BDNF-TrkB signaling in oxytocin neurons contributes to maternal behavior

  1. Kristen R Maynard
  2. John W Hobbs
  3. BaDoi N Phan
  4. Amolika Gupta
  5. Sumita Rajpurohit
  6. Courtney Williams
  7. Anandita Rajpurohit
  8. Joo Heon Shin
  9. Andrew E Jaffe
  10. Keri Martinowich  Is a corresponding author
  1. Lieber Institute for Brain Development, Johns Hopkins Medical Campus, United States
  2. Johns Hopkins University, United States
  3. Johns Hopkins Bloomberg School of Public Health, United States
  4. Johns Hopkins University School of Medicine, United States
5 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
Disruption of BDNF from promoters I and II leads to impaired maternal care.

(a) Schematic of Bdnf transcript production. Transcription is initiated from promoters upstream of individual 5’-untranslated regions (UTRs) and spliced to the common coding exon IX. Each transcript …

https://doi.org/10.7554/eLife.33676.002
Figure 1—figure supplement 1
Bdnf-e1 -/- females show abnormal mating behaviors.

(a-d) Representative images depicting male genitalia injuries observed in Bdnf-e1 breeder cages. Breeder males frequently show inflamed, infected, and/or mutilated genitalia (b–d) compared to normal …

https://doi.org/10.7554/eLife.33676.003
Figure 1—figure supplement 2
Bdnf-e1 -/- females have a normal estrous cycle.

Bdnf-e1 -/- virgin females enter all stages of the estrous cycle, including proestrous (P), estrous (E), diestrous (D), and metestrous (M), similar to WT females. Bdnf-e1 -/- virgins enter estrous ~e…

https://doi.org/10.7554/eLife.33676.004
Oxytocin transcripts are reduced in Bdnf-e1 and -e2 -/- with impaired maternal care.

(a–b) qPCR demonstrating relative expression levels of oxytocin (a) and oxytocin receptor (b) transcripts in the HYP of 4 week old Bdnf-e1, -e2, -e4, and -e6 female mice (n = 4–5 per genotype; 1-way …

https://doi.org/10.7554/eLife.33676.005
Figure 3 with 3 supplements
Loss of TrkB in OXT neurons leads to parenting deficits in postpartum mothers and virgin females.

(a) Confocal z-projections of Oxt and Ntrk2 transcripts in brain slices containing the PVN from an adult female visualized with RNAscope in situ hybridization. TrkB transcripts (green) are highly …

https://doi.org/10.7554/eLife.33676.006
Figure 3—figure supplement 1
Ablation of TrkB protein in OXT neurons in OxtCre; TrkBflox/flox mice.

(a-d) Confocal z-projections of PVN from OxtCre; TrkBflox/flox (a, d), TrkBflox/flox (b), and OxtCre (c) adult mice. Immunolabeling of OXT (a, b, c, d), TrkB (a’, b’, c’), and merged images (a”, b”, …

https://doi.org/10.7554/eLife.33676.007
Figure 3—figure supplement 2
Selective loss of TrkB mRNA in OXT neurons in OxtCre; TrkBflox/flox mice.

(a-c) Confocal z-projections of Oxt (a, b, c) and Ntrk2 (a’, b’, c’) transcripts in brain sections containing the PVN from OxtCre; TrkBflox/flox (a), OxtCre (b), and TrkBflox/flox (c) visualized …

https://doi.org/10.7554/eLife.33676.008
Figure 3—figure supplement 3
OxtCre mice show normal maternal behavior.

(a-c) Maternal (a), abnormal parenting (b), and retrieval (c) indices for WT and OxtCre virgins during foreign pup retrieval test. OxtCre virgin females show no differences on these indices compared …

https://doi.org/10.7554/eLife.33676.009
Figure 4 with 2 supplements
Identification of a unique molecular profile for OXT neurons using ribosomal tagging.

(a) Locus of the ribosomal protein Rpl22 in the Ribotag mouse and breeding strategy used to obtain OxtCre; Rpl22HA mice. (b) qPCR analysis validating significant enrichment of Oxt transcripts in IP …

https://doi.org/10.7554/eLife.33676.010
Figure 4—figure supplement 1
Validation of select genes enriched or de-enriched in OXT neurons.

