NEUR 303 - Lecture 20

Where is prolactin receptor (Prlr) expression especially high in the brain?

Prlr is widespread throughout the brain but is especially enriched in the hypothalamus, which controls reproduction, metabolism, thermoregulation, and maternal behaviour.

Why is widespread Prlr expression in the brain biologically meaningful?

Because prolactin levels rise strongly in pregnancy/lactation, widespread Prlr expression allows prolactin to coordinate multiple physiological adaptations (maternal behaviour, metabolism, fertility suppression, thermoregulation).

What does heterogeneity of prolactin-responsive cells imply?

Prolactin likely acts across many different neural circuits, not just one “maternal circuit,” allowing broad regulation of body systems during pregnancy and lactation.

What is single-cell RNA sequencing used for?

To determine which genes are expressed in individual cells, allowing identification of distinct neuronal and non-neuronal cell types.

What are the main steps of scRNAseq?

Tissue dissection → tissue dissociation into single cells → FACS sorting → sequencing RNA from each cell → building expression profiles → clustering cells into types

What is FACS and why is it important in scRNAseq?

Fluorescence-activated cell sorting separates individual cells from a tissue suspension, enabling sequencing of single-cell gene expression.

Why is scRNAseq powerful compared to bulk tissue sequencing?

It reveals cell-type-specific gene expression, instead of averaging gene expression across many mixed cell types.

What is “Hypomap”?

A combined database integrating 18 mouse hypothalamus scRNAseq datasets, containing ~400,000 cells.

How many neuronal clusters are identified in mouse Hypomap?

Around 130 neuronal clusters.

Why might prolactin be considered a “system-wide pregnancy signal”?

Because prolactin reaches uniquely high levels during pregnancy, making it a strong indicator of the pregnant state.

When does placental lactogen begin rising during human pregnancy?

Near the end of the first trimester and increases exponentially through the second and third trimesters.

What happens to pituitary prolactin levels during pregnancy?

Pituitary prolactin levels rise during pregnancy, alongside placental lactogen.

What percentage of mouse hypothalamic neurons express Prlr?

About 15% of all neurons.

In how many neuronal clusters is Prlr expressed in mouse hypothalamus?

Prlr is expressed in 54% of clusters (71 out of 130 clusters)

What does widespread Prlr expression across clusters suggest?

Prolactin signalling likely influences many hypothalamic cell types and behaviours, not one single pathway.

What is UMAP used for in scRNAseq analysis?

To reduce high-dimensional gene expression data into a 2D/3D plot so similar cells cluster together.

What does clustering on a UMAP plot indicate?

Cells close together share similar gene expression, meaning they likely represent the same cell type or functional population.

How can UMAP distinguish broad neuronal types?

Excitatory glutamatergic neurons, inhibitory GABAergic neurons, and non-neuronal cells cluster into separate regions based on gene expression.

What is NucSeq (nuclear sequencing)?

A single-cell-like sequencing method using cell nuclei rather than whole cells, often used in human brain tissue.

How many clusters were identified in human hypothalamus NucSeq data?

Around 458 clusters.

How many human hypothalamus clusters show >50% Prlr expression?

About 122 out of 458 clusters.

Which non-neuronal population shows high Prlr expression in humans?

Choroid plexus cells.

Why is high Prlr expression in the choroid plexus important?

It suggests prolactin may influence CSF-related brain regulation and hormonal transport/signalling beyond neurons.

What is spatial transcriptomics?

Whole-genome expression mapping done on small “spots” across a tissue section, preserving anatomical location.

How does spatial transcriptomics differ from scRNAseq?

scRNAseq measures genes in single cells, while spatial transcriptomics measures gene expression in multi-cell tissue spots, giving lower resolution but anatomical mapping.

What does spatial transcriptomics show about Prlr distribution?

It shows Prlr expression patterns in humans that resemble rodent distribution, supporting rodents as useful models.

What are two major reasons prolactin signalling is high during pregnancy/lactation?

(1) Suppression of dopamine negative feedback and (2) secretion of placental lactogen.

What is placental lactogen?

A prolactin-like hormone produced by the placenta that binds the prolactin receptor and increases prolactin signalling.

When does human placental lactogen rise during pregnancy?

It appears near the end of the first trimester and rises exponentially through the 2nd and 3rd trimesters.

Why is prolactin considered a strong “pregnancy signal”?

Because extremely high prolactin/lactogen levels occur mainly during pregnancy, allowing the brain/body to detect and adapt to the pregnant state.

Which brain region is especially critical for prolactin effects on maternal behaviour?

The medial preoptic area (MPOA).

What experiment shows MPOA prolactin receptors are essential for maternal behaviour?

Prlr floxed mice injected with AAV-Cre into the MPOA (deleting Prlr locally) showed loss of maternal behaviour.

What does this MPOA knockout experiment demonstrate?

It provides causal evidence that prolactin signalling in MPOA neurons is required for maternal behaviour.

What is fibre photometry used for?

Measuring real-time neuronal activity in living animals by recording fluorescence from calcium indicators.

What does GCaMP measure?

Intracellular calcium, which increases when neurons fire (activity marker).

Why is fibre photometry useful for maternal behaviour studies?

It links activity of specific neurons (e.g., Prlr+ MPOA neurons) to specific behaviours in real time.

