Neurolucida 7

Coronal slices between bregma +2.8 and +1.7 were surveyed for MSNs within the core and shell of the NAc, using the lateral ventricle and the anterior commissure as landmarks with the aid of a rat brain atlas (Paxinos and Watson, 2007 ) (Figure 1). The contour function in Neurolucida 7 (MBF Bioscience, VT, USA) was used to demarcate the NAc core and NAc shell in each slice (Figure 2). Between 2 and 9 neurons per region per animal were traced for dendritic length parameters using a 63x objective or for spine densities (reported as spines per 100 μm) using a 100x objective on a Zeiss Axioskop II (Carl Zeiss, Germany) using an automated xyz stage driven by Neurolucida® 7 software (MBF Biosciences, VT, USA). All tracing was performed in a blinded fashion with respect to treatment. Morphological parameters of Golgi-Cox impregnated neurons were analyzed in a manner similar to previous reports (Klenowski et al., 2015 (link)).

As described in detail previously [18 (link)], coronal slices between bregma -2.54 and -3.24 were surveyed for principal neurons within the BLA, using the internal capsule and the external capsule as landmarks with the aid of a rat brain atlas [34 ]. The contour function in Neurolucida 7 (MBF Bioscience, VT, USA) was used to demarcate the BLA and the LA in each slice. Between 2 and 6 neurons were sampled from the anterior and posterior basolateral amygdaloid nuclei within the BLA from each animal (Fig 1) and were traced for dendritic length parameters using a 63x objective or for spine densities (reported as spines per 100 μm) using a 100x objective on a Zeiss Axioskop II (Carl Zeiss, Germany) using an automated xyz stage driven by Neurolucida^® 7 software (MBF Biosciences, VT, USA). All tracing was performed in a blinded fashion with respect to treatment. Morphological parameters of Golgi-Cox impregnated neurons were analyzed in a manner similar to previous reports [35 (link)].

Biocytin Fills: To reliably reconstruct the fine axonal branches of cortical neurons, dedicated experiments were performed following the classical avidin-biotin-peroxidase method. Biocytin (Sigma) was added to the intracellular solution at a high concentration (5–10 mg/ml), which required extensive sonication. At the end of recordings, the patch pipette was removed carefully until obtaining an inside out patch. The slice was then left in the recording chamber for at least further 5–10 min to allow further diffusion. Slices were then fixed with 4% paraformaldehyde in phosphate buffer saline (PBS, Sigma) for at least 48 hr. Following fixation, slices were incubated with the avidin-biotin complex (Vector Labs) and a high concentration of detergent (Triton-X100, 5%) for at least two days before staining with 3,3′Diaminobenzidine (DAB, AbCam). Cells were then reconstructed and cortical layers delimited using Neurolucida 7 (MBF Bioscience) and the most up to date mouse atlas (Allen Institute).