PSYC Call for Commentators: BIOCHEMICAL BASIS OF COMA (969 lines)

Stevan Harnad harnad at cogito.ecs.soton.ac.uk
Sat Sep 18 08:40:06 EST 1999


            Smythies: BIOCHEMICAL BASIS OF COMA

    The target article below has just appeared in PSYCOLOQUY, a
    refereed journal of Open Peer Commentary sponsored by the American
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    neural or cognitive scientists are hereby invited to submit Open
    Peer Commentary on it. Please email or see websites for
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    AUTHORS' RATIONALE FOR SOLICITING COMMENTARY: This target article
    reviews the biochemical basis of various types of coma, including
    general anesthesia. This is a subject of considerable current interest
    in its own right in neuroscience and anesthesiology and may throw some
    light on the biochemical mechanisms of consciousness. It also concerns
    the interrelations among several biochemical and microanatomical
    systems whose investigators normally do not have much contact. This
    interdisciplinary communication could lead to interesting new
    developments in these diverse fields (e.g., concerning the role of
    redox reactions at the glutamate synapse, the role of endocytosis in
    postsynaptic functions, the role of dismuting catecholamine-iron
    complexes in the brain, etc.). Commentary would accordingly be welcome
    from specialists in the following fields: the neurochemistry of the
    glutamate receptor and of synaptic plasticity, reactive oxygen species
    and antioxidants, trafficking mechanisms for iron and possibly
    catecholamines at postsynaptic sites in the brain, the role of
    catecholamines as antioxidants (in particular in the case of their
    complexes with iron), oxidative pathways of catecholamine metabolism in
    the brain, the function of endocytosis in neurons, and the
    neuropharmacology of general anesthesia.

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psycoloquy.99.10.026.coma-biochemistry.1.smythies       Fri Sep 17 1999
ISSN 1055-0143        (30 paragraphs, 73 references, 1 note, 924 lines)
PSYCOLOQUY is sponsored by the American Psychological Association (APA)
                Copyright 1999 John Smythies

                THE BIOCHEMICAL BASIS OF COMA
                Target Article on Coma-Biochemistry

                John Smythies
                Division of Neurochemistry
                Brain and Perception Laboratory
                Center for Brain and Cognition
                University of California, San Diego
                La Jolla CA 92093-0109 
                and
                Department of Neuropsychiatry
                Institute of Neurology
                Queen Square, London
                smythies at psy.ucsd.edu

    ABSTRACT: Current research on the neural basis of consciousness is
    based mainly on neuroimaging, physiology and psychophysics. This
    target article reviews what is known about biochemical factors that
    may contribute to the development of consciousness, based on loss
    of consciousness (i.e., coma). There are two theories of the
    biochemical mode of action of general anaesthetics. One is that
    anaesthesia is a direct (i.e., not receptor-mediated) effect of the
    anaesthetic on cellular neurophysiological function; the other is
    that some alteration of receptor function occurs. General
    anaesthetics are mainly GABA agonists but some (such as ketamine)
    are glutamate antagonists. They also affect other systems,
    particularly cholinergic ones. There are various comas of metabolic
    origin. For example, a combination of small doses of the iron
    chelators desferrioxamine and prochlorperazine induce a profound
    and long lasting coma in humans. The mechanisms that might mediate
    this include redox mechanisms at the glutamate synapse,
    post-synaptic endocytosis of dopamine and iron, and intracellular
    iron-dopamine complexes, which are powerful dismuters of the
    superoxide anion. New findings in cell biology relating to
    endocytosis and recycling of receptors are discussed in a wider
    context. These biochemical events may induce coma by two
    mechanisms: (i) Consciousness may depend on widespread cortical (or
    cortico-thalamic) activation.  (ii) Whereas these biochemical
    changes are widespread, only the changes in a subset of
    'consciousness' neurons may count. An experimental program to
    distinguish between these two alternatives is proposed.

    KEYWORDS: anaesthetics, coma, consciousness, desferrioxamine
    dopamine, GABA, general glutamate iron, redox mechanisms, synapses

I. INTRODUCTION.

1. Francis Crick (1994) has suggested that one way of discovering the
neural basis of consciousness is to identify (i) those neurons that are
active during conscious states and inactive during states of loss of
consciousness, and (ii) those neurons whose activity is unaffected by
the state of consciousness, and then see if we can determine
differences between the two populations. Given our increasing knowledge
of the plasticity of neurons, it may also be possible that within a
single population of neurons, one type of activity may be associated
with conscious states and another with unconscious ones. Most current
work stimulated by this strategy concentrates upon brain imaging that
identifies the macroanatomical areas involved, on physiological studies
that look at such factors as synchronization of 40 Hz rhythms, or on
psychophysical investigations using such phenomena as retinal rivalry.

