A Device for Stereotaxic Viral Delivery into the Brains of Neonatal Mice

Abstract

The increasing interest in manipulating neural circuits in developing brains has created a demand for reliable and accurate methods for delivering viruses to newborn mice. Here we describe a novel 3D-printed mouse neonatal stereotaxic adaptor for intracerebral viral injection that provides enhanced precision and reliability. Using this device, we injected A2a-Cre mice with a Cre-dependent hM4D-mCherry viral construct at postnatal day 1 (P1) and demonstrated selective expression in the striatal indirect pathway neurons on days P7, P11 and P25. Similarly, dopaminergic midbrain neurons were selectively targeted with a Cre-dependent green fluorescent protein virus in Dat-IRES- Cre neonates and expression examined at P25. Our open-source neonatal stereotaxic mouse adaptor facilitates neonatal neuronal targeting, which should improve the ability to label and modify neural circuits in developing mouse brains.

3D‐printed design of a stereotaxic adaptor for the precision targeting of brain structures in infant mice

“…Existing solutions take advantage of cryoanaesthesia (i.e., the cooling of the entire body; Tang et al, 2020), yet with drawbacks similar to bolus injection methods particularly in transgenic mice with altered heat perception, tolerance or ability to generate body heat for recovery. Notably, cooling was found insufficiently reproducible for mice older than P4 (Olivetti et al, 2020).…”

“…Even if head fixation is within tolerable limits using existing adaptors, including stages tailored to the small size of neonatal mice (Mathon et al, 2015), stable anaesthesia certainly poses another notable challenge, particularly in infant mice where overdosing is common and drug tolerance is variable (Olivetti et al, 2020). Existing solutions take advantage of cryoanaesthesia (i.e., the cooling of the entire body; Tang et al, 2020), yet with drawbacks similar to bolus injection methods particularly in transgenic mice with altered heat perception, tolerance or ability to generate body heat for recovery.…”

“…These include (i) the lack of stereotaxic atlases for many strains of mice, particularly because reference points at their skulls are less clearly discernible (but see Paxinos & Ashwell, 2018), (ii) relatively high mortality because of anaesthetic overdoses due to the lack of integrated adaptors and (iii) permanent damage to craniofacial bones and superficial brain structures because of the excessive pressure arising upon head fixation. To circumvent experimental difficulties, a number of methodological modifications (Ho et al, 2020;Mathon et al, 2015), including cone-shaped cradle adaptors for ice-anaesthetized newborn rodents (Olivetti et al, 2020), were recently introduced. Nevertheless, a pressing need persists for improved surgical methods in infant mice during the postnatal (P) 6-to 10-day period, when major cerebral reorganization occurs (Hooks & Chen, 2020).…”

A low-cost device for cryoanesthesia of neonatal rodents

“…While this procedure is routinely used in adults, it is suboptimal for neonates, which are increasingly used to study how neural circuits develop postnatally [4][5][6][7][8]: inhalation anesthetics can negatively and lastingly affect the brain of neonatal rodents, in particular areas associated with memory [9][10][11][12]. Instead, cryoanesthesia, i.e.…”

“…anesthesia by deep hypothermia, has been commonly used in such young animals. This is typically carried out by submerging neonates in crushed ice or using simple cooling pads [7,8,[13][14][15]. These approaches, however, do not provide accurate or consistent temperature control, and questions about the safety and appropriateness of hypothermic anesthesia for neonates have thus been raised [16].…”