(a) qPCR analysis validating significant enrichment (Myo5a, Ank2, Peg3, and Kmt2a) and de-enrichment (Agrp, Cartpt) of select transcripts in IP compared to Input fractions. (b–d) Representative …

https://doi.org/10.7554/eLife.33676.011
Figure 4—figure supplement 2
Validation of Ovation RNA-seq V2 system with SoLo RNA-seq system.

(a) Plot depicting log2 fold changes of DEGs in Oxt IP vs. Oxt Input using RNA-seq V2 system compared to the SoLo RNA-seq system. Fold changes are highly correlated between kits. (b) Plot depicting …

https://doi.org/10.7554/eLife.33676.012
Figure 5 with 1 supplement
Perturbations in BDNF signaling impact gene pathways critical for plasticity in OXT neurons.

(a) Strategy for TRAP in OXT neurons from control and Bdnf-e1 -/- mice (n = 3 per genotype) using AAV-DIO-RiboTag and RNA-seq. (b) Confocal image showing HA (white) and tdTom (red) expression in …

https://doi.org/10.7554/eLife.33676.013
Figure 5—figure supplement 1
DEGs in Oxt Input vs. Oxt IP do not overlap with DEGs in control IP vs. Bdnf-e1 -/-IP.

(a) Plot depicting log2 fold changes of DEGs in Oxt IP vs. Oxt Input compared to the log2 fold changes of DEGs in control IP vs. Bdnf-e1 -/- IP. As expected, there is no correlation between the two …

https://doi.org/10.7554/eLife.33676.014

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
Information
Genetic reagent
(Mus musculus)
Bdnf-e1; Bdnf-e2 micePMID: 26585288
Genetic reagent
(Mus musculus)
Oxt-Cre miceJackson LabsRRID:IMSR_JAX:024234
Genetic reagent
(Mus musculus)
tdTomato miceJackson LabsRRID:IMSR_JAX:007914
Genetic reagent
(Mus musculus)
Ntrk2 flox/
flox mice
PMID: 18511296
Genetic reagent
(Mus musculus)
Cort-Cre miceJackson LabsRRID:IMSR_JAX:010910
Genetic reagent
(Mus musculus)
RPL22-HA miceJackson LabsRRID:IMSR_JAX:011029
Genetic reagent
(Adeno-assoicated virus)
AAV1-DIO-RPL22HA-GFPPMID: 25855171
Antibodyanti-mouse HACovanceRRID:AB_291262
Antibodygoat anti-mouse Alexa 647Thermo-Fisher
Scientific
RRID:AB_141693
Antibodyanti-mouse OXTPMID: 3880813
Antibodyanti-rabbit cFOSMilliporeRRID:AB_2631318
Antibodydonkey anti-rabbit Alexa 488Thermo-Fisher
Scientific
RRID:AB_141708
Antibodyanti-rabbit
TrkB H-181
Santa CruzRRID:AB_2155274
Commercial
assay or kit
Ovation RNA-Seq V2 kitNugenCat#: 7102
Commercial
assay or kit
Ovation SoLo RNA-seq
System Mouse
NugenCat#: 0502–32
Commercial
assay or kit
KAPA Library
Quantification Kit
KAPA
Biosystems
Cat#: KR0405
Commercial
assay or kit
Miseq Reagent Kit v3IlluminaCat#: MS-102–3001
Commercial
assay or kit
Ribogreen RNA assay kitInvitrogenCat#: R11490
Commercial
assay or kit
RNAscope
Fluorescent
Multiplex V1
Advanced Cell
Diagnostics
Cat#: 320850
Chemical
compound, drug
A/G magnetic
beads
PierceCat#: 88803
Chemical
compound, drug
10% NBFSigmaCat#: HT501128
Chemical
compound, drug
Fluoromount GSouthern
Biotechnology
Cat#: 0100–01
Sequence-
based reagent
Taqman probe
Oxt
Life TechnologiesCat#: Mm01329577_g1
Sequence-
based reagent
Taqman probe
Oxtr
Life TechnologiesCat#: Mm01182684_m1
Sequence-
based reagent
Taqman probe
Gapdh
Life TechnologiesCat#: 4352932E
Sequence-
based reagent
Taqman probe
Myo5a
Life TechnologiesCat#: Mm00487823_m1
Sequence-
based reagent
Taqman probe
Ank2
Life TechnologiesCat#: Mm00618325_m1
Sequence-
based reagent
Taqman probe Peg3Life TechnologiesCat#: Mm01337379_m1
Sequence-
based reagent
Taqman probe Kmt2aLife TechnologiesCat#: Mm01179235_m1
Sequence-
based reagent
Taqman probe
Agrp
Life TechnologiesCat#: Mm00475829_g1
Sequence-
based reagent
Taqman probe CartptLife TechnologiesCat#: Mm04210469_m1
Sequence-
based reagent
RNAscope probe OxtAdvanced Cell
Diagnostics
Cat#: 493171
Sequence-
based reagent
RNAscope probeNtrk2Advanced Cell
Diagnostics
Cat#: 423611
Sequence-
based reagent
RNAscope probeGabra2Advanced Cell
Diagnostics
Cat#: 435011
Sequence-
based reagent
RNAscope probeCckarAdvanced Cell
Diagnostics
Cat#: 313751
Sequence-
based reagent
RNAscope probeNtrk2 (Exon S)Advanced Cell
Diagnostics
Cat#: 539481
Sequence-
based reagent
RNAscope probe
Kmt2a
Advanced Cell
Diagnostics
Cat#: 408951
Sequence-
based reagent
RNAscope probePeg3Advanced Cell
Diagnostics
Cat#: 492581
Sequence-
based reagent
RNAscope probe
Ank2
Advanced Cell
Diagnostics
Cat#: 413221

