Excitatory GABA: “Maybe It’s Not So Exciting After All!”

Energy Substrates,Excitatory GABA — Tags: , , — 4:34 am

The Epilepsy Currents journal, Volume 10, Issue 5, pages 128–130, September/October 2010, published an article titled “Another Look at Early GABAergic Neurotransmission: Maybe It’s Not So Exciting After All!” by Jong M. Rho MD.

Pros and Cons of reconsidering the excitatory GABA dogma according to Dr Rho, in direct quotes *

PROs:

  • “Collectively, these studies cast doubt on the biological relevance of GABA-induced depolarization as evidenced by a multitude of cellular electrophysiological studies.” [1, 2]
  • “Certainly, these authors make a compelling case for a thoughtful re-examination of the time-honored use of ACSF formulations that solely employ glucose as an energy substrate.” **
  • “If the observations of Rheims et al. and Holmgren and colleagues are ultimately validated, then a couple of generations of in vitro studies are likely to be at risk for relegation to the murky domain of artifact.”
CONs

  • “Intriguing as their findings are, the authors have not yet firmly established a mechanism for their general observation ofmetabolic substrate-induced reversal of GABA excitation, despite preliminary evidence invoking the bicarbonate–chloride exchanger.”
  • “Although Rheims et al. and Holmgren and colleagues indicate that their results may be similar to the mechanism of ketogenic action, this link remains speculative at best.”
  • “Whether GABA-evoked depolarization is merely a developmental aberration that is compensated for by differential and age-dependent utilization of energy substrates or whether it is still a fundamental physiological phenomenon important for neuronal maturation, and possibly seizure genesis, remains unclear.” ***

————–

*  all six points are direct quotations of Dr Rho selected to demonstrate his opinion of the papers (1, 2)

** see recent review: Neuronal activity in vitro and the in vivo reality: the role of energy homeostasis

*** There’s a growing body of evidence in favor  the first part of the statement and against its second part showing that the excitatory GABA phenomenon exists only in certain conditions: either brain slices supported by standard ACSF or, if in vivo, during blockade of ketogenesis (3). In healthy immature animals in vivo (3, 4) , as well as in hippocampal preparations in toto (5-7), GABA is shown to always be inhibitory.

References

1. GABA Action in Immature Neocortical Neurons Directly Depends on the Availability of Ketone Bodies. Rheims S, Holmgren CD, Chazal G,Mulder J, Harkany T, Zilberter T, Zilberter Y. J Neurochem 2009;110(4):1330–1338

2. Energy Substrate Availability as a Determinant of Neuronal Resting Potential, GABA Signaling and Spontaneous Network Activity in the Neonatal Cortex In Vitro. Holmgren CD,MukhtarovM,Malkov AE, Popova IY, Bregestovski P, Zilberter Y. J Neurochem 2010;112(4):900–912

3. Rheims S., PhD thesis, Universite de la Mediterranee, 2008

4.  Bremner L, Fitzgerald M & Baccei M. (2006). Functional GABAA-Receptor-Mediated Inhibition in the Neonatal Dorsal Horn. J Neurophysiol 95, 3893-3897

5. Wong, T., et al. (2005) Postnatal development of intrinsic GABAergic rhythms in mouse hippocampus. Neuroscience 134, 107-120

6. Derchansky, M., et al. (2008) Transition to seizures in the isolated immature mouse hippocampus: a switch from dominant phasic inhibition to dominant phasic excitation. J Physiol 586, 477-494

7. Dzhala V et. al., Progressive NKCC1-Dependent Neuronal Chloride Accumulation during Neonatal Seizures The Journal of Neuroscience, (2010) 30(35):11745–11761 • 11745

How does GABA behave in the intact brain?

Excitatory GABA,Methodology,Neuroscience FAQ, Q&A — Tags: , — 9:26 am

Original Q&A :: About these Q&A :: Other Q&A

Q: Dr. Zilberter,

In your post at brainfuels.com, you cited several researchers and the closing phrase was: “The work undermined the role of depolarizing GABA”, commented Dr. Jean-Marc Goaillard from the Mediterranean University in Marseille”.

First, does depolarizing equals inhibitory? Second, I just wondered:  why nobody directly measured GABA properties in the whole brain, in natural conditions? Wouldn’t it be the final proof of how GABA behaves in its natural environment?

