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

23 Sep 2020-bioRxiv (Cold Spring Harbor Laboratory)-

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.

AbstractBumetanide 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.

Topics: Bumetanide (57%)

Summary (2 min read)

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.

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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 [35].
Multiple lines of evidence implicate disrupted E/I balance in the neurobiology of Autism
Spectrum Conditions (ASC; autism henceforth) [612]. 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 [2123], 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 [2530]. 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, 3335],
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

Citations
More filters

Journal Article
Abstract: The dynamics of binocular rivalry may be a behavioral footprint of excitatory and inhibitory neural transmission in visual cortex. Given the presence of atypical visual features in Autism Spectrum Conditions (ASC), and the growing evidence in support of the idea of an imbalance in excitatory/inhibitory neural transmission in animal and genetic models of ASC, we hypothesized that binocular rivalry might prove a simple behavioral marker of such a transmission imbalance in the autistic brain. In support of this hypothesis, we previously reported a slower rate of rivalry in ASC, driven by longer transitional states between dominant percepts. We tested whether atypical dynamics of binocular rivalry in ASC are specific to certain stimulus features. 53 participants (26 with ASC, matched for age, sex, and IQ) participated in a binocular rivalry experiment in which the dynamics of rivalry were measured at two levels of stimulus complexity, low (grayscale gratings) and high (colored objects). Individuals with ASC experienced a slower rate of binocular rivalry, driven by longer transitional states between dominant percepts. These exaggerated transitional states were present at both low and high levels of stimulus complexity (gratings and objects), suggesting that atypical binocular dynamics in autism are robust with respect to stimulus choice. Interactions between stimulus properties and rivalry dynamics in autism indicate that achromatic grating stimuli produce stronger group differences. These results confirm the finding of atypical dynamics of binocular rivalry in ASC. These dynamics were present for stimuli of both low and high levels of visual complexity, suggesting a pervasive imbalance in competitive interactions throughout the visual system of individuals with ASC.

5 citations


References
More filters

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Abstract: In the immature brain, GABA (gamma-aminobutyric acid) is excitatory, and GABA-releasing synapses are formed before glutamatergic contacts in a wide range of species and structures. GABA becomes inhibitory by the delayed expression of a chloride exporter, leading to a negative shift in the reversal potential for choride ions. I propose that this mechanism provides a solution to the problem of how to excite developing neurons to promote growth and synapse formation while avoiding the potentially toxic effects of a mismatch between GABA-mediated inhibition and glutamatergic excitation. As key elements of this cascade are activity dependent, the formation of inhibition adds an element of nurture to the construction of cortical networks.

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TL;DR: Although inhibition is typically as strong as excitation, it is not necessary to establish tuning, even in the receptive field surround, and Balanced inhibition might serve to increase the temporal precision and thereby reduce the randomness of cortical operation, rather than to increase noise as has been proposed previously.
Abstract: Neurons in the primary auditory cortex are tuned to the intensity and specific frequencies of sounds, but the synaptic mechanisms underlying this tuning remain uncertain. Inhibition seems to have a functional role in the formation of cortical receptive fields, because stimuli often suppress similar or neighbouring responses, and pharmacological blockade of inhibition broadens tuning curves. Here we use whole-cell recordings in vivo to disentangle the roles of excitatory and inhibitory activity in the tone-evoked responses of single neurons in the auditory cortex. The excitatory and inhibitory receptive fields cover almost exactly the same areas, in contrast to the predictions of classical lateral inhibition models. Thus, although inhibition is typically as strong as excitation, it is not necessary to establish tuning, even in the receptive field surround. However, inhibition and excitation occurred in a precise and stereotyped temporal sequence: an initial barrage of excitatory input was rapidly quenched by inhibition, truncating the spiking response within a few (1-4) milliseconds. Balanced inhibition might thus serve to increase the temporal precision and thereby reduce the randomness of cortical operation, rather than to increase noise as has been proposed previously.

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"A causal study of bumetanide on a m..." refers background in this paper

  • ...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]....

    [...]


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20 Oct 2011-Neuron
TL;DR: Current views of how inhibition regulates the function of cortical neurons are discussed, and a number of important open questions are pointed to.
Abstract: Cortical processing reflects the interplay of synaptic excitation and synaptic inhibition. Rapidly accumulating evidence is highlighting the crucial role of inhibition in shaping spontaneous and sensory-evoked cortical activity and thus underscores how a better knowledge of inhibitory circuits is necessary for our understanding of cortical function. We discuss current views of how inhibition regulates the function of cortical neurons and point to a number of important open questions.

1,214 citations


"A causal study of bumetanide on a m..." refers background in this paper

  • ...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]....

    [...]


Journal ArticleDOI
09 Jun 2011-Neuron
TL;DR: A genome-wide analysis of rare copy-number variation in 1124 autism spectrum disorder families, each comprised of a single proband, unaffected parents, and, in most kindreds, an unaffected sibling, finds significant association of ASD with de novo duplications of 7q11.23, where the reciprocal deletion causes Williams-Beuren syndrome.
Abstract: We have undertaken a genome-wide analysis of rare copy-number variation (CNV) in 1124 autism spectrum disorder (ASD) families, each comprised of a single proband, unaffected parents, and, in most kindreds, an unaffected sibling. We find significant association of ASD with de novo duplications of 7q11.23, where the reciprocal deletion causes Williams-Beuren syndrome, characterized by a highly social personality. We identify rare recurrent de novo CNVs at five additional regions, including 16p13.2 (encompassing genes USP7 and C16orf72) and Cadherin 13, and implement a rigorous approach to evaluating the statistical significance of these observations. Overall, large de novo CNVs, particularly those encompassing multiple genes, confer substantial risks (OR = 5.6; CI = 2.6-12.0, p = 2.4 × 10(-7)). We estimate there are 130-234 ASD-related CNV regions in the human genome and present compelling evidence, based on cumulative data, for association of rare de novo events at 7q11.23, 15q11.2-13.1, 16p11.2, and Neurexin 1.

1,114 citations


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"A causal study of bumetanide on a m..." refers background in this paper

  • ...Looking forward, a major impediment to drug development in psychiatric research is the lack of robust, objective markers of the underlying targeted neural processes [54, 55]....

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