A causal study of bumetanide on a marker of excitatory-inhibitory balance in the human brain

TL;DR: It is shown that, contrary to expectation, acute administration of bumetanide does not alter binocular rivalry dynamics in neurotypical adult individuals, and this results raise important questions about the efficacy of acute bumeteanide administration for altering E/I balance in the human brain.

Abstract: Bumetanide has received much interest as a potential pharmacological modulator of the putative imbalance in excitatory/inhibitory (E/I) signaling that is thought to characterize autism spectrum conditions. Yet, currently, no studies of bumetanide efficacy have used an outcome measure that is modeled to depend on E/I balance in the brain. In this manuscript, we present the first causal study of the effect of bumetanide on an objective marker of E/I balance in the brain, binocular rivalry, which we have previously shown to be sensitive to pharmacological manipulation of GABA. Using a within-subjects placebo-control crossover design study, we show that, contrary to expectation, acute administration of bumetanide does not alter binocular rivalry dynamics in neurotypical adult individuals. Neither changes in response times nor response criteria can account for these results. These results raise important questions about the efficacy of acute bumetanide administration for altering E/I balance in the human brain, and highlight the importance of studies using objective markers of the underlying neural processes that drugs hope to target.

Summary

Introduction

• Excitatory and inhibitory (E/I) activity is balanced in neural systems at multiple spatial scales [1, 2], and this balance is thought to be critical for typical neural function [3–5].
• In particular, studies in both humans and in animal models suggest that altered inhibitory signaling, mediated by the neurotransmitter GABA, may characterize the condition [10, 11].
• During development, the polarity of GABAergic action transitions from excitatory to inhibitory due to a progressive reduction in intracellular chloride (Cl-) concentration in principal neurons [14, 15] -- a developmental sequence that may be disrupted in animal models of autism [16, 17].
• Importantly, to date, direct evidence that bumetanide increases inhibition in the human brain is lacking, which complicates linking the reported symptomatic benefits to the drug’s predicted physiological effects.
• The authors tested this hypothesis in a within-subjects drug-placebo, crossover design pharmacological study of rivalry dynamics in neurotypical adults.

Materials and Methods

• Written consent was obtained from all participants, and all studies were approved by the Massachusetts Institute of Technology Institutional Review Board.
• Bumetanide is an FDAapproved loop-diuretic known to antagonize sodium-potassium-chloride cotransporters, NKCC1 and NKCC2, which modulate intracellular chloride concentration.
• For each participant and trial, the frequency of perceptual transitions as well as the duration of any perceptual event (red, green, or mixed) were calculated.
• Binocular rivalry replay trial stimuli were identical to those used in the main rivalry experiment, and the paradigm was identical to their previously published studies [31, 33].

Results

• The authors predicted that bumetanide, a drug known to alter intracellular Cl- concentration and, by proxy, posited to increase GABAergic inhibition, would increase perceptual suppression during rivalry.
• The authors also assessed performance on rivalry replay control trials to establish whether any observed changes were due to non-perceptual effects on response latencies or response criteria [39, 40].
• To test whether bumetanide affects the depth of perceptual suppression during rivalry, the authors calculated the drug effect on the proportion of suppression for each individual (Proportion of Suppression on Drug - Placebo days) using a Wilcoxon signed-rank test.
• Drug effects are not confounded by shifts in response latency or response criteria.

Test-retest reliability

• To examine the stability of their primary measure, perceptual suppression, the authors calculated test-retest reliability by correlating performance on drug versus placebo days across individuals in each study.
• Bumetanide does not affect self-reported drowsiness Participants did not report significant differences in drowsiness between placebo and drug days (mean: 0.35 questionnaire points +/- 1.69 points, p = 0.367).

Discussion

• The authors have shown that acute administration of bumetanide does not alter binocular rivalry dynamics in neurotypical adult individuals.
• Indeed, the effects the authors observed (lower perceptual suppression) here trended in the opposite direction as predicted from previous studies of the impact of GABA modulators on rivalry dynamics [31, 32].
• Previous studies examining the longitudinal effects of bumetanide in individuals with autism have often demonstrated success in modulating social processing.
• It is thought that bumetanide may affect neural processing by modulating E/I balance in the brain.
• By this measure (and excluding self-citations to the and last authors of their current paper), their references contained 3.8% woman/ woman(last), 3.8% man/woman, 22.6% woman/man, 69.8% man/man, and 0% unknow n categorization.