Additional files

Supplementary file 1

Table Legends (Excel tables attached as separate tabs in a single file).

Table S1. Differential gene expression analysis of OXT Input vs. IP samples for all expressed genes. Table S2. Comparison of significant OXT neuron gene enrichment using Nugen Ovation RNA-seq System V2 kit vs. Nugen Ovation SoLo RNA-seq system. Table also includes comparison of differential gene expression between OXT neurons, Cort inhibitory interneurons, and Ntsr1 layer VI corticothalamic neurons versus their respective input RNA samples, as well as an overall OXT-IP versus pooled Cort-IP and Ntsr1-IP samples to assess specificity for these genes that were enriched in OXT neurons. Table S3. Gene Ontology analysis in OXT neurons. Enriched and depleted GO terms from differential expression analysis of OXT Input vs. IP. Table S4. Differential gene expression analysis of OXT-IP vs. Cort-IP and Ntsr1-IP samples for all expressed genes. Table S5. Gene ontology enrichment analysis for genes differentially expressed in OXT-IP vs. Cort-IP and Ntsr1-IP samples Table S6. OXT enriched mouse model genes in SFARI. List of genes enriched in OXT neurons implicated in genetic animal models of autism spectrum disorder (ASD) by the Simons Foundation Autism Research Initiative (SFARI). Table S7. OXT enriched human genes in SFARI. List of genes enriched in OXT neurons implicated in ASD by the SFARI Human Gene Module using the subset of mouse-expressed genes with human homologs. Table S8. Differential gene expression analysis of control IP vs. Bdnf-e1 -/- IP samples. Table S9. Gene Ontology analysis in OXT neurons with disruption of BDNF signaling. GO terms enriched from differential expression analysis of control IP vs. Bdnf-e1 -/- IP samples. Table S10. BDNF-dependent OXT genes in SFARI. List of genes perturbed in OXT neurons following disruption of BDNF signaling that are implicated in autism spectrum disorder (ASD) by the Simons Foundation Autism Research Initiative (SFARI).

https://doi.org/10.7554/eLife.33676.015
Transparent reporting form
https://doi.org/10.7554/eLife.33676.016

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