Techniques for histological reconstruction of brain stem recording sites

The masseter muscle of Sprague-Dawley rats was injected with 10 mg of horseradish peroxidase. The electrical activity of the trigeminal motor nucleus was investigated 24-36 hours later using micropipettes filled with a 2.0 M NaCl-7% fast green FCF solution. A parallel series of penetrations at 200 micron intervals was made through the motor complex at known depths. The grid formed from these penetrations was reconstructed using the following techniques. Marks were made at known depths in one or more of the tracts by iontophoresing fast green from the microelectrode tip with a hyperpolarizing current of 10 muA for 10 minutes. To assure proper alignment of the tissue containing the green marks, two agar-India ink plugs were placed caudal and parallel to the recording sites. The frozen tissue was sectioned on a microtome, first through the agar plugs and then through the tissue containing the green marks. The tissue could be incubated for the peroxidase reaction product or stained for Nissl substance. These combined procedures offer a means to correlate the structure and function of brain stem nuclear groups.