Figures

Figure 1. Experimental paradigm. Participants took part in a double-blind crossover study to test the effects of bumetanide, a drug thought to alter GABAergic action, on binocular rivalry, a noninvasive marker of excitatory-inhibitory balance in visual cortex. After an initial study visit (Day 1; see Materials and Methods), participants returned for two experimental testing days (Day 2 and Day 3). On each experimental day, participants were administered either a placebo or the drug. After waiting for drug absorption, participants completed binocular rivalry and control rivalry replay trials, so that the effect of bumetanide on rivalry dynamics could be assessed.

Figure 3. High test-retest reliability of rivalry dynamics. Individual rivalry dynamics were stable across bumetanide and placebo days (Rs = 0.657, p = 0.002). For example, a fast rivaler on Day 2 remained a fast rivaler on Day 3 relative to the group. Dashed lines represent the 95% confidence interval of the regression line.

Figure 2. Experimental results. a) No effect of bumetanide administration was found on perceptual suppression (Z = -1.634, p = 0.10, r = 0.367), although this effect trended in the opposite direction as predicted. (top) This trend was driven by less time in dominant percepts (Z = -2.315, p = 0.021, r = 0.518); (bottom) no differences in mixed percept durations were observed (Z = -0.597, p = 0.55, r = 1.133). b) No effect on switch-rate was observed (Z = -0.28, p = 0.779, r = 0.062).

Full text

RUNNING HEAD: BUMETANIDE EFFECTS ON BINOCULAR RIVALRY
1
A causal study of bumetanide on a marker of excitatory-inhibitory balance in the
human brain
Thomas L. Botch
1
, Alina Spiegel
2
, Catherine Ricciardi
3
, Caroline E. Robertson
1+
1
Department of Psychological and Brain Sciences, Dartmouth College, Hanover, NH
03755, USA
2
School of Medicine, Johns Hopkins University, Baltimore, MD, 21205
3
Clinical Research Center, Massachusetts Institute of Technology, Cambridge, MA
02139, USA
+
Corresponding author: Dr. Caroline Robertson, caroline.e.robertson@dartmouth.edu
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.22.304279doi: bioRxiv preprint

BUMETANIDE EFFECTS ON BINOCULAR RIVALRY
2
Abstract
Bumetanide has received much interest as a potential pharmacological modulator of the
putative imbalance in excitatory/inhibitory (E/I) signaling that is thought to characterize
autism spectrum conditions. Yet, currently, no studies of bumetanide efficacy have used
an outcome measure that is modeled to depend on E/I balance in the brain. In this
manuscript, we present the first causal study of the effect of bumetanide on an objective
marker of E/I balance in the brain, binocular rivalry, which we have previously shown to
be sensitive to pharmacological manipulation of GABA. Using a within-subjects placebo-
control crossover design study, we show that, contrary to expectation, acute
administration of bumetanide does not alter binocular rivalry dynamics in neurotypical
adult individuals. Neither changes in response times nor response criteria can account
for these results. These results raise important questions about the efficacy of acute
bumetanide administration for altering E/I balance in the human brain, and highlight the
importance of studies using objective markers of the underlying neural processes that
drugs hope to target.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.22.304279doi: bioRxiv preprint

