Golgi Staining

The Golgi fixative consisted of three solutions that were prepared separately and combined in the final step of the process. Solution A required heating 750 ml of distilled water to 90°C prior to the addition of 37.5 grams (g) of mercuric chloride (Hg), the solution was then allowed to cool. Solution B required the heating of 750 ml of dH₂O to 60°C followed by the addition of 37.5 g of potassium dichromate (K₂Cr₂O₇), this solution was then allowed to cool. Solution C was made by dissolving 30 g of potassium chromate (K₂Cr₂O₄) in 750 of dH₂O. Solutions A, B, and C were then combined in a new flask and 1500 ml of room temperature dH₂O was added. The solution was stored in a glass jar and covered in tinfoil for a least 6 days prior to use.

Following 14 days of incubation in the Golgi fixative, whole brains underwent a series of dH₂O and sucrose washes in preparation for tissue sectioning and development. Initially, brains were immersed in dH₂O for 4 h then immersed in fresh dH₂O for another 3 h followed by fresh dH₂O overnight. On the second day of washes, brains were immersed in 10% sucrose for 8 h followed by 20% sucrose overnight. On the third day, brains were immersed in 30% sucrose for a minimum of 4 days prior to sectioning on a Vibratome.

A humidified box with a plastic grate was created 24 h prior to sectioning to prevent the tissue from drying when it was slide mounted. Tissue was sectioned on a Vibratome at 200 μm and floated in a 30% sucrose solution prior to mounting. Twenty sections were taken from each brain through the NAc and placed immediately onto double dipped (2%) gelatinized slides. Slides were then placed in the humidified box in a dark cupboard for no longer than 24 h.

A desiccated box was created 24 h prior to development by placing Drierite pellets in the bottom of a plastic box that contained a plastic grate. Golgi staining was developed by immersing tissue mounted slides in several solutions, beginning with a 1-min rinse in dH₂O. Next, slides were submerged in 28% ammonium hydroxide for 40 min and then dH₂O for 1 min. Slides were then submerged in Kodak Film Fix A (diluted 1:1 with dH₂O) for 40 min where the solution was kept protected from light. Next, slides were washed twice in dH₂O prior to dehydration. For dehydration, slides were submerged in 50% ethyl alcohol (ETOH) for 1 min, 75% ETOH for 1 min, and 95% ETOH for 1 min. The remaining solutions were desiccated with molecular sieve for at least 24 h prior to use. Slides underwent three 5-min washes in 100% desiccated ETOH following by a 10-min wash in a desiccated solution of 33%ETOH, 33% Clearene and 33% chloroform. The final wash consisted of slides being placed in desiccated Clearene for 15 min. Slides were then coverslipped with Permount and placed in a desiccated box for a minimum of 3 days to allow the Permount to harden.

For each of the three behavioral groups (operant, classical, control), three MSNs within the NAcSh per rat, with a minimum of three rats per abstinence period were analyzed. This resulted in a minimum of nine neurons per abstinence period within each of the behavioral groups. The NAcSh was constricted in the anterior-posterior plane from approximately +1.7 to +3.2 mm from bregma according to Paxinos and Watson (1998). Neurons were picked at random but had to meet specific criteria to be selected. Neurons had to be entirely impregnated, staining had to be uniform and complete, the cell body had to be within the 200 μm section depth, and the neuron had to be relatively isolated from surrounding neurons. Neurons were reconstructed at 100× magnification using Neurolucida software (MicroBrightField, Williston, VT, USA). The cell body was traced first, followed by a minimum of three primary dendrites and subsequent branches, in their entirety, and all visible spines were marked. For each neuron, the total number of spines were analyzed and divided by total dendritic length to obtain the spine density of each neuron.