Bumetanide hyperpolarizes GABA reversal potential in immature neurons (A) and (B) voltage-ramps obtained from P0 cortical neurons of pups treated with intraperitoneal injections of either saline or bumetanide under gramicidin-perforated patch-clamp conditions. E_GABA reversed at more hyperpolarized potentials in bumetanide-treated mice than in controls (as indicated by the x-intercept). (C) Blocking NKCC1 with bumetanide effectively changed the chloride gradient in newborn cortical neurons, making GABA hyperpolarizing to the resting potential instead of depolarizing as in control cells. Bar graphs indicate mean ± SEM (number of recorded cells are indicated in parenthesis in bar graphs, n = 3 animals per condition,***P < 0.0001, t-test). (D) Comparison of E_GABA values for control- and bumetanide-treated cells recorded at RT and at 32 °C. Increasing the recording temperature did not significantly alter the E_GABA. Bar graphs indicate mean ± SEM (number of recorded cells are indicated in parenthesis in bar graphs, n = 2 animals at 32 °C, ***P < 0.0001, t-test). (E) Timeline of different drugs treatments. Black line represents length of control (PBS) treatment, and colored lines represent different bumetanide (Bum) exposure windows.

Bumetanide treatment alters morphology of cortical neurons. (A) and (B) 3D reconstructions of 2 cortical neurons from 4-week-old control or bumetanide-treated (E15–P7) mice. Animals were electroporated with GFP plasmids at E15 for visualization of the cells. Photographs on the left show flattened confocal stacks of neurons that were semiautomatically traced to produce dendritic diagrams on the right. Multiple rotational views of the same 2 neurons are shown on the right. Quantification of the soma size (C), number of primary dendrites (D), number of secondary dendrites (E), and total branching points (F) of traced cortical neurons. Compared with control, bumetanide-treated pyramidal neurons have less developed dendritic morphology. Scale bar, 20 μm. Bar graphs indicate mean ± SEM (PBS: n = 3 animals, bumetanide n = 4 animals; ***P < 0.0001, t-test).

Bumetanide treatment causes developmental delay in young mouse pups. (A) Average weights of animals treated with either saline or bumetanide from E15 to P7. Stars above data point represent those differences that are statistically significant. (B) Count of each motion (no movement, head raise, forelimb raise, or walking) when an animal is observed for 5 min. Stars in bars represent those movements that are significantly different from those of controls. (C) Percent of animals in each group that are able to turn around on a negative slope when initially placed head down. (D) Percent of animals able to hang from a bar for longer than 10 s (E) Percent of animals able to raise head and forelimbs when lowered to a surface by tail. Data points indicate mean ± SEM (n = 30 animals per condition, *P < 0.05, **P < 0.001, ***P < 0.0001, t-tests for A and B, logrank tests for C–E).

Blocking NKCC1 with bumetanide during a critical period disrupts the balance of excitatory and inhibitory synapses in the adult. (A) and (B) current traces of mPSCs of layer II cortical neurons recorded from 4-week-old and adult mice treated with either saline (PBS) or bumetanide from E15 to P7. Traces on the right represent expanded segments of traces on the left. Cells were recorded at –70 mV holding potential and in ACSF containing 0.5 μM TTX. AMPA mPSCs were isolated with bath application of 100 μM bicuculline and GABA mPSCs with 20 μM DNQX. (C) Frequency of AMPA (left) and GABA (middle) mPSCs for different windows of bumetanide exposure as described in Figure 1D. Ratio of GABA to AMPA mSPCs (right) shows that bumetanide treatment from E15 to P7 and E17 to P7 resulted in significant increases due to defects in forming excitatory AMPA synapses (D) same parameters analyzed in (C) but in 8- to 12-week-old adult mice. Bar graphs indicate mean ± SEM, number of recorded cells is indicated in each bar graph. Number of animals per condition are as follows: 4 week: control = 3, E15–E19 = 2, E15–P5 = 2, E15–P7 = 3, E17–P7 = 2, P0–P7 = 3, P7–P14 = 5; adult: control = 7, E15–E19 = 8, E15–P5 = 6, E15–P7 = 11, E17–P7 = 11, P0–P7 = 10, P7–P14 = 11; (*P < 0.01, **P < 0.001, ***P < 0.0001 compared with control; t-test).

Bumetanide treatment decreases spine density of cortical pyramidal neurons. (A) and (B) images of secondary dendrites from 4-week-old cortical pyramidal neurons from control or bumetanide-treated mice electroporated with GFP at E15. (C) Quantification of the average spine density shows that neurons from bumetanide-treated animals exhibited a significant spine decrease compared with controls. Scale bar, 20 μm. Bar graphs indicate mean ± SEM (PBS: n = 56, 3 animals; bumetanide n = 47, 4 animals; ***P < 0.0001, t-test). Quantification of reconstructed cells demonstrated that total dendrite length (D), total dendrite volume (E), total number of dendrite segments (F), number of dendrite terminal points (G), and full branch level (H) are all significantly decreased in bumetanide-treated pyramidal neurons compared with control. Bar graphs indicate mean ± SEM (PBS: n = 17 cells, 3 animals, bumetanide n = 18 cells, 4 animals, ***P < 0.0001, t-test).

Bumetanide treatment causes alterations in anxiety-related behavior and sensorimotor gating. (A) Elevated plus maze testing for anxiety-related behavior and emotionality. Graphs show percent of time spent in the open arms (left), percent of distance traveled in the open arms (middle), and number of entries into the open arms (right). (B) Same parameters shown for the closed arms. (C) PPI of the startle response testing for sensorimotor gating functions. Different startle amplitudes (in newtons) in response to the 120-dB stimulus (stim) are graphed against the different prepulse (pp) values. (D) PPI is measured by the degree to which the maximal startle response is inhibited by the prepulse stimulus. Bar graphs indicate mean ± SEM (n = 29 animals per condition). (E) Post hoc analysis of mice with similar startle response amplitudes demonstrates that even in mice matched for maximal startle, bumetanide-treated animals exhibit significant decrease in their PPIs (F) (PBS: n = 21 animals, bumetanide n = 29 animals; *P < 0.05, **P < 0.001, ***P < 0.0001, t-tests for A and B, 2-way ANOVA for C–F).