BUMETANIDE EFFECTS ON BINOCULAR RIVALRY
3
Introduction
Excitatory and inhibitory (E/I) activity is balanced in neural systems at multiple spatial
scales [1, 2], and this balance is thought to be critical for typical neural function [3–5].
Multiple lines of evidence implicate disrupted E/I balance in the neurobiology of Autism
Spectrum Conditions (ASC; autism henceforth) [6–12]. In particular, studies in both
humans and in animal models suggest that altered inhibitory signaling, mediated by the
neurotransmitter GABA, may characterize the condition [10, 11]. Despite the
accumulating evidence, the intricacies of autism neurobiology are poorly understood,
hindering efforts to develop treatment strategies for the condition.
One prominent developmental account of autism proposes a disruption of an important
neurobiological milestone, known as the GABA-switch, as a potential explanation for
disturbed inhibitory action in the autistic brain [13]. During development, the polarity of
GABAergic action transitions from excitatory (depolarizing) to inhibitory (hyperpolarizing)
due to a progressive reduction in intracellular chloride (Cl-) concentration in principal
neurons [14, 15] -- a developmental sequence that may be disrupted in animal models of
autism [16, 17]. In light of these accounts, it has been posited that augmenting GABAergic
action might provide a promising therapeutic for some symptoms associated with autism
[13, 18].
Bumetanide, a loop diuretic, has proven hopeful in rectifying GABA polarity in valproic
acid and Fragile X animal models of autism [16, 19]. Bumetanide is thought to increase
the hyperpolarizing potential of GABA by blocking NKCC1 receptors, which are
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.22.304279doi: bioRxiv preprint

BUMETANIDE EFFECTS ON BINOCULAR RIVALRY
4
responsible for Cl- entrance into the cell [20]. Further, some studies of bumetanide in
humans, specifically children with autism, have shown evidence for attenuation of social
symptom severity and improvement of emotion recognition [21–23], although, notably,
these benefits are not universally observed [24]. Importantly, to date, direct evidence that
bumetanide increases inhibition in the human brain is lacking, which complicates linking
the reported symptomatic benefits to the drug’s predicted physiological effects.
Therefore, we sought to test the effects of bumetanide on a robust behavioral index of E/I
balance, binocular rivalry. Rivalry is a simple visual phenomenon that is modeled to rely
on the on the balance of inhibition and excitation in visual cortex [25–30]. Prior
pharmacological studies in humans reveal a causal link between rivalry dynamics and
GABAergic inhibition using both GABA
A
and GABA
B
modulators [31, 32], as well as a
dependence of rivalry dynamics on tonic levels of GABA in visual cortex [11, 32]. Given
these links between rivalry dynamics and E/I balance in visual cortex, as well as recent
evidence showing altered rivalry dynamics in adult individuals with autism [11, 33–35],
rivalry has been suggested as a noninvasive perceptual marker of E/I signaling in visual
cortex, and its putative disturbance in psychiatric conditions, including autism.
Here, we asked whether acute bumetanide administration would alter rivalry dynamics.
We hypothesized that bumetanide would increase the degree to which individuals
predominantly perceive one image fully suppressed from awareness (“perceptual
suppression”), which computational and empirical data suggest is gated by GABAergic
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.22.304279doi: bioRxiv preprint

BUMETANIDE EFFECTS ON BINOCULAR RIVALRY
5
inhibition [31, 36, 37]. We tested this hypothesis in a within-subjects drug-placebo, cross-
over design pharmacological study of rivalry dynamics in neurotypical adults.
Materials and Methods
Participants. 21 healthy adults (N = 15 female; mean age 22.5 +/- 3.68 SD years)
participated in the study. Written consent was obtained from all participants, and all
studies were approved by the Massachusetts Institute of Technology Institutional Review
Board. All participants had normal or corrected-to-normal vision, were neither pregnant
nor nursing, and were free from: (1) any known history of psychiatric or neurological
conditions; (2) any other diagnosed medical conditions, including a history of heart failure;
(3) any psychiatric medications; and (4) any known drug allergies (including bumetanide).
All studies took place at the MIT Clinical Research Center, under the constant observation
of a research nurse/nurse practitioner (C.R.) and nursing team.
Study drugs: bumetanide (loop-diuretic). Participants participated in a study investigating
the effects of bumetanide (1 mg) on binocular rivalry dynamics. Bumetanide is an FDA-
approved loop-diuretic known to antagonize sodium-potassium-chloride cotransporters,
NKCC1 and NKCC2, which modulate intracellular chloride concentration. At low
concentrations, bumetanide has a high affinity to block NKCC1, thereby reducing
intracellular chloride concentration and, by proxy, altering GABAergic action potentials
[14, 20]. Bumetanide dosage was chosen to fall within the standard prescribed range.
Experimental design: placebo-controlled crossover design. Each participant took part in
a 3-day study, comprised of: a health assessment/practice session (Day 1) and 2
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.22.304279doi: bioRxiv preprint