What are DREADDs?

Designer GPCR receptors inserted into neurons that can be activated or inhibited by synthetic drugs (e.g., CNO).

What does hM3Dq do?

It is an excitatory DREADD that activates neurons via Gq signalling and increased intracellular Ca²⁺.

What happens when MPOA Prlr+ neurons are artificially activated in non-parental animals?

They did not show parental behaviour; instead they became inactive and curled up, revealing these neurons have additional roles.

What unexpected finding was discovered about MPOA prolactin-responsive neurons?

Many are warm-sensing neurons involved in thermoregulation.

Why did activation of Prlr+ MPOA neurons cause inactivity/hypothermia?

Activating warm-sensing neurons mimicked “heat,” triggering cooling mechanisms, causing the animal’s body temperature to drop.

What does this reveal about prolactin receptor expression in MPOA?

MPOA Prlr neurons are functionally diverse and include thermoregulatory circuits, not just parenting circuits.

How does body temperature normally change during pregnancy?

Basal temperature rises during pregnancy, but in late pregnancy there is an adaptive dip to help prevent overheating.

Why does late pregnancy show an adaptive drop in temperature?

To promote heat loss during peak fetal growth/metabolic demand and prevent dangerous overheating for mother and fetus.

What happens to temperature during early lactation?

Temperature rises again, reflecting high metabolic activity needed for milk production.

What happens when prolactin receptors are deleted from the rostral POA in pregnancy?

The adaptive late-pregnancy dip disappears and temperature continues to rise.

What does this show about prolactin action during pregnancy?

Prolactin signalling is required for pregnancy-specific thermoregulatory adaptation.

What happens when Prlr is knocked out in glutamatergic neurons under warm conditions (30°C)?

Pregnancy outcomes are severely impaired and many pups do not survive.

Why was Vglut2-Cre used in these experiments?

Because warm-sensing thermoregulatory neurons are largely glutamatergic, so Vglut2-Cre targets that population.

What does this experiment demonstrate?

Prolactin signalling provides resilience to environmental heat stress, supporting reproductive success.

How does in situ hybridisation (ISH) work and what does it measure?

ISH uses a labelled probe that binds to a specific mRNA sequence in tissue sections, showing where a gene is expressed anatomically.

How does immunohistochemistry (IHC) work and what does it measure?

IHC uses antibodies to bind a specific protein in tissue sections, allowing visualization of protein localisation in cells/brain regions.

How does Cre-Lox recombination work and what is it used for?

Cre recombinase cuts DNA at loxP sites, allowing conditional gene knockout, gene activation, or cell labelling/ablation in specific tissues or cell types.

What is the difference between breeding-based Cre-Lox and AAV-Cre delivery?

Breeding Cre x flox targets a broad cell population genetically, while AAV-Cre stereotaxic injection deletes genes in a specific brain region.

How does single-cell RNA sequencing (scRNAseq) work?

Tissue is dissociated into single cells, cells are sorted (often by FACS), then RNA is sequenced per cell to identify cell types and gene expression patterns.

How does spatial transcriptomics work?

It sequences RNA from defined “spots” on tissue sections, mapping gene expression to specific anatomical locations (lower resolution than scRNAseq).

How does a UMAP plot help in transcriptomics?

UMAP reduces complex gene-expression data into 2D/3D so similar cells cluster together, helping identify cell populations.

How does Western blotting work?

Proteins are extracted, separated by gel electrophoresis, transferred to a membrane, then detected with antibodies to measure protein presence and fold change.

How does RT-qPCR work?

RNA is extracted and converted to cDNA, then amplified using PCR to measure relative mRNA expression levels quantitatively.

How do DREADDs (chemogenetics) work?

Genetically engineered GPCRs (e.g. hM3Dq/hM4Di) are expressed in targeted neurons and activated by CNO to increase or decrease neuronal activity.

How does optogenetics work?

Light-sensitive proteins (e.g. channelrhodopsin activates, halorhodopsin inhibits) allow precise millisecond control of neuronal firing using light.

How does anterograde tracing work?

Viral tracers label neurons and track where axons project forward from the injection site to downstream targets.

How does retrograde tracing work?

Retrograde viruses are taken up by axon terminals and transported back to cell bodies, identifying which neurons project into a target region.

What is dual immunohistochemistry used for?

It uses two antibodies with different fluorophores to test co-localisation, showing whether two proteins are expressed in the same cells.

What is electrophysiology (ex vivo patch clamp) used for?

Brain slices are recorded to measure neuronal firing and membrane properties, often used to confirm if DREADDs/optogenetics successfully alter activity.

What is telemetry recording used for in physiology experiments?

Implanted devices measure internal physiology (e.g. core body temperature) continuously in freely moving animals.

What is a radioimmunoassay (RIA) and what is it used for?

An old but sensitive hormone assay where radiolabelled hormone competes for antibody binding, allowing quantification of hormone levels (e.g. prolactin).

What is a bioassay using Nb2 cells and what does it measure?

Nb2 lymphoma cells proliferate in response to lactogens; proliferation (via WST-1 absorbance) measures biological activity of prolactin/lactogens.

What is stereotaxic surgery and why is it important?

A surgical method using brain coordinates to inject viruses/drugs precisely into specific brain nuclei (e.g. MPOA, arcuate nucleus).