC-324; Warner Instruments, USA). LC neurons were visualized using a Nikon E600 upright microscope (Optical Apparatus, USA) fitted with a 40× water-immersion objective, differential interference contrast and infrared filter. The image from the microscope was enhanced using a CCD camera and displayed on a monitor. Recording pipettes were fashioned on a P-97 micropipette puller (Sutter Instruments, USA) using borosilicate glass capillary tubing (1.2 mm o.d., 0.69 mm i.d.; Warner Instruments). Pipettes were filled with electrolyte of the following composition (in mm): K-gluconate 70, KCl 70, NaCl 2.0, EGTA 4.0, Hepes 10, MgATP 4.0, Na2GTP 0.3, and 0.1% biocytin (pH 7.3).
A visualized cell was approached with the electrode, a GΩ seal established and the cell membrane ruptured to obtain a whole-cell recording using a Multiclamp 700B amplifier (Molecular Devices, USA). Series resistance was monitored throughout the experiment. If the series resistance of the electrode was unstable or exceeded four times the electrode resistance, electrophysiological data from the cell were discarded. The main criteria for accepting a recording were an action potential amplitude of 65–70 mV, action potential shape characteristic of an LC neuron and membrane potential between −50 and −60 mV. If the cell retained a stable baseline and resistance and did not depolarize over time, the cell was retained for analysis. Signals were digitized by Digidata 1320-series analogue-to-digital converter and stored online using pClamp 9 software (Molecular Devices). Only one cell per slice was recorded. The experimental protocol involved recording baseline cell characteristics in current clamp, including firing rate (Hz), input resistance (derived from the linear portion of a voltage-current plot of hyperpolarizing current steps MΩ), resting membrane potential (mV), membrane time constant (τ
, ms), action potential amplitude (mV) and duration (ms) and afterhyperpolarization (AHP) amplitude (mV) and AHP t1/2
duration (measured from the peak of the AHP to half the amplitude of the AHP, in ms) (Beck et al. 2004
). After determining baseline characteristics, CRF (final concentration of 11 nm
) was bath-applied for at least 3 min and then the cell characteristics were measured again. In some experiments, the selective CRF1
antagonist, antalarmin (final concentration of 3 μm
) was bath-applied after determining baseline cell characteristics. After antalarmin was in the bath for at least 10 min, the firing rate and membrane properties in the presence of antalarmin were recorded. CRF was then bath-applied as described above and cell characteristics determined again in the presence of both CRF and antalarmin. Data were analysed with Clampfit software (Molecular Devices).
Following recordings, slices were transferred to a vial containing 4% paraformaldehyde in phosphate buffer (PB, 100 mm, pH 7.4) and kept overnight in fixative. Immunohistochemistry for tyrosine hydroxylase (to identify the LC) and visualization of biocytin (to identify the labelled cell) were performed as follows. Slices were permeabilized with Triton X-100 (0.3%), non-specific binding was blocked with normal donkey serum (5%) for 1 h and slices incubated overnight at 4 °C with a mouse monoclonal antibody against tyrosine hydroxylase (1:1000; ImmunoStar, USA). After washing, the slices were incubated with donkey anti-mouse IgG Alexa Fluor 488 (1:1000) and streptavidin conjugated to Alexa Fluor 594 (1:1000) (Molecular Probes, USA) for 2 h. The slices were mounted on glass slides with Fluoromount-G (SouthernBiotech, USA) and visualized by fluorescence microscopy using a Leica DMRXA microscope. Images were captured with a Hamamatsu ORCA-ER digital camera (Bridgewater, USA) using Open Laboratory software (Improvision, UK).
In preliminary experiments the morphological characteristics of LC neurons that were patched for different durations were compared and it was determined that after 10 min there was no difference. For all of the neurons for which morphological characteristics were reported, cells were recorded for at least 30 min. Labelled LC neurons were photographed with a high-resolution charge-coupled device camera at 200× magnification. The morphological analysis was performed by an investigator blind to the treatment group of the specimen. Only cells explicitly located within the LC were included for analysis. Well-established methods of analysis were used to characterize and quantify dendritic morphology of the recorded cells (Swinny et al. 2004
; Swinny & Valentino, 2006
). The entire contours of dendrites for each cell were traced using the morphometric program supplied by OpenLab (Improvision). Dendritic projections arising from the cell soma were counted as primary dendrites. The branch number was defined as the number of times these primary dendrites gave rise to side branches. The length of the longest process was defined as the distance between the cell body and the most distal dendritic ending. Finally, the total dendritic length was calculated by the addition of the lengths of all dendritic arbors.
Data are presented as means±standard errors of the mean (s.e.m.) unless otherwise stated. Statistical differences between baseline electrophysiological characteristics and morphological parameters of the different treatment groups were assessed using parametric and non-parametric one-way analysis of variance (ANOVA) and Tukey–Kramer post-hoc test. A paired t test was used to test for significance of the CRF or antalarmin effect.
Solutions and drugs
All chemicals used for making the aCSF and electrolyte were obtained from Sigma (USA). Ovine CRF was supplied by Dr J. Rivier (The Salk Institute, San Diego, CA, USA) and used at a final concentration of 11 nm. Antalarmin (dissolved in DMSO; final DMSO concentration in bath, 0.015%) was provided by Kenner C. Rice (Laboratory of Medicinal Chemistry, NIH/NIDDK, Bethesda, MD, USA) and used at a final concentration of 3 μm.