Koppen et al, 1997 - ACh Release and Choline Availability in Rat Hippocampus: Effects of Exogenous Choline and Nicotinamide

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Study Objectives & Hypotheses

• Objective: Investigate how choline availability affects acetylcholine (ACh) release in the hippocampus of awake rats.

• Hypothesis: Enhancing choline availability (via acute choline, dietary choline, or nicotinamide) facilitates ACh release, particularly under stimulated conditions.

Background

• ACh is critical for hippocampal and cortical function, implicated in memory and learning.

• The “cholinergic hypothesis of geriatric memory dysfunctions” posits ACh deficits contribute to cognitive dysfunction in dementia.

• Previous studies showed choline administration increases ACh levels in the striatum and nucleus accumbens (NAc).

  • Striatum - high cholinergic innervation density and ACh turnover rate

  • NAc - brain region that contains cholinergic interneurons with primarily nAChRS and mAChRs, plays a role in modulating dopamine signaling, crucial for motivation and reward processing. 

• Increased choline may increase synthesis and release of ACh in the hippocampus under conditions of increased neuronal firing

  • The decrease in ACh function caused by atropine, pentylenetetrazol, and fluphenazine can be buffered by pretreatment with choline or phosphatidylcholine in the striatum and hippocampus.

    • Atropine: Blocks mAChR autoreceptors (self-regulatory mech on the presynaptic neuron)

    • Fluphenazine: Direct mAChRs antagonists

    • Pentylenetetrazol: Alters ACh levels and release in the brain

  • Choline in non-stimulated hippocampus has no effect on ACh function

• Hippocampal, but not striatal slices, can store and mobilize choline for ACh synthesis

  • Brain extracellular choline removal: rapid cellular uptake, phosphorylation of choline, and CSF

Methods

• Subjects: ~50 Male Wistar rats

• In vivo microdialysis:

  • Extracellular concentrations of choline and ACh in the right ventral hippocampus

  • Anesthetized with pentobarbital, recover from surgery for 24 hr, testing for 2 days

  • Probes perfused with Ringer’s solution containing neostigmine

  • Extracellular levels of choline and ACh collected every 15 min, analyzed via High-performance liquid chromatography (HPLC)

    • basal efflux = average output of three consecutive samples that varied <5%

• Stimulated activity via Atropine

Choline availability enhancement methods:

1. Acute administration of choline:

   1. Nicotinamide → ACh release: SC nicotinamide + mannitol (sugar), 2 hrs later atropine + saline

   2. Choline → ACh release: Simultaneous IP choline Cl, atropine + saline

2. Nicotinamide pretreatment:

   • Nicotinamide + choline → ACh release: SC nicotinamide + mannitol, 2 hr later IP atropine, choline Cl, saline

3. Dietary choline supplementation:

   • In the drinking water for 15–18 days, 5x normal diet of choline

   • Supplementation terminated 12-16 hr before the microdialysis experiment to ensure measurement of chronic, not acute choline supp.

Results

• Basal rates in the hippocampus of control rats:

  • 114 fmol/min ACh

  • 1.38 pmol/min choline

• Atropine-Only administration

  • Max ACh at 30 min (365 ± 61%), elevated up to 3 hr (167 ± 13%)

• Choline-Only Administration

  • 15 min after injection: small and short-lasting increasez

    • basal choline (25 ± 13%)

    • ACh (126 ± 5%)

• Concurrent Choline + Atropine Administration

  • Longer duration (2 hours) and a higher maximum (570 vs. 365% of controls) than atropine alone

• Nicotinamide + Mannitol Administration

  • Maximum choline at 2 hours (205 ± 7%), elevated up to 5 hr

  • Mannitol did not significantly affect ACh concentration

  • Nicotinamide did not affect basal rates

  • Small increase 15-min post-injection (125 ± 7%)

• Nicotinamide pretreatment (Atropine 2 hr post-Nicotinamide)

  • This time-point: Max effect of nicotinamide and plateaued extracellular choline

  • Atropine only: Max ACh (524 ± 92%) 1 hour after atropine, elevated past 3 hours

  • Atropine + choline: Max ACh (557 ± 96%) 30 mins after injection

• Dietary Supplementation

  • 38% basal choline increase, 1.92 pmol/min

  • non-sig basal ACh increase, 140 fmol/min

    • control, 114 fmol/min

  • Atropine induced ACh increase after 30 min (max 563 ± 32%) in choline supp. rats

  • Saline induced ACh increase after 15 min (57 ± 26%)

• Comparative evaluation

  • ACh release in the 3 hours post-atropine injection

  • Choline, atropine, and nicotinamide all increased ACh activity

    • not significantly different between groups

    • Nicotinamide seems to be particularly effective at increasing ACh activity, with choline co-administration further increasing efficiency

Discussion

• Increased choline availability enhances stimulated (not basal) ACh release in the hippocampus.

  • A basal effect may be masked by neostigmine: prevents the breakdown of ACh

  • Short-term (15 min post-injection) increases by saline, choline, and nicotinamide may be an unspecific arousal reaction (artifact)

  • The increase was similar irrespective of mechanism for both basal and max rates

    • synthesis and release of hippocampal ACh is influenced by choline supplementation, irrespective of route of administration

• Mechanism varies by treatment:

  • Acute choline: Likely involves a precursor pool (e.g., phosphocholine).

    • when ACh demand is high, choline can be scavenged from phosphocholine

    • Very little metabolism into phosphatidylcholine

  • Choline diet: May promote release from lipid-bound stores, possibly not reflected in hippocampal/brain extracellular choline levels.

    • Choline taken up by the brain is slowly (24–72 hr) incorporated into

      phospholipids (mainly phosphatidylcholine)

    • There are increases in brain and CSF concentrations of phosphatidylcholine in rats on a choline-enriched diet

    • The observed increase in ACh activity is beyond the observed 30% increase in choline, so it is more likely that the brain scavenged choline lipid stores.

      • unsure if this process can occur fast enough to be “on-demand”

  • Nicotinamide: Directly doubles extracellular choline, enhancing ACh synthesis.

    • A vitamin of the B group, it can elevate extracellular and CSF choline via inhibiting choline clearance from the brain or maybe via actively metabolizing phosphatidylcholine

    • This may mark the max increase in choline that reflects an increase in ACh, where additional (beyond double) choline does not result in increased cholinergic activity.

• HACU transporter (high-affinity choline uptake) may not be fully saturated under stimulated conditions, allowing for enhanced choline use.

  • Or the Low-affinity choline carrier way facilitate uptake

  • The HACU is not required for ACh synthesis from choline

• Findings support targeting choline availability as a potential therapeutic strategy for central cholinergic dysfunctions, especially in conditions with elevated neuronal firing.