Hippocampal Dependent Neurogenesis and Behaviours

Hippocampal Dependent Neurogenesis

  • Adult hippocampal neurogenesis (AHN) is the process of generating new neurons in the adult brain that contribute to normal brain function.

  • It affects brain structure/plasticity and contributes to normal brain function and spatial memory.

  • AHN occurs in the subventricular zone (SVZ) of the lateral ventricles and the dentate gyrus (DG) of the hippocampus.

  • AHN declines with age and can be affected by exercise, environmental enrichment, and dietary manipulation.

Why Investigate Neurogenesis?

  • Increasing aging population.

  • Increase in neurological disease/disorders that affect memory function.

  • Examples:

    • Alzheimer’s: 900,000 people with dementia in the UK.

    • Parkinson’s: 153,000 people currently living with PD in the UK.

    • Epilepsy: 630,000 people in the UK.

    • Depression: 1 in 6 people (16% of the UK population).

    • Anxiety: 8.2 million cases in the UK (2013).

Adult Neurogenesis

  • The process of generating new neurons in the adult brain that contribute to normal brain function

  • First discovered by Joseph Altman in 1965.

  • Altman used autoradiographic evidence of post-natal neuronal cell differentiation.

  • Experiment:

    • Long Evans rats treated with H3-Thymidine (various ages) and terminated after 2 weeks.

    • Labelled cells found in the basal area of the granule layer of the dentate gyrus (DG).

    • Results:

      • 10-day-old rats: 35 labelled cells.

      • 30 to 40 days old: ~5 cells.

      • 4 months or older (adults): ~2 labelled cells or less.

      • A decline of 19% to 3% labelled cells.

Stages of Hippocampal Neurogenesis and Cell Markers

BrdU - Bromodeoxyuridine

  • 5-bromo-2′-deoxyuridine.

  • Thymidine analogue that incorporates into the DNA of dividing cells (S-phase of cell cycle).

  • Used for birth-dating and monitoring cell proliferation – labels new-born cells.

  • Can be administered orally (drinking water) or via i.p. injection for in vivo labelling.

  • The timing of BrdU administration is important for interpreting the outcome of an experiment/treatment.

Immunohistochemistry (IHC)

  • Process of detecting the presence of proteins and antigens on tissue sections using antibodies.

  • Antibody-antigen interactions are visualised either chromogenically (coloured enzyme substrate) or fluorescently (fluorescent probe).

  • Provides information on the location of proteins in a whole tissue context.

  • Tissue processing (fixation – perfusion vs. immersion) can impact staining outcomes.

Types of IHC

  • DAB-IHC:

    • Primary antibody (1° Ab) binds to the antigen of interest.

    • Secondary antibody (2° Ab) with HRP (horseradish peroxidase) binds to the primary antibody.

    • DAB (diaminobenzidine) substrate is used, resulting in a brown/black precipitate.

  • Fluorescent-IHC:

    • Primary antibody (1° Ab) binds to the antigen of interest.

    • Secondary antibody (2° Ab) with a fluorescent tag binds to the primary antibody.

Assessing Hippocampal and Neurogenesis-Dependent Behaviours

  • Contextual Fear Conditioning:

    • Learned fear memory (survival response).

    • Requires inputs from the hippocampus and CA3 region.

    • Rodents learn to associate a neutral context with an aversive stimulus, e.g., foot shock.

    • Display fear response, e.g., freezing behaviour.

Spontaneous Location Recognition (SLR) Task

  • Pattern separation memory – DG dependent

  • Highly sensitive to changes in hippocampal neuroplasticity.

  • SLR manipulates the similarity of spatially landmarked locations during the sample phase when memory is encoded, and pattern separation processes are active.

  • Takes less than 1 month to run this task.

Touchscreen Tasks – Location Discrimination

  • Operant box task, same concept as SLR hand task.

  • Rodents are required to discriminate between 2 white squares on the screen.

  • The distance between squares can be varied.

  • Neural systems used:

    • Hippocampus.

    • Neurogenesis.

Behavioral Tasks - Anxiety

  • Open Field Test.

  • Elevated Plus Maze.

  • Elevated Zero Maze.

Theory in Practice

  • Experiment:

    • Day 1: BrdU and Ghrelin administration.

    • Days 10-20: Cognitive task.

    • Day 28: Analysis.

  • SLR Task:

    • Sample phase with small separation (A2, A3) and X-small separation (A1, A4, A5) between landmarks.

    • Choice phase 24 hours later. 'Novel' location.

  • Discrimination ratio:

    • DiscriminationRatio=Time spent at novel location−Time spent at familiar locationTotal time spentDiscrimination Ratio = \frac{Time \ spent \ at \ novel \ location - Time \ spent \ at \ familiar \ location}{Total \ time \ spent}

  • Results:

    • Ghrelin improves discrimination ratio in small separation condition.

  • DCX staining:

    • Ghrelin increases DCX+ cells in the dentate gyrus.

  • BrdU/NeuN staining:

    • Ghrelin increases the number of BrdU+ cells and BrdU/NeuN+ cells in the dentate gyrus.