Neuronal activity in vitro and the in vivo reality
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
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!”)
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
The astrocyte–neuron ketone body shuttle
http://brainfuels.com/category/theories/astrocyte–neuron-ketone-shuttle/
2.
The astrocyte–neuron lactate shuttle
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
Ben-Ari, Y., et al. (2007) GABA: a pioneer transmitter that excites
immature
neurons and generates primitive oscillations. Physiol Rev 87, 1215-
neurons and generates primitive oscillations. Physiol Rev 87, 1215-
1284
4.
Rheims, S., et al. (2009) GABA action in immature neocortical neurons
Rheims, S., et al. (2009) GABA action in immature neocortical neurons
directly
depends on the availability of ketone bodies. J Neurochem 110, 1330-
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.
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
Wong, T., et al. (2005) Postnatal development of intrinsic GABAergic
rhythms
in mouse hippocampus. Neuroscience 134, 107-120
in mouse hippocampus. Neuroscience 134, 107-120
7.
Derchansky, M., et al. (2008) Transition to seizures in the isolated
Derchansky, M., et al. (2008) Transition to seizures in the isolated
immature
mouse hippocampus: a switch from dominant phasic inhibition to
mouse hippocampus: a switch from dominant phasic inhibition to
dominant
phasic excitation. J Physiol 586, 477-494
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
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
The History of Artificial Cerebrospinal Fluid (ACSF)
For the ACSF updates in 2009 to 2011 -> see Sweet & sour recipes for the brain
ACSF from 1934 to 1950.
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Events
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Comments
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References
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1934
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Alexis F. Hartmann developed several solutions for replacement of lost physiological fluids in clinics | The solutions didn’t include glucose but one of them contained 27 mM Na-lactate. This solution is still in clinical use. | Hartmann, A. F. (1934) Theory and practice of parenteral fluid administration. JAMA, J. Am. Med. Assoc., 103, 1349±1354.
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1949
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A modification of one of Hartman’s solutions was developed, mimicking the cerebro-spinal fluid | This solution mimicked natural CSF: 21.7 mM HCO3 was close to 21.1 mM observed in CSF although it contained 4.5 mM glucose, almost 1.5 times higher than in CSF | Elliott, K. A. and Jasper, H. H. (1949) Physiological salt solutions for brain surgery; studies of local pH and pial vessel reactions to buffered and unbuffered isotonic solutions. J Neurosurg, 6, 140-152. |
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1950- current
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The term “artificial cerebrospinal fluid” (ACSF) introduced | ACSF is used in clinics and in experiments on brain slices | Elliott, K. A.and Lewis, R. C. (1950) Clinical uses of an artificial cerebrospinal fluid. J Neurosurg, 7, 256-260. |
Amazingly, but in 1950, the history of ACSF stops and in the neuroscience labs, researchers working with brain slices still use the same ACSF (although since that time, the knowledge about neuronal biochemical needs significantly progressed).
“The natural cerebrospinal fluid (CSF)… attempts to simulate not the milieu surrounding the brain’s cells but rather the more easily accessible (and hence analyzable) fluid in the ventricular system. Recipes for artificial CSF (ACSF) vary, often quite widely, between labs. Moreover, commercially available ‘ACSF’… has a composition that is known to be different from that of the CSF” (E.C. McNay, R.S. Sherwin / Journal of Neuroscience Methods 132 (2004) 35–43).
Amazingly, but in 1950, the history of ACSF stops and in the neuroscience labs, researchers working with brain slices still use the same ACSF (although since that time, the knowledge about neuronal biochemical needs significantly progressed).
“The natural cerebrospinal fluid (CSF)… attempts to simulate not the milieu surrounding the brain’s cells but rather the more easily accessible (and hence analyzable) fluid in the ventricular system. Recipes for artificial CSF (ACSF) vary, often quite widely, between labs. Moreover, commercially available ‘ACSF’… has a composition that is known to be different from that of the CSF” (E.C. McNay, R.S. Sherwin / Journal of Neuroscience Methods 132 (2004) 35–43).
Related reading: Barriers and fluids that connect and divide blood, brain, and neurons
On the theory of excitatory GABA
As described in the post “On the mechanisms of brain protection by ketones“, GABA, the principal brain chemical, normally acts to prevent hyperactivity in the neuronal networks. However, in the immature neurons, it acts to promote hyperactivity, at least this is how a twenty-year theory goes. This phenomenon is used to explain many properties of the developing brain (1). The “excitatory GABA” or “depolarizing GABA” (which are not the same, but it can become rather technical to explain) results were obtained in the in vitro experiments when a very thin slice of the brain survives in an artificial solution (ACSF) — the same solution for for both mature and immature brains. (more…)
On the mechanisms of brain protection by ketones
Neuronal activity in immature neocortical neurons depends on the availability of ketone bodies in ACSF
The provoking findings of Rheims et al. suggest that an important caveat of previous electrophysiological experiments is that they were carried out with artificial cerebrospinal fluid (ACSF) added with energy sources that can only be metabolized through glycolytic pathways (e.g. glucose).
Researchers studied how naturally occurring ketones influenced activity of brain cells during development. They showed that a shortage of ketones caused pathological changes in brain cells resulting in abnormal behavior of GABA, the principal brain chemical helping to resist hyperactivity. It was repeatedly reported earlier that, normally working as a “break pedal”, GABA did not do the job in the immature brain and acted as a “gas pedal” instead. To imagine the devastating consequences, picture a car having two gas pedals and no brakes.
To make things worse, the energy deficit during hyperactivity is usually combined with increased energy demands thus starting a vicious circle — demands/deficit/demands — a well known feature of many neurodegenerative diseases including Alzheimer’s, Parkinson’s, epilepsy, encephalopathies, dementia, or multiple sclerosis. For many of them, the ketogenic diet was shown to be of a significant help. In the new article, the French and UK researchers offered an explanation. When there was enough of ketone bodies, GABA displayed its natural “break” properties and parameters of brain cells were also normal — as it happens in real life, in real animals and babies.
Researchers suggest that sufficient supply of appropriate brain fuels can break the vicious circle and prevent brain’s hyper-excitation. They now look into other natural energy substrates possibly having greater potential as a “diet in a bottle” than the costly ketones while being as efficient as the overly-stringent ketogenic diet.
Source: J Neurochem. 2009 Aug;110(4):1330-8. Epub 2009 Jun 22. 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.
To make things worse, the energy deficit during hyperactivity is usually combined with increased energy demands thus starting a vicious circle — demands/deficit/demands — a well known feature of many neurodegenerative diseases including Alzheimer’s, Parkinson’s, epilepsy, encephalopathies, dementia, or multiple sclerosis. For many of them, the ketogenic diet was shown to be of a significant help. In the new article, the French and UK researchers offered an explanation. When there was enough of ketone bodies, GABA displayed its natural “break” properties and parameters of brain cells were also normal — as it happens in real life, in real animals and babies.
Researchers suggest that sufficient supply of appropriate brain fuels can break the vicious circle and prevent brain’s hyper-excitation. They now look into other natural energy substrates possibly having greater potential as a “diet in a bottle” than the costly ketones while being as efficient as the overly-stringent ketogenic diet.Source: J Neurochem. 2009 Aug;110(4):1330-8. Epub 2009 Jun 22.
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.
http://starturl.com/GAGA-ketones
