Article Figures & Data
Figures
- Figure 1.
GCaMP3 Ca2+ imaging faithfully correlates to electrophysiologically recorded membrane potential oscillations in TIDA neurons. A, Fluorescence micrograph in the coronal plane of a male DAT–GCaMP3 mouse brain at the level of the arcuate nucleus. Converted to grayscale and inverted for better contrast. Inset, Higher magnification of marked region. Ba–c, Confocal micrographs of marked region in A, immunostained for Tyrosine Hydroxylase (TH) (red; Ba), GFP (green; Bb), and merged (Bc). Yellow represents colocalization of signals. C, Photomicrograph of a fluorescent DAT-GCaMP3 neuron in the dmArc during whole-cell patch-clamp recording. Dashed line outlines recording pipette. Da, Whole-cell current-clamp recording from a DAT-GCaMP3 neuron. Downsampled trace overlaid in red. Db, Downsampled trace from Da aligned with the simultaneously recorded Ca2+ imaging activity (green). Note coincident activity peaks in both signals. Dc, Gray box in Db expanded to illustrate high peak-to-peak correlation (black arrow) and the slow decay kinetics of the Ca2+ signal compared with the membrane depolarization. E, Cross-correlogram between the downsampled membrane potential and the Ca2+ activity of trace (Dd). Scale bars: (in Ba) Ba–c, 100 µm; C, 25 µm. 3V, Third ventricle; ME, median eminence.
- Figure 2.
The frequency of TIDA neuron Ca2+ oscillations increases during lactation. A, Representative concomitant traces of Ca2+ fluctuations in DAT-GCaMP3 neurons in the dmArc from the same slice in adult males (Aa), female in diestrus/metestrus (D/M; Ab), female in estrus (from the proestrus+estrus group; P + E; Ac), and lactating dam (Ad). B, Box plot represents the frequency of the Ca2+ oscillations in DAT-CGaMP3 neurons in the dmArc recorded from female mice in proestrus and estrus. There is no significant difference between the two groups (unpaired Student's t test, p = 0.14). n indicates the number of animals/neurons. Box plots represent the interquartile ranges (square) and median (line). Values with whiskers indicate minimum and maximum values. C, Scatter plots represent the mean frequency and SEM of oscillating DAT-GCaMP3 neurons in the dmArc (i.e., all oscillating cells, from all slices of a single mouse). The mean frequency was significantly different between the lactating group and the male, D/M, and P + E groups. ***p< 0.001 (one-way ANOVA with Tukey's multiple comparison as post hoc test). **p > 0.01 (one-way ANOVA with Tukey's multiple comparison as post hoc test). All other comparisons between groups are nonsignificant. n indicates the number of animals/cells. D, The number of oscillating cells in a slice plotted against the mean oscillation frequency of the same slice. No correlation (Pearson correlation: R2 < 0.05) is found, suggesting that the number of active cells within a preparation does not influence the speed of the activity. E, Serum prolactin concentration measured by immunoassay in the experimental groups shown in C. *p > 0.05 (one-way ANOVA with Tukey's multiple comparison as post hoc test). **p > 0.01 (one-way ANOVA with Tukey's multiple comparison as post hoc test). All other comparisons between groups are nonsignificant. n indicates the number of samples/animals.
- Figure 3.
Rhythmicity, but not functional connectivity, of TIDA neurons is affected in lactating dams. A, Representative network maps of the functional connectivity in DAT-GCaMP3 neurons in the dmArc of adult male (Aa), female in diestrus/metestrus (D/M; Ab), female in estrus (from the proestrus+estrus group; P + E; Ac), and lactating dam (Ad). Color coding represents the degree of correlation between neuron pairs. Ba–d, Representative autocorrelation from one of the neurons from maps shown in A for the respective groups. Ba, Autocorrelogram illustrates the oscillation period (T), the amplitude of the first trough (α), and the amplitude of the second peak (β); the RI was defined as RI = β/α. C, Scatter plots represent mean values of the correlation matrix between groups and SEM. There are no significant differences (p = 0.31-0.87). One-way ANOVA with Tukey's multiple comparison used as post hoc test. n indicates the number of network maps per hemisphere. D, Box plots represent the interquartile ranges (square) and median (line). Values with whiskers indicate minimum and maximum values of the RI. The RI of the lactating group was significantly lower than the RI of the other groups. ****p< 0.001 (Kruskal–Wallis with Dunn's multiple comparison as post hoc test). n indicates the number of cells. All other comparisons between groups are nonsignificant. Scale bar: (in Ad) Aa–d, 50 µm. 3V, Third ventricle. E, The correlation between the Ca2+ oscillation frequency and the RI is low, suggesting that the speed of the oscillation is independent of the rhythmicity.
