NEUR 303 - lecture 8

What is the principle behind lesion studies in neuroendocrinology, and what do cytotoxic lesions specifically target?

Lesion studies determine whether a brain region is necessary for a function by observing behavioural deficits after damage. Cytotoxic lesions selectively destroy neuronal cell bodies while sparing fibres of passage, allowing more precise identification of functional regions.

What does lesioning the MPOA reveal about its role in maternal behaviour?

Lesioning the MPOA abolishes maternal behaviour, demonstrating it is essential for integrating hormonal and sensory inputs and coordinating maternal responses.

Why is the MPOA considered a “central hub” in maternal behaviour circuits?

It integrates olfactory, hormonal, and sensory inputs and directs both motivational (approach) and avoidance-inhibiting pathways.

What is cFos and why is it classified as an immediate early gene?

cFos is a nuclear protein rapidly expressed in response to neuronal activation. It is an immediate early gene because it is transcribed quickly without requiring prior protein synthesis.

How is cFos used as a proxy for neuronal activity?

Increased cFos expression indicates that neurons were recently active, allowing researchers to map brain regions engaged during behaviours like maternal care.

Why is timing critical when measuring cFos expression?

cFos mRNA and protein peak at different times (~60–90 minutes for protein), so incorrect timing can miss or misrepresent neuronal activation.

What have cFos studies shown about maternal behaviour?

Pup exposure induces strong cFos expression in the MPOA, especially in experienced mothers, highlighting its importance in maternal circuits.

What is in situ hybridisation and what does it measure?

ISH is a technique used to detect specific mRNA sequences in tissue, providing both gene expression and spatial localisation.

What is the main advantage of ISH in neuroscience?

It allows precise localisation of gene expression within specific brain regions.

Compare radioactive, fluorescent, and colorimetric ISH.

• Radioactive: very sensitive but slow and hazardous

• Fluorescent: fast and multiplex but signal fades

• Colorimetric: stable but limited multiplexing

What makes RNAscope superior to traditional ISH methods?

It is highly sensitive, allows multiplexing, and provides quantitative measurement of RNA levels, though it is expensive.

What does immunohistochemistry detect and how does it work?

IHC detects proteins using antibodies; a primary antibody binds the protein and a labelled secondary antibody enables visualisation.

What is the difference between direct and indirect IHC?

Direct uses a labelled primary antibody; indirect uses a secondary antibody to amplify the signal.

What are the two main types of IHC visualisation?

Chromogenic (colour change) and fluorescent (multi-colour imaging).

What are the strengths and limitations of IHC?

Strengths: relatively cheap, quick, multiplexing possible
Limitations: semi-quantitative, dependent on antibody availability

Key distinction between ISH and IHC?

ISH detects mRNA; IHC detects protein.

How does pup exposure affect MPOA activity?

It strongly increases neuronal activation (cFos expression), especially in experienced mothers.

What is the maternal “approach pathway”?

MPOA → ventral tegmental area (VTA) → nucleus accumbens (NA) → ventral pallidum (VP)

What role does dopamine play in maternal behaviour circuits?

Dopamine from the VTA inhibits GABAergic neurons in the nucleus accumbens, disinhibiting the ventral pallidum and promoting behaviour.

Why is disinhibition important in this pathway?

Removing inhibitory signals allows activation of downstream motor and behavioural responses.

What happens if MPOA or VTA is inactivated?

Animals lose preference for pups, indicating disrupted maternal motivation.

How does the ventral pallidum influence behaviour?

It connects to motor systems and drives behavioural output when activated.

What defines a rewarding stimulus in neuroscience?

A stimulus that produces pleasure, motivates behaviour, and increases the likelihood of repeated exposure.

How does dopamine function in reward circuits?

It is released in anticipation of reward and reinforces behaviours associated with that reward.

What is conditioned place preference (CPP)?

A behavioural test measuring preference for environments associated with rewarding stimuli.

What does CPP reveal about pups?

Pups are inherently rewarding, as animals prefer pup-associated environments.

What does the T-maze test measure in maternal studies?

Motivation to retrieve pups, particularly in novel environments.

How do lactating females differ from virgins in behavioural tests?

They show increased motivation and faster pup retrieval, indicating enhanced reward sensitivity.

What surprising finding occurs in early lactation regarding reward?

Mothers may prefer pups over drugs like cocaine, indicating strong natural reward value.

What are mouse lines in neuroendocrinology?

Genetically modified mice used to study gene function and neural circuits.

What is a knockout mouse?

A mouse in which a gene has been deleted or inactivated.

What is a knock-in mouse?

A mouse with an inserted or modified gene, often for tracking or functional studies.

How does the Cre-Lox system work?

Cre recombinase cuts DNA at LoxP sites, allowing deletion or inversion of specific gene sequences.

Why is Cre-Lox powerful?

It enables cell-type and region-specific gene manipulation.

Why must both Cre and LoxP be introduced transgenically?

Neither occurs naturally in mammals.

How are viruses (e.g., AAV) used with Cre-Lox?

They deliver Cre recombinase to specific brain regions, enabling spatial and temporal control of gene expression.

What is GCaMP and how does it work?

A genetically encoded calcium indicator that fluoresces when calcium enters neurons, indicating activity.

Why is calcium used as a proxy for neuronal firing?

Calcium influx occurs during action potentials and synaptic activity.

Why is KCl applied at the end of slice experiments?

to depolarise neurons and confirm tissue viability - proves the slice is healthy (the tissue isn't dead and that’s why its not producing a response)

What does fibre photometry measure?

Bulk (population-level) calcium activity in living animals.

What are the advantages and limitations of fibre photometry?

Advantages: real-time, can track behaviour
Limitations: no single-cell resolution

What are GRIN lenses and what do they allow?

Implanted optical lenses that allow imaging of individual neurons in vivo.

What are the trade-offs of GRIN lens imaging?

High resolution but expensive, invasive, and technically challenging.

What are GRAB sensors and what do they measure?

Genetically encoded sensors that detect extracellular neurotransmitter levels in real time.

How are GRAB sensors different from GCaMP?

GRAB measures neurotransmitter release (extracellular); GCaMP measures neuronal activity via calcium (intracellular).