Thank you,

Theo

A: Dear Theo,

I believe you mean the post On the theory of excitatory GABA, correct me if I am wrong.

Depolarization of neuronal membrane is a change making it more electrically positive, or less negative. In neurons it may result in an action potential if the depolarization is larger than certain threshold. If this event repeats on a regular basis, the neuron or a network of connected neurons becomes more active (more frequently generating action potentials). In the case of GABA, the primary inhibitory neurotransmitter, depolarization means that it is in a less inhibitory state often failing to prevent the excitatory transmitter(s) from hyperactivity, which in clinical sense can mean seizures.

Hyper-polarization, on the other hand, inhibits the occurrence of an action potential, in his respect, when GABA is inhibitory, the odds of hyperactivity decrease. Now, regarding your second (excellent!) question, I can give you a reference to a PhD thesis, where the student reported this exact result (1). He investigated GABA properties in  whole mice (in vivo) and demonstrated that GABA was always inhibitory – unlike experimental results obtained on brain slices where GABA was depolarizing in young animals. Since in this project, researchers (the student, S. Rheims and his supervisor, Dr. Y. Zilberter) were interested in ketone bodies as the usual suspect when it comes to the anti-epileptic effects of the ketogenic diet, they chemically blocked the ketogenesis preventing ketone bodies from being produced to work as brain fuel.


In this condition, GABA behaved pretty much as it does in brain slices. This and the in toto (see footnote) experiment results, tell us that the “excitatory GABA” is, perhaps: 1) result of experimental limitation of the brain slice preparation; 2) GABA action depend on metabolic status of not only brain slice but also of a whole intact animal in a bad metabolic condition.

Footnote. Since this Q&A, I posted results on in toto experiments (2, 3, 4), where GABA was also inhibitory in the preparation of a whole hippocampus of immature animals : Studies of GABA action: in vivo, in toto, and in vitro and in vivo experiments showing inhibitory GABA-action (5).

Sources:

1. S. Rheims; PhD Thesis,2008. Faculté des Sciences de Luminy Ecole Doctorale de la Vie et de la Santé. Initiation et modulation des oscillations physiologiques et pathologiques dans le neocortex immature: role de la transmission GABAergique.

2. Wong, T., et al. (2005) Postnatal development of intrinsic
GABAergic rhythms in mouse hippocampus. Neuroscience 134, 107-120

3. Derchansky, M., et al. (2008) Transition to seizures in the
isolated immature mouse hippocampus: a switch from dominant phasic
inhibition to dominant phasic excitation. J Physiol 586, 477-494

4. Dzhala V et. al., Progressive NKCC1-Dependent Neuronal Chloride Accumulation during Neonatal Seizures The Journal of Neuroscience, (2010) 30(35):11745–11761 • 11745

5. Bremner L, Fitzgerald M & Baccei M. (2006). Functional GABAA-Receptor-Mediated Inhibition in the Neonatal Dorsal Horn. J Neurophysiol 95, 3893-3897.

Comparison of lactate kinetics in vitro and in vivo is to be done

Astrocyte–neuron lactate shuttle,Lactate,Methodology — Tags: , , — 6:23 am

Glucose is an energy source for both neurons and glia in the adult brain but lactate, one of the monocarboxylic acids, being converted from pyruvate in astrocytes and supplied to neuron (“astrocyte-neurona lactate shuttle“) is an important energy fuel alternative to glucose as well. The role of exogenously added lactate as a viable energy substrate has remained controversial although recent data showed that exogenous lactate might be selectively taken up by neuron in intact rat brain (1) and that lactate works as an energy substrate supporting neuronal activity in presence or absence of glucose in vitro (2, 3) and in vivo (4, 5).

