Rodent Behavior Unveiled: Two Studies Dissect Vocalization and Parental Care

Two separate research papers have published detailed methodologies used to study distinct behaviors in different rodent species—one exploring vocal communication in lab mice, the other investigating the complex spectrum of parental and infanticidal behavior in African striped mice.

Vocalization Study in Laboratory Mice

The vocalization study, approved by the Cold Spring Harbor Laboratory Institutional Animal Care and Use Committee, involved adult (>3 months) male and female C57BL/6J mice from The Jackson Laboratory.

Animal Husbandry

Mice were maintained at a temperature of 20–22 °C, 30–70% humidity, and a 12-hour light/12-hour dark cycle.

Behavioral Recordings

To elicit vocalizations, a female mouse was first rendered mute via synaptic silencing of the caudolateral periaqueductal gray (PAG) using tetanus-toxin light chain. After two weeks, this muted female was placed with a conspecific male in a clean cage.

Audio was recorded for 1–2 hours at a 250 kHz sampling rate using two Avisoft CM16/CMPA microphones. Vocalizations were segmented with USVSEG software and manually curated. "Songs" were defined as sequences of loud notes lasting ≥1.5 seconds with gaps ≤0.5 seconds. Note rate, pitch, and amplitude were then calculated.

Surgical and Imaging Procedures

Stereotaxic viral injections were performed under 1–2% isoflurane, following a subcutaneous dose of meloxicam (2 mg/kg). Injections targeted the orofacial motor cortex (OMC) using specific coordinates.

Various viral constructs (AAV, Sindbis) were used for projection mapping, tracing, and silencing. Brains were processed for imaging using either confocal microscopy (Zeiss LSM 980 Airyscan2) or serial two-photon tomography (STPT) on a TissueCyte 1000 microscope, with images aligned to the Allen Mouse Brain Common Coordinate Framework.

MAPseq and Data Analysis

For mapping axonal projections, barcode libraries were injected into the OMC. Barcode RNA from microdissected brain sections was extracted, amplified, and sequenced.

Data analysis included modeling projection probabilities and classifying neurons (IT, CT, PT). Bootstrap resampling was used to match neuron numbers across species, and previously collected OMC cooling data were reanalyzed to assess the structure's causal contribution to song rhythm.

Parental Behavior Study in African Striped Mice

The parental behavior study, adhering to guidelines from Princeton University's Institutional Animal Care and Use Committee, used laboratory-bred African striped mice originating from Goegap Nature Reserve, South Africa.

Animal Subjects and Rearing

Mice were maintained at 22.2 °C, 40–55% humidity, on a 12/12 light/dark cycle. Standard rodent chow was restricted to 4g per mouse per day.

Male offspring, after being reared with their biological parents, were assigned at weaning to either Group-Housed (GH) conditions (3–4 similarly aged conspecifics) or Socially Isolated (SI) conditions.

Behavioral Assays

From postnatal day (PND) 80, sexually naive males underwent social and exploratory testing. A primary assay was the 20-minute pup interaction test, where a novel pup (PND 1–6) was introduced after a 40-minute habituation period.

Behaviors such as inspection, licking/grooming, lateral contact, huddling, and attack were recorded. Males were categorized as:

• Infanticidal: Displayed aggressive pup-directed behaviors.
• Allopaternal: Showed significant caring contact.
• Ambivalent: Behaviors falling between the two categories.

Initial characterization involved GH (n=24) and SI (n=23) males, as well as primiparous fathers (n=18) and mothers (n=7). A subsequent intervention study exposed GH and SI males to varying rehousing and food restriction conditions over 16 days.

Neural and Molecular Analyses

Brain-wide cFos Expression: Pup-exposed males (n=11 GH, n=11 SI) and empty cage controls (n=10 GH, n=10 SI) were euthanized 60 minutes after exposure. Brains were processed for immunohistochemistry to quantify cFos+ density.

Single-Nucleus RNA Sequencing (snRNA-seq): Medial preoptic area (MPOA) tissue from 17 mice (infanticidal, allopaternal, and ambivalent males, plus dams and sires) was processed. Libraries were prepared using 10X Genomics Chromium and sequenced on an Illumina NovaSeq 6000.

Fluorescence in situ Hybridization (FISH): RNAscope technology was used with custom probes for Agouti, eGFP, and Mc4r.

Quantitative PCR (qPCR): MPOA tissue from 23 sexually naive GH males was analyzed for relative expression of Agouti and Agrp genes.

Enzyme-Linked Immunosorbent Assay (ELISA): Leptin concentration was measured from trunk blood serum.

Viral Manipulation Study

Sexually naive males were pre-screened for pup interaction and randomly assigned to an Agouti overexpression group or a control eGFP group. AAV vectors carrying either pAAV-hSyn-Agouti-T2A-eGFP-WPRE or pAAV-hSyn-eGFP-WPRE were bilaterally infused into the MPOA.

Pup interaction tests were conducted 1 and 3 weeks post-injection, allowing researchers to assess the causal role of Agouti signaling in parental behavior.

Statistics

Data were analyzed using R (v.4.4.1) and GraphPad Prism (v.9.4.1), with a significance threshold of P ≤ 0.05. Power analyses were used to determine sample sizes.