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Date of Award

Spring 2012

Degree Type

Thesis

Degree Name

Master of Science (MSc)

Department

Medical Sciences (Division of Physiology/Pharmacology)

Supervisor

Jan D. Huizinga

Co-Supervisor

Wolfgang Kunze

Language

English

Committee Member

Elena Verdu

Abstract

The small intestine holds an intrinsic ability to digest and absorb nutrients from the food we intake without intervention from the central nervous system. This ability is made possible by the population of cells that inhabit the gut, particularly interstitial cells of Cajal of the myenteric plexus and sensory primary intrinsic neurons (AH cells), which ultimately influence muscle function and motility. The AH cells are the first neurons in the hierarchy of sensory neurons in the gut and are therefore a perfect candidate to test the effects of Bifidobacterium longum NCC3001 supernatant since in a physiological setting the metabolites secreted by this bacterium can interact with the AH cells directly or indirectly through absorption by the mucosa.

The probiotic Bifidobacterium longum NCC3001 has been shown to normalize anxiety-like behaviour and hippocampal brain derived neurotropic factor (BDNF) levels in mice infected with Trichuris muris in a model of infectious colitis. Utilizing a chronic model of colitis, a study was conducted to decipher whether or not the anxiolytic effects of Bifidobacterium longum NCC3001 involved the vagus. My specific objective in this study was to find evidence for interaction between B.longum NCC3001 and myenteric neurons as a potential route for B.longum NCC3001 to influence CNS function. We assessed a cell’s electro-responsiveness through spike discharge, which is the number of action potentials elicited in response to a supra-threshold depolarizing current injection.

The electro-responsiveness of neurons perfused with B. longum NCC3001 supernatant (conditioned medium; n = 4) was significantly reduced compared to the control group (those perfused with Krebs solution; n = 5; P = 0.016). The electro-responsiveness of neurons perfused with the conditioned medium was also significantly lower than that of neurons perfused with unconditioned group (MRS growth medium alone) group (n = 4; P = 0.029). In comparing the excitabilities of the neurons in the control group with that of the control media group, there was no statistical difference (P = 0.29).

In subsequent studies, the objective was to identify the AH cells and to determine the effect of B. longum NCC3001 conditioned medium on this population of cells. The electro-responsiveness as measured through spike discharge of AH cells perfused with the conditioned medium (n = 5) was significantly reduced compared to neurons perfused with the unconditioned medium (n = 5; P = 0.02). Sensory neurons perfused with the conditioned medium (n = 9) exhibited a significant reduction in their instantaneous input resistances compared to neurons perfused with the unconditioned medium (n = 8; P = 0.01). There was also a significant reduction in the time-dependent input resistance of neurons perfused with the conditioned medium (n = 9) compared to neurons perfused with the unconditioned medium (n = 8; P = 0.02). In addition, perfusion of the conditioned medium over sensory neurons (n = 9) significantly reduced the magnitude of the hyperpolarization-activated cationic current (Ih) compared to neurons perfused with the unconditioned medium (n = 8; P = 0.0003). Furthermore, there was also a significant reduction in the action potential half width duration of myenteric sensory neurons perfused with conditioned medium (n = 5) compared to that exhibited by neurons perfused with the unconditioned medium (n = 5; P = 0.008).

In later experiments, we wanted to gain a more comprehensive understanding of the effect of this bacterium on the gut so we evaluated its effects on the gut musculature. Upon full immersion, the supernatant of Bifidobacterium longum NCC3001 (conditioned medium) caused an initial depolarization of the circular smooth muscle cell. This depolarization continued until the slow wave oscillations in these cells ceased and membrane potential would plateau. Several minutes after this plateau, the slow wave oscillations reappeared and the cell was significantly hyperpolarized relative to the conditions before conditioned medium was added. The resting membrane potential of circular smooth muscle cells in Krebs solution was -54.3 mV and -70.3 mV approximately two minutes after full immersion by the supernatant when the cell was hyperpolarized and a stable recorded was achieved (n = 7; P = 0.02). The average time of onset of depolarization was 18.6 s and the average change in membrane potential (depolarization) from onset of effect to its plateau was 14.0 mV (n = 7). Occasionally, the addition of the conditioned medium only caused an immediate but slight depolarization (n = 3) and in other cases caused only a hyperpolarization of the cell (n = 3) with no significant changes in any slow wave characteristics in either case. Furthermore, any cells that exhibited the waxing and waning of the slow wave lost this pattern upon the addition of the conditioned medium (n = 10).

In attempts to understand the role of neurotransmission in this system, we conducted several experiments whereby carbachol (acetylcholine agonist) and L-NNA (nitric oxide synthase inhibitor) were administered to the muscle. Prior to the addition of 1μM carbachol or 2e-4 M L-NNA, we would only observe the pacemaker slow wave associated with the interstitial cells of Cajal of the myenteric plexus during the perfusion of Krebs solution. Upon the addition of carbachol (n = 3) or L-NNA (n = 4), we would observe a second slower frequency pattern appear, referred to as a waxing and waning pattern.

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