Notwithstanding experimental data supporting the existence of astrocyte-neurona lactate shuttle, the opponents (for contrasting views see 6, 7) basing on mathematical modeling, challenge the very phenomenon, strongly insisting on the following experiments that should be done before jumping to conclusions:

“In this perspective, we suggest that highly relevant experiments to help the experimental-theoretical interaction could be: (i) comparison of lactate kinetics in vitro, in vivo using biosensors, and in vivo using 1H MRS, in the same brain areas, and using various stimulation protocols; (ii) comparison, in vitro and in vivo, of NADH kinetics in astrocytes and neurons, and extracellular lactate and pH kinetics; (iii) this approach would be reinforced if the kinetics of oxygen concentration and intracellular pH and sodium were also measured, using for instance microelectrodes and fluorescent dyes.” (8)

  1. Yamada A, et. al., Lactate is an alternative energy fuel to glucose in neurons under anesthesia. NeuroReport 20:1538–1542, 2009
  2. Ivanov A, Mukhtarov M, Bregestovski P and Zilberter Y (2011). Lactate effectively covers energy demands during neuronal network activity in neonatal hippocampal slices. Front. Neuroenerg. 3:2.
  3. Kasischke K (2011). “Lactate fuels the neonatal brain”. Front. Neuroenerg. 3:4.
  4. Wyss M, Jolivet R, Buck A, Magistretti P, and Weber B. (2011). In Vivo Evidence for Lactate as a Neuronal Energy Source. J Neuroscience, 31(20):7477-7485
  5. Suzuki A, Stern SA, Bozdagi O, Huntley GW, Walker RH, Magistretti PJ, Alberini CM. Astrocyte-neuron lactate transport is required for long-term memory formation. Cell. 2011 Mar 4; 144(5):810-23 Evaluation access here
  6. Korf J. 2006. Is brain lactate metabolized immediately after neuronal activity through the oxidative pathway? J Cereb Blood Flow Metab 26:1584–1586.
  7. Schurr A. 2006. Lactate: The ultimate cerebral oxidative energy substrate? J Cereb Blood Flow Metab 26:142–152.
  8. Aubert AS, Costalat R, Compartmentalization of Brain Energy Metabolism Between Glia and Neurons: Insights from Mathematical Modeling. GLIA 55:1272–1279, 2007

Neuronal activity in vitro and the in vivo reality

Energy Homeostasis,Energy Substrates,Excitatory GABA,Methodology — Tags: , , , , , , , , , — 1:15 pm
In the brain, neuronal electrical activity and intricate metabolic energy provisions are closely related. Although both functions have been painstakingly researched by electrophysiologists and biochemists, insufficient interaction between the two domains leads to difficulty in extrapolating the properties observed in the in vitro studies to the properties of the whole in vivo brain. In this paper, we hope to clarify the relationships between neuronal energy status and neuronal electrical function.

“A man with his head is something much more then a man’s body plus his separate head” – J. Miller (1965)

Whole is equal to more than the sum of its parts (on some interdisciplinary methodological problems)

In the history of life sciences, perhaps beginning with Aristotle’s time, reductionism prevailed leaving the opposite philosophical approach, holism, outside scientific paradigm. Reductionism and reductionists are concerned with at least two dominant themes: a) the interactions between different domains of knowledge; b) the place of a part in the whole (1). (more…)

Studies of GABA action: in vivo, in toto, and in vitro

Astrocyte–neuron ketone shuttle,Astrocyte–neuron lactate shuttle,Energy Homeostasis,Energy Substrates,Excitatory GABA,Methodology — Tags: , , , , , , , — 6:36 am

On discrepancies of data from experiments on brain slices, in toto, and in vivo

Let's start with the fundamental differences between environments depending on the types of experiments.

(See Commentary: Excitatory GABA: “Maybe It’s Not So Exciting After All!”)

neuroscience, brain energy homeostasis, set point, control system, in vivo, in vitro

Brain energy homeostasis versus energy supply in the brain slice preparation.

A. Energy homeostasis of the brain serves to meet brain’s energy needs in spite of environmental and metabolic disturbances. It continuously compares the correlates of energy requirements (SET POINT) with the actual energy supply (OUTPUT) through the negative feedback loop sending information about OUTPUT to the brain metabolic sensors. When the difference between SET POINT and OUTPUT exceeds certain value, a correction signal is sent to the control centers responsible for the initiation of the executive systems. The goal of executive system is fixing the consequences of disturbances. As the result, within the homeostatic margins, the required and actual energy supplies are maintained close to each other.

B. Brain slice is lacking all mandatory elements of a homeostatic system receiving instead a constant and arbitrary energy supply, which is independent of both qualitative and quantitative needs of cells.

