Digital midgard precision current source

After the final recording, small electrolytic lesions were made by passing an electric current (4 μA, 150 s using an Elgiloy electrode and a lesion maker (Digital Midgard Precision Current Source, Stoelting Co., USA) to locate the recording sites. Then, the monkeys were deeply anesthetized with an overdose of pentobarbital sodium and were perfused with 0.5 M phosphate buffer saline (PBS) followed by 8% formalin in 0.5 M PBS. The lesions were identified in 100 μm-thick coronal sections cut on a freezing microtome, and these lesions were used as reference points to determine the recording sites. Figure 2 shows Nissl-stained sections at anteroposterior (AP) + 26 mm in the left hemisphere of monkey P and AP + 31 mm in the left hemisphere of monkey C.

Experiments involving neural pathway-tracing were as follows: (Exp. 2) Retrograde tracing from the dpLHA following unilateral pressure injection of either Fluorogold (FG, Fluorochrome LLC, Denver, CO; 2% in 0.9% NaCl), or cholera toxin B subunit conjugated to (red fluorophore) Alexa Fluor 594 (CTB-AF594, 0.25% in 0.9% NaCl, Life Technologies, Carlsbad, CA; Cat. No. C-22842). Pressure injections were performed using a microinfusion pump as described above; injection volumes were 100 nl (Exp. 4). Anterograde tracing from the vHP field CA1 following unilateral iontophoretic injection of Phaseolus vulgaris-leucoagglutinin (PHAL, Vector Labs, Burlingame, CA; 2.5% in 0.1 M sodium phosphate-buffered saline, pH 7.4) was performed using a precision current source (digital midgard precision current source, Stoelting) as described previously (Hahn and Swanson, 2010 (link)). PHAL immunoreactive axons in the LHA confirmed the exclusively ipsilateral nature of vHP to LHA projections (Figure 6—figure supplement 1; representative vHP PHAL injection site, Figure 6).

For retrograde pathway tracing, rats were anesthetized and sedated with a ketamine (90 mg/kg)/xylazine (2.8 mg/kg)/acepromazine (0.72 mg/kg) cocktail and placed in a stereotaxic apparatus. Rats received a unilateral iontophoretic injection of FG (Fluorochrome LLC; 2% in 0.9% NaCl) targeting either vHP (n = 4): −4.7 mm AP, 4.6 mm ML, −6.4 mm DV (from the dura at the injection site) or the ACB (n = 4): 1.2 mm AP, 1.0 mm ML, −6.75 mm DV (from the dura at the injection site) (coordinates from ref. ⁵⁷ ). Iontophoresis was performed using a precision current source (Digital Midgard Precision Current Source, Stoelting, Wood Dale, IL, USA) as described previously^{14 (link)}. Following a 12-day survival period, animals were fixation-perfused and tissue was harvested and processed for immunofluorescence.