Q&A Report: Developing Prefrontal Circuits and Their Role in Health and Disease

Dr. Jastyn A. Pöpplau and Marilena Hnida answer questions from their webinar on prefrontal networks in developing head-fixed mice, and the role of these networks in cognition and models of neurodevelopmental diseases.

These answers were provided by:

Jastyn A. Pöpplau, PhD
Institute for Developmental Neurophysiology, Center for Molecular Neurobiology (ZMNH)
University Medical Center Hamburg-Eppendorf (UKE)

Marilena Hnida, MSc
PhD Student
Institute for Developmental Neurophysiology, Center for Molecular Neurobiology (ZMNH)
University Medical Center Hamburg-Eppendorf (UKE)

If microglia re-populate the cortex after drug application, then what causes the long-term cognitive defects?

We conducted similar experiments in adult (postnatal day 70) mice and found no lasting alterations. The long-term cognitive and behavioral deficits after adolescent microglia ablation suggest that the time point of application is critical. As discussed during the presentation, we found that in particular during the adolescent period microglia activity is increased. Preventing or delaying this developmental activation most likely interferes with the developmental wiring of neuronal circuits, which ultimately results in the observed deficits.

How was surgery performed on juvenile mice?

We performed the surgery no earlier than postnatal day 14. The mice were anesthetized with the inhalation anesthetic isoflurane and received an injection of the opioid buprenorphine (0.1mg/kg bw, s.c. (hind leg)) 30 minutes before the surgery. The mice were fixed in a stereotactic surgery frame with the blunt ends of the ear bars facing the mouse and applying light pressure. The hair on the head was wet with ethanol (70%) and removed with scissors. The skin was then opened with a small V-cut which was prolonged rostrally and caudally, and the skin was removed so that an oval spanning from between the eyes to the ears and caudal part of the skull, exposing the prefrontal cortex, hippocampus and cerebellum was formed. The connective tissue was removed and the skull dried with pressurized air. The skull was roughened with drilling lines made with a dental drill applying very little pressure. The drilling coordinates above recording sites were marked with a permanent marker and framed with a 3D custom printed window, in order to prevent cement from running over it, and covered with tissue compatible silicon (Kwik-Sil, Kwik-Cast, WPI). A small hole was drilled above the cerebellum and a silver wire with a connector, serving as a reference electrode, was inserted, so that it was placed between the skull and the brain. A first layer of dental cement (Super-Bond Universal Kit K058E, Hentschel-Dental) was applied, and the reference electrode as well as the headplate (Neurotar) were fixed with this cement. After drying, a second layer of cement (Paladur, Henry Schein Dental) was applied to further stabilize the headplate, window and reference electrode. The mice were injected with isotonic saline solution (0.01ml/g bw, s.c. (hind leg)) to prevent dehydration and were placed in a separate cage on a heating pad. They woke and recovered quickly, and after 30-45 min could be placed back to the dam and their litter mates. The whole litter received wet food and lactose free cat milk or coffee whitener with Meloxicam (1-2 mg/kg). The craniotomy was performed 2 days later under isoflurane anesthesia in the same stereotactic frame. After drilling, cranial windows were covered with tissue compatible silicon (Kwik-Sil, Kwik-Cast, WPI). The mice were weaned and received wet food and lactose-free, high-caloric milk.