C. When brain structures are preserved as in the in toto preparation  (not shown on the figure), there’s the possibility that energy substrates can come from the glial depots (1, 2)

As an example, let’s consider these four situations with GABA action:

1. In vitro (brain slice) using standard ACSF (footnote a)

GABA is consistently reported to be excitatory in the neonatal brain slices (e.g., 3 for review)

2. In vitro (neonatal’s brain slice) using energy substrate-fortified ACSF

GABA is inhibitory  (4, 5)

3. In toto, hippocampus (footnote b) preparation

In the intact neonatal hippocampus preparation, standard ACSF, intensive oxygenation (!), GABA is inhibitory (6, 7, 8 )

4. In vivo (neonates)

GABA is inhibitory in the normal neonates (9, 10,11) but it is much less inhibitory during chemical blockade of ketone’s production (blockade of ketogenesis) (9).

Sources

Sources:
1.
The astrocyte–neuron ketone body shuttle
http://brainfuels.com/category/theories/astrocyte–neuron-ketone-shuttle/
2.
The astrocyte–neuron lactate shuttle
http://brainfuels.com/category/theories/astrocyte–neuron-lactate-shuttle/
3.
Ben-Ari, Y., et al. (2007) GABA: a pioneer transmitter that excites
immature
neurons and generates primitive oscillations. Physiol Rev 87, 1215-
1284
4.
Rheims, S., et al. (2009) GABA action in immature neocortical neurons
directly
depends on the availability of ketone bodies. J Neurochem 110, 1330-
1338
5.
Holmgren CD, et. al., (2010) Energy substrate availability as a
determinant of neuronal resting potential, GABA signaling and
spontaneous network activity in the neonatal cortex in vitro. J
Neurochem. Feb;112(4):900-12. Epub 2009 Nov 24.
6.
Wong, T., et al. (2005) Postnatal development of intrinsic GABAergic
rhythms
in mouse hippocampus. Neuroscience 134, 107-120
7.
Derchansky, M., et al. (2008) Transition to seizures in the isolated
immature
mouse hippocampus: a switch from dominant phasic inhibition to
dominant
phasic excitation. J Physiol 586, 477-494
8.
S. RHEIMS. THESE DE DOCTORAT. Pour obtenir le grade de DOCTEUR DE
L’UNIVERSITE AIX MARSEILLE II. Spécialité : neurosciences. Le 31
octobre 2008

1. The astrocyte–neuron ketone body shuttle

2. The astrocyte–neuron lactate shuttle

3. Ben-Ari, Y., et al. (2007) GABA: a pioneer transmitter that
excites immature neurons and generates primitive oscillations. Physiol
Rev 87, 1215-1284

4. Rheims, S., et al. (2009) GABA action in immature neocortical
neurons directly depends on the availability of ketone bodies. J
Neurochem 110, 1330-1338

5. Holmgren CD, et. al., (2010) Energy substrate availability as a
determinant of neuronal resting potential, GABA signaling and
spontaneous network activity in the neonatal cortex in vitro. J
Neurochem. Feb;112(4):900-12. Epub 2009 Nov 24.

6. Wong, T., et al. (2005) Postnatal development of intrinsic
GABAergic rhythms in mouse hippocampus. Neuroscience 134, 107-120

7. Derchansky, M., et al. (2008) Transition to seizures in the
isolated immature mouse hippocampus: a switch from dominant phasic
inhibition to dominant phasic excitation. J Physiol 586, 477-494

8. Dzhala V et. al., Progressive NKCC1-Dependent Neuronal Chloride Accumulation during Neonatal Seizures The Journal of Neuroscience, (2010) 30(35):11745–11761 • 11745

9. Rheims S., PhD thesis, Universite de la Mediterranee, 2008

10. Bremner L, Fitzgerald M & Baccei M. (2006). Functional GABAA-Receptor-Mediated Inhibition in the Neonatal Dorsal Horn. J Neurophysiol 95, 3893-389

Footnotes

a) ACSF – artificial cerebrospinal fluid (CSF). This solution closely matches the electrolyte concentrations of CSF – A Harvard Bioscience Company

b) Hippocampus – a complex neural structure shaped like a sea horse, has a central role in the formation of memories


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