- Figure 4.
Lactation-associated network changes reverse on weaning of pups. A, Representative concomitant traces of Ca2+ fluctuations in DAT-GCaMP3 neurons in the dmArc from the same slice in nulliparous female in estrus (Aa), primipara lactating (L1) dam (Ab), dam in estrus, 3 weeks after weaning (Ac), and lactating dam following a second pregnancy (L2; Ad). B, Box plot represents the interquartile ranges (square) and median (line). Values with whiskers indicate minimum and maximum values of the oscillation frequency. Note significantly lower frequency in weaned dams compared with L1 and L2. **p < 0.01 (one-way ANOVA with Tukey' multiple comparison as post hoc test). ****p < 0.0001C, There is no significant difference between the average frequency of the Ca2+ oscillations between the estrus (nulliparous) group and the weaned females during estrus (unpaired Student's t test, p = 0.06). D, There is no significant difference between the correlation matrix mean between the estrus (nulliparous) group and the weaned females during estrus (unpaired Student's t test, p = 0.14). n indicates the number of network maps per hemisphere. E, There is no significant difference between the rhythmicity index between the estrus (nulliparous) group and the weaned females during estrus (Mann–Whitney, p = 0.33). n indicates the number of cells. Aa, Ab, Same traces as Figures 2Ac, d, respectively. The number of animals is as follows: L1, n = 5; L2, n = 5; nulliparous, n = 7; weaned, n = 4.
- Figure 5.
Lactation-associated increase in oscillation frequency is paralleled by depolarization and changes in TIDA membrane properties. A, Whole-cell current-clamp in vitro recording of a DAT-GCaMP3 neuron in the dmArc from a nulliparous female. Membrane potential alternates between hyperpolarized DOWN states and depolarized UP states crowned by action potential discharge. Note increasing frequency of oscillations as depolarizing current of increasing amplitude is injected (10 pA steps). B, Current-clamp recording of a DAT-GCaMP3 neuron from a lactating female. Note faster oscillation frequency that slows down on injection of hyperpolarizing current (−15 pA step). C, The mean DOWN state potential (nadir) of DAT-GCaMP3 dmArc neurons from lactating females is more depolarized (−54.00 ± 1.87 mV; n = 10) than in virgin females (−60.00 ± 1.15 mV; n = 11). **p < 0.01 (unpaired Student's t test). D, Nadir potential plotted against oscillation frequency (data from whole-cell recordings in DAT-GCaMP3 neurons). Note linear relationship in both nulliparous and lactating females, and overall higher oscillation frequencies in lactating dams. Ea, Electrophysiological recordings of DAT-GCaMP3 neuron from a nulliparous (top) and lactating (below) female in the presence of TTX (500 nm), to abolish oscillations and establish a stable baseline, in response to a series of hyperpolarizing square current steps (−10/−20 to −50 pA). Blue arrow indicates outwardly rectifying (A-like) current. Note depolarizing sag in lactating female (absent from nulliparous control), indicative of the h-current (Ih, magenta arrow). Eb, Representative traces from a whole-cell voltage-clamp recording from DAT-tdTomato neuron in the dmArc from a lactating dam before and after 4-AP application (4 mm) application. Command voltage protocol to elicit A-like K+ current shown above. A-type currents were evoked in response to 10 mV membrane potential steps from −80 to 70 mV after a −100 mV prepulse. Ec, Current–voltage curves from nulliparous and lactating dams in the presence or absence of 4-AP. No significant difference was found between control and 4-AP conditions (p = 0.08, F test; n = 5 for nulliparous; n = 6 for lactating).
- Figure 6.
