Suppl Data1.xlsx Supplementary materials to Ozawa et al. (2023) [DOI: 10.3390/ijms241713590]

Suppl Data1.xlsx Supplementary materials to Ozawa et al. (2023) [DOI: 10.3390/ijms241713590]</p

Supplementary materials to Ozawa et al. (2023) [DOI: 10.3390/ijms241713590]

Supplementary materials to Ozawa et al. (2023) [DOI: 10.3390/ijms241713590] with captions.</p

Neuron densities and abundance ratios of oriented and unoriented neurons.

<p>(A) Histograms of neuron density for NR and GR mice. The difference in the neuron density between NR and GR mice was statistically significant for the vertical orientation (p<0.05, Student t-test). Error bars indicate SE. (B) Ratios of the numbers of oriented, unoriented and unresponsive neurons to the total number of neurons identified by OGB-1 signals for the NR group (left) and GR group (right). The absolute numbers of respective types of neurons are shown in parentheses. The abundance ratio of oriented neurons as well as that of unoriented neurons did not differ between the two groups.</p

Typical results of two-photon imaging for juvenile NR and GR mice.

<p>(A) Image of Ca²⁺ signals in a slice of the VI of an NR mouse. Fluorescence signals from OGB-1 (green) and SR101 (orange) identify neurons and astrocytes, respectively. Dark spots and strips indicate blood vessels. The scale bar indicates 100 µm. (B) Traces of Ca²⁺ responses of four neurons to stimulus orientations of grating stimuli, where stimulus orientations are indicated by the inclinations of the black bars placed below. These four neurons were located at positions indicated by the numbered arrows in (A). Gray columns represent 5 s stimulation periods. Vertical and horizontal bars respectively indicate 5% () and 5 s. (C) Color-coded images of vigorously responsive neurons in single slices of an NR mouse (left) and a mouse GR from P23 to P31 (right), which were reconstructed from two-photon Ca²⁺ imaging conducted at P42 and P31, respectively. The left image was reconstructed from the Ca²⁺ signals in the slice shown in (A). The color of the dots indicates the preferred orientation, whereas gray dots indicate responsive but unoriented neurons. The color code is shown below the left image. (D) Group-averaged orientation distributions for NR (n = 3) and GR (n = 3) mice. Error bars indicate SE. The scale bar indicates 100 µm.</p

Orientation bias indices (OBIs) for NR and GR mice and sensitivity profile for orientation plasticity.

<p>(A) Averaged OBIs obtained from intrinsic signal imaging of NR and GR groups for juvenile and adult mice. (B) Averaged OBIs obtained from two-photon imaging of juvenile NR and GR groups. (C) Age dependence of OBIs averaged among animals of the same age for NR and GR mice. The dashed gray line indicates OBI = 0.5, corresponding to the absence of any representation bias towards the vertical or horizontal orientation. Faint dots indicate OBIs for individual mice. Error bars indicate SE. Significant differences in OBIs for GR and NR mice are shown as *p<0.05, **p<0.01 and ***p<0.001. (D) Sensitivity profile for orientation plasticity induced by the one-week of exposure to vertical orientation from 3w to 15w. The critical period for orientation plasticity is suggested to lie between 4w and 7w, during which the sensitivity index (SI) is prominently large. Also, orientation plasticity is found to be preserved to some extent even after 12w.</p

Cortical activation patterns and orientation distributions.

<p>(A) Single-orientation maps of an NR mouse and a mouse GR from P21 to P28, which were reconstructed from intrinsic signal optical imaging conducted at P29 and P28, respectively. The darkness indicates the strength of intrinsic signals evoked by oriented grating stimuli. The dotted rectangles show the region of interest. R: rostral, L: lateral. (B) Orientation distributions obtained from the single-orientation maps shown in (A). The ordinate indicates the relative size of activation areas eliciting stimulus-related responses stronger than 2SD for each stimulus orientation. (C) Group-averaged orientation distributions for NR (n = 45) and GR (n = 44) groups of juvenile mice. Error bars indicate SE (standard error). All scale bars indicate 1 mm.</p

Orientation-restricting goggles and experimental setup for intrinsic signal optical imaging.

<p>(A) Photograph of goggles (left) and picture of a goggle-mounted mouse (right). The scale bar indicates 5 mm. (B) The monitor screen presenting visual stimuli was placed 25 cm apart from either eye of a mouse. The angle between the center of the screen and the animal's midline was set at 45°. Optical imaging was performed transcranially in a rectangular region in the hemisphere contralateral to the stimulated eye, which included the primary visual cortex. (C) Images of blood vessels in the skull and cortical surface (left), and stimulus-evoked intrinsic signals whose strength is indicated by their darkness (right). A detailed analysis was performed using the signals evoked inside the dashed rectangle. The scale bar indicates 1 mm.</p

Whisker-evoked CBF changes measured by laser Doppler flowmetry from the barrel cortex.

<p>(<b>A</b>) Single example trace of 10 Hz air puff stimulation experiment. The recording was made from a WT mouse. (<b>B</b>) Proportion of barrel cortex astrocytes that elicited Ca²⁺ elevations after whisker stimulation (10 Hz, 20 s) for WT (n = 3) and IP₃R2 KO (n = 3). (<b>C</b>) Mean CBF of WT (black) and IP₃R2 mice (white) during 20 s whisker stimulation was compared. The number of tested animal for each stimulation paradigm is shown in parenthesis. Whisker stimulation with 5 or 10 Hz significantly increased cerebral blood flow in WT and KO when compared with prestimulus period. No significant differences in CBF response were observed between WT and KO in any of the stimulation paradigms.</p

Averaged traces of laser Doppler flowmetry in C57BL/6J and IP₃R2-KO mice.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066525#s2" target="_blank">Results</a> for C57BL/6J and IP₃R2-KO mice are represented in blue and red traces, respectively. Each experimental condition has N>5 animals. Upon 200 µA stNBM, the cerebral blood flow (CBF) showed an immediate increase, followed by a transient decrease that overshot the baseline both in WT (<b>A</b>) and KO (<b>E</b>). CBF change by the stNBM is attenuated by the muscarinic receptor antagonist atropine (ATR) (<b>B</b>). Weak stNBM stimulation (50 µA) resulted in negative laser Doppler flowmetry signal (<b>C</b>) and similar changes were observed in KO (<b>F</b>). Stimulation outside the NBM failed to induce CBF increase (<b>D</b>). Shaded areas represent s.e.m.</p

Overview of the stNBM experiment.

<p>(<b>A</b>) Sketch for experimental set up. A laser Doppler probe is placed above the thinned skull at the primary somatosensory cortex. A bipolar stimulation electrode is inserted to target the nucleus basalis of Meynert (NBM) in the ipsilateral side. (<b>B</b>) An example trace of laser Doppler flowmetry from a wild type mouse in response to a single train NBM stimulation (stNBM, arrow). (<b>C</b>) <i>In vivo</i> two-photon imaging of Fluo-4 AM loaded astrocytes in the somatosensory cortex of C57BL/6J (WT, upper panels) and IP₃R2-KO (lower panels). (<b>D</b>) Proportion of barrel cortex astrocytes that elicited Ca²⁺ elevations upon stNBM.</p