Dietary Restriction Affects Neuronal Response Property and GABA Synthesis in the Primary Visual Cortex

<div><p>Previous studies have reported inconsistent effects of dietary restriction (DR) on cortical inhibition. To clarify this issue, we examined the response properties of neurons in the primary visual cortex (V1) of DR and control groups of cats using <i>in vivo</i> extracellular single-unit recording techniques, and assessed the synthesis of inhibitory neurotransmitter GABA in the V1 of cats from both groups using immunohistochemical and Western blot techniques. Our results showed that the response of V1 neurons to visual stimuli was significantly modified by DR, as indicated by an enhanced selectivity for stimulus orientations and motion directions, decreased visually-evoked response, lowered spontaneous activity and increased signal-to-noise ratio in DR cats relative to control cats. Further, it was shown that, accompanied with these changes of neuronal responsiveness, GABA immunoreactivity and the expression of a key GABA-synthesizing enzyme GAD67 in the V1 were significantly increased by DR. These results demonstrate that DR may retard brain aging by increasing the intracortical inhibition effect and improve the function of visual cortical neurons in visual information processing. This DR-induced elevation of cortical inhibition may favor the brain in modulating energy expenditure based on food availability.</p></div

Poles with error bars represented the average optical density (OD) value of GABA immunoreactivity in the primary visual cortex of each normal control cat (NC1-4) and DR cat (DR1-4).

<p>The mean OD of GABA immunoreaction in each DR cat was significantly larger than that in any individual control cat (** p<0.001).</p

Immunohistochemical labeling of GABAergic neurons in the primary visual cortex of DR cats (A&C) and normal control cats (B&D).

<p>(A&B) show the distribution of GABA neurons across different cortical layers (layer I, II-III, IV, V and VI) at a low amplification. (C&D) show GABA neurons at a higher amplification. The scale bar equals to 25 μm.</p

Percentile value of neurons showing different orientation bias (OB) (A) and motion direction bias (DB) (B) for DR cats (open circle) and normal control cats (solid circle).

<p>The total number of neurons was 75 and 88 respectively for DR cats and control cats. A percentile value indicated the percentage of neurons whose OBs or DBs were lower than the corresponding OB or DB value on the horizontal axis. DR cats showed significantly increased OB and DB value compared with control cats (p<0.0001; p<0.0001).</p

The response property of two typical neurons from the normal control (A, C, E) and DR (B, D, F) group of cats respectively.

<p>(A&B) The voltage trace of the neuron’s response to its preferred stimulus orientation and motion direction. A spike with amplitude above the horizontal broken line was counted as an action potential. Spontaneous activity was acquired during 1s pre-stimulus period. The neuron’s visually-driven response was evoked by 5 cycles of drifting grating stimulus with the preferred orientation, equivalent to a stimulus duration of 1.7s. (C&D) Mean response (pole with error bar) of the neuron to different stimulus orientations. The maximum response represented the neuron’s response to the preferred stimulus orientation and motion direction. (E&F) Circle variance showed the neuron’s response selectivity for stimulus orientations and motion directions, with orientation bias (OB) of 0.275 and 0.747 respectively, motion direction bias (DR) of 0.133 and 0.487 respectively.</p

Measures of the cell number (CN), mean orientation bias (OB), motion direction bias (DB), maximum response (MR) to the preferred stimulus orientation, average response (AR) to all stimulus orientations, baseline response (BR) and signal-to-noise ratio (STN) for studied neurons of each normal control cat (NC1, NC2, NC3, NC4) and DR cat (DR1, DR2, DR3, DR4).

<p>Measures of the cell number (CN), mean orientation bias (OB), motion direction bias (DB), maximum response (MR) to the preferred stimulus orientation, average response (AR) to all stimulus orientations, baseline response (BR) and signal-to-noise ratio (STN) for studied neurons of each normal control cat (NC1, NC2, NC3, NC4) and DR cat (DR1, DR2, DR3, DR4).</p

The quantity of GAPDH protein measured with enzyme linked immunosorbent assay.

<p>The quantity was expressed as a relative value of GAPDH (μg) to total proteins (g) in V1 samples of each normal control cat (NC1, NC2, NC3, NC4) and DR cat (DR1, DR2, DR3, DR4). Assays for each subject were performed in triplicate. The mean content in DR cats showed no significant difference from that in control cats (F(1,8) = 0.02, p>0.5).</p

Comparison of the expression levels of GABA-synthesizing enzyme subunit, GAD67, between DR and normal control cats.

<p>The top panel shows a typical sample of a Western blot result from each DR cat (DR1-4) and control cat (NC1-4) using GAD67 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibodies. The bottom panel shows the average optical density (OD) of GAD67 bands normalized against the corresponding GAPDH in both groups of cats. The average normalized optical density of GAD67 in DR cats was significantly higher than that in the control (** p<0.01).</p

Body weight (BW) changes of each DR cat (open circle) and normal control cat (open square) during the period of DR.

<p>The BW of normal control cats (NC1: red; NC2: blue; NC3: cyan; NC4: green) showed an insignificant increase (p = 0.07), whereas the BW of DR cats (DR1: red; DR2: blue; DR3: cyan; DR4: green) were significantly decreased (p<0.01).</p

Record of body temperature (A), heart rate (B), femoral artery contraction blood pressure (C) and blood oxygen saturation (D) for each DR cat and control cat during the period of DR.

<p>Open squares represented normal control cats (NC1: red; NC2: blue; NC3: cyan; NC4: green). Open circles denoted DR cats (DR1: red; DR2: blue; DR3: cyan; DR4: green).</p