Mice were bred in-house by mating one parent homozygous for a Met
allele in which exon 16 is flanked by loxP (floxed) sites (Metfx/fx
, provided by S. Thorgeirsson; (Judson et al., 2009
)) with another parent carrying an Emx1
-cre transgene (provided by K. Jones; (Gorski et al., 2002
)) and a single floxed Met
). The Metfx/fx
breeding lines were both fully back-crossed onto the C57BL/6J
background. The genotypes of their offspring (i.e., Metfx/fx
/WT) were determined by PCR as described previously (Judson et al., 2009
) or with real-time PCR by Transnetyx (Cordova, TN). Electrophysiological data collected at different institutions were consistent with each other. Data from male and female mice were pooled, as initial analysis of data from individual animals did not reveal any sex differences. All procedures using mice were approved by the Institutional Animal Care and Use Committee of University of Southern California, Vanderbilt University, or Northwestern University and conformed to NIH guidelines.
Retrograde labeling was performed in anesthetized mice as described (Anderson et al., 2010
). Contralaterally projecting corticostriatal neurons in AFC were selectively labeled by stereotaxically injecting ~50 nL of red or green fluorescent microspheres (Lumafluor) into the (left) dorsolateral striatum (coordinates, relative to bregma: 0.0 mm posterior, 2.0 mm lateral, 2.5 mm ventral). Corticopontine neurons were labeled by injecting the (right) ventral pons in the region of the pontine nuclei, approached at a 50° angle off the vertical axis through a craniotomy 3.2 mm posterior to lambda, 0.5 mm lateral, and 4.9 mm ventral from the surface of the brain. In all cases, animals were injected ≥16 hr prior to brain slice preparation.
Brain slices were prepared as described (Anderson et al., 2010
). Mice (male or female) were sacrificed at P21–32 by decapitation under deep isoflurane anesthesia. Brains were dissected and sectioned in ice-cold, carbogenated choline solution (in mM: 110 choline chloride, 25 NaHCO3
, 2.5 KCl, 1.25 NaH2
, and 0.5 CaCl2
, 7 MgSO4
, 25 D-glucose, 11.6 sodium ascorbate, 3.1 sodium pyruvate). Parasagittal slices (300 µm thick) of the right cerebral hemisphere containing the AFC were cut, approximately 1–2 mm lateral to the midline, using a slice angle that was rolled rightward ~15° to align the slice plane optimally with pyramidal neuron apical dendrites and descending axons. This angle yielded 2–3 contiguous slices suitable for recording. We routinely verified that the recorded neurons had intact apical dendrites, based on epifluorescence visualization of the dye-filled neuron after recording. Slices were transferred to and incubated in carbogenated ACSF (in mM: 126 NaCl, 2.5 KCl, 26 NaHCO3
, 2 CaCl2
, 2 MgCl2
, 1.25 NaH2
, and 10 D-glucose) for 30 min at 35 °C, and then maintained at 22 °C until recording.
Electrophysiological recordings and LSPS mapping were performed as described (Anderson et al., 2010
; Shepherd, 2011
), with the experimenter blind to genotype. Slices were transferred to the recording chamber of a custom-built LSPS microscope, and fluorescently labeled neurons were patched with internal solution (in mm: 126 K-gluconate, 4 KCl, 10 HEPES, 4 ATP, 0.3 GTP, 10 mM phosphocreatine, and either 0 or 0.05 Alexa 594, 488, or 350). LSPS mapping was performed at 22 °C in ACSF with 0.2 mM MNI-caged glutamate (Tocris), 4 mM CaCl2
, 4 mM MgCl2
, and 5 µM CPP (Tocris). Intrinsic properties were measured in current clamp mode immediately before mapping, by presenting families of current steps (−100 to +500 pA in 50 pA increments, duration 0.5 sec). Traces were analyzed offline to identify action potentials (APs). Frequency-current relationships were calculated based on the numbers of APs per current step, and the frequency-current slopes were calculated by linear regression. Spike frequency adaptation was calculated by averaging the ratio of the third and fifth inter-spike interval for all responses with >5 APs. Signals were filtered at 2–4 KHz and sampled at 10 KHz. For LSPS we used Ephus
software to control data acquisition (www.ephus.org
; (Suter et al., 2010
)) and custom routines for offline analysis. In some cases, neurons were filled with biocytin during recording, and subsequently stained with streptavidin-conjugated fluorophores and imaged on a two-photon laser scanning microscope system. Paired recordings were made in plain ACSF at 32 °C.
Morphometric measurements were made from slice images captured during experiments. Cortical thickness (dcortex
) was measured as the distance from the cortical surface (pia) to the border of layer 6 and white matter (WM). The depth of the soma in the cortex was measured as the distance from the pia to the soma (dsoma
), and the normalized soma position was calculated as dsoma
. Similarly, we measured the distance from the pia to the layer 5A/B border (d5A/B
) and calculated the normalized distance as dL5A/B
. Identification of this border was guided by a previous quantification of the laminar profile of optical density in video micrographs of agranular cortex (Weiler et al., 2008
). Neuronal density was measured in Nissl-stained slices of AFC, by counting the number of neurons in a defined region of interest in layer 2/3 (a 0.1 by 0.2 mm box).
Synaptic input maps were analyzed using custom routines and published approaches (Anderson et al., 2010
). These are described further in the Results. We note here that black pixels in input maps, representing dendritic stimulation, are ignored in calculating averages (i.e., averages across map planes, for calculating ‘front views’; or averages across map rows, for calculating ‘side views’). There are fewer black pixels in front view average maps than in individual maps, in which black pixels occur at various locations. On the one hand this is a positive effect: more details (pixels) emerge as more neurons are pooled for averaging. On the other hand, it introduces an under-sampling effect, because for these emergent pixels the number of neurons contributing to their average pixel will be less than the total number of neurons in the group. This effect is strongest near the soma, where one or more pixels usually remain black even after averaging, but also affects pixels around the apical dendrite. The latter is potentially problematic because this is also a region of synaptic input from layer 2/3. However, because black pixels tended to be sparse and variable in location, this effect was generally minor. For layer 2/3 pixels, in the most severe case, the fraction of neurons contributing to the average, relative to total neurons, was 0.3, but on average this fraction was ~0.9. We emphasize that average maps are used as a graphical tool to aid in the visualization of these complex three dimensional data sets.
Unless otherwise indicated, group data including bars in graphs represent the mean ± s.e.m. Differences in the means of two or more groups were tested by Student’s t-test (for normally distributed data) or Wilcoxon rank-sum test (for non-normally distributed data), as indicated. For all comparisons, significance was defined as p < 0.05.