TIDA neurons express HCN channel mRNA. A, Confocal micrograph from the arcuate nucleus of a female nulliparous mouse in estrus after ISH (RNAScope) performed to detect DAT mRNA (white); section counterstained with DAPI (blue) to visualize anatomy. Note distribution of DAT-expressing TIDA cells in the dmArc (arrow). Insets, High-magnification examples of TIDA cells coexpressing DAT (white) and (in clockwise order): the HCN channel 1 (HCN1; magenta) and HCN2 (green); the HCN channel auxiliary subunit, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b; green); HCN3 (magenta); and HCN4 (magenta). B, Confocal micrograph organized as in A, from the arcuate nucleus of a lactating dam. All RNA transcripts detected in the nulliparous female TIDA neurons can also be found in the same neurons from a lactating female. Scale bars: A, B, 100 μm; Inset, 20 μm. 3V, Third ventricle; ME, median eminence. C, Histogram displaying the H-score (see Materials and Methods) of DAT-expressing neurons in the dmArc that coexpress HCN1-4 mRNA. The individual values represent the score from slices from 3 animals in each group. The H-score is not statistically different for either HCN mRNA between the two experimental groups (unpaired Student's t test: HCN1, p = 0.43; HCN2, p = 0.45; HCN3, p = 0.13; HCN4, p = 0.05).
- Figure 7.
An increase in h-current contributes to increased TIDA oscillation frequency during lactation. Aa, Whole-cell current-clamp in vitro recording of a DAT-GCaMP3 neuron in the dmArc from a lactating dam; negative current step injected to reveal depolarizing sag indicative of h-current (Ih; magenta trace). Superimposed gray trace represents the same cell recorded in the presence of the Ih-blocker, ZD7288 (50 μm). Sag abolished after pharmacological blockade. Ab, The voltage difference between the sag and the steady state of a hyperpolarizing step is significantly larger in TIDA neurons from lactating dams compared with nulliparous females. ***p < 0.001 (unpaired Student's t test). Ac, The blockage of Ih by ZD7288 decreased the nadir potential in lactating dams (Lac) but not in nulliparous females (Nulli). *p < 0.001 (paired t test). Ba, Representative trace from a whole-cell voltage-clamp recording from DAT-tdTomato neuron in the dmArc from a lactating dam (in the presence of TTX, 500 nm) before (magenta) and during (gray) ZD7288 application (50 μm). Bb, Application of ZD7288 decreased the sag amplitude of Ih in both nulliparous and lactating dam TIDA neurons. *p < 0.05 (paired Student's t test). Bc, I-V curves from nulliparous and lactating dams in the presence or absence of ZD7288. Bd, The comparison of the slope obtained by linear fitting of the I-V curves reveals a difference in lactating dams with respect to nulliparous, which is abolished during Ih blockade by ZD7288. *p < 0.05 (two-way ANOVA with Sidak's multiple comparison as post hoc test). C, Representative traces of Ca2+ fluctuations in a DAT-GCaMP3 neuron before and during ZD7288 application in nulliparous and lactating dam. Box plot represents the effect of bath-applied ZD7288 (50 μm) on the frequency of Ca2+ oscillations in DAT-GCaMP3 neurons from nulliparous and lactating females. In nulliparous, the frequency remains unchanged during pharmacological blockade of Ih (p = 0.77, paired Student's t test); whereas in lactating females, the frequency decreased during ZD7288 application (p = 0.01, paired Student's t test) **p = 0.01. The frequency was significantly different between control conditions without ZD7288 (p = 0.03, unpaired t test).
Tables
- Table 1.
Summary of basic electrophysiological properties of TIDA neurons in nulliparous and lactating femalesa
Nulliparous Lactating State p Cell capacitance 19.7 ± 2.7 20.3 ± 3.0 p = 0.88 Membrane potential (mV) Condition Control −51.4 ± 0.9 −42.74 ± 3.3 Main effect of states: p = 0.018*
Conditions: p = 0.041*ZD −51.2 ± 0.5 −44.6 ± 2.8 Interaction: p = 0.046* Input resistance (GΩ) Condition Control 0.7 ± 0.2 −2.1 ± 0.5 Main effects of state: p = 0.046* ZD 0.6 ± 0.2 −3.5 ± 1.4 Conditions: p = 0.262
Interaction: p = 0.19
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