2. It cannot merely be assumed, however, that only the electrical
activity of neurons is necessary and sufficient for the generation of
the conscious state. It is theoretically possible that some of the
relevant differences that Crick is looking for between 'consciousness'
and 'non-consciousness' neurons may be biochemical. If so, it might be
useful to consider what these biochemical factors might possibly be.
The purpose of this review is to see whether any useful leads can be
obtained by examining the biochemical changes which produce coma. This
in turn may suggest mechanisms relevant to the conscious state, always
making the Jacksonian caveat that, if a certain biochemical change A
leads to loss of function X, one cannot assume that the normal function
of A is to produce X. Nevertheless, a study of the biochemical
mechanisms underlying coma, if cautiously interpreted, may throw some
light on the sort of mechanisms to investigate during the conscious
state.

II. THE BIOCHEMISTRY OF GENERAL ANAESTHESIA.

3. There are two schools of thought with regard to the way anaesthetics
work. The earlier theory is that these agents, which are lipophilic
molecules, act by infiltrating the lipid plasma membrane and disrupting
its function mechanically (Janoff et al, 1981). A more modern version
of this classical 'general' theory (i.e., non-receptor mediated) has
been presented by Reis and Puil (1999a, b). Using thalamic brain slices
from rats, they showed that isoflurane hyperpolarizes thalamic neurons
by increasing K+ conductance (leak). This inhibits neuronal firing by
short circuiting the effectiveness of depolarizing pulses and shunting
voltage dependent Na+ and Ca2+ channels. This effect still occurs in
the presence of GABA receptor antagonists. The authors note that the K+
mechanism is ubiquitous in cells and that anaesthetics affect a number
of other systems, but they also claim that the effect they report on
the cortico-thalamic-cortical system could by itself account for the
general anaesthesia. Anoxia (which also induces coma) hyperpolarizes
CNS neurons by increasing K+ conductance, and in deep slow-wave sleep
thalamo-cortical neurons hyperpolarize and show spike-bust rhythms, but
the basic mechanism is different.

4. In anaesthesia there is an increase in Na+ -spike thresholds and in
decrease in low threshold calcium spikes not seen in deep sleep.
Furthermore, in deep sleep thalamo-cortical neurons hyperpolarize
because of cholinergic disfacilitation leading to oscillations. In
anaesthesia the hyperpolarization is due to the increase in K+
conductance and there is no concomitant shift in either tonic or burst
firing. Reis and Puil (1999) conclude that their results fit in with
the 'classical' (non-receptor mediated) theory of the action of
anaesthetics. Further support for the 'specific neural network' (i.e.,
local) theory has recently been supplied by Fiset et al (1999). They
carried out PET studies on normal volunteers given propofol at three
doses. They report that anaesthesia is associated with a generalized
decrease in cortical blood flow, but there is a preferential decrease
in blood flow (bilateral) in the medial thalamus, cuneus and precuneus,
and in the posterior cingulate and orbitofrontal cortices as well as in
the right angular gyrus. These are all brain regions previously
associated with arousal, associative functions and autonomic control.
These authors conclude that their data supports the specific network
theory of the action of general anaesthetics rather than the
non-specific generalized theory.

5. The alternative theory is that general anaesthesia is mediated by
local receptors. Most general anaesthetics (such as barbiturates and
halothane) inhibit GABAergic transmission by enhancing the receptor
binding of GABA and increasing the receptivity of the GABA receptor
channel (Franks and Lieb, 1994). They probably bind directly to
lipophilic pockets in the receptor protein (Eckenhoff and Johansson,
1997). There is evidence to suggest that they do not affect presynaptic
transmitter release (Liachenko et al, 1998). However, the general
anaesthetic ketamine, like nitrous oxide ('laughing gas'), is a
glutamate NMDA receptor blocker, and acts differently (Franks and
Lieb,1998). It modulates the action of GABA on the GABA receptor, but
has no action by itself on this receptor (Dzoljic & van Duijn, 1998).

6. It would be naive to suggest that anaesthetics act by only one
mechanism. The biochemistry of synaptic systems is enormously
complicated and anaesthesia could affect a number of these.

    (i) Numerous studies have been made of the effect of general
    anaesthetics on the cholinergic systems. General anaesthetics have
    been shown to increase rates of desensitization at the nicotinic
    ACh receptor (Raines et al, 1995; Scheller et al, 1997; Liu et al,
    1995), and to act as antagonists (Andoh et al, 1997; Franks and
    Lieb, 1997). Ether increases the burst frequency at this receptor
    by promoting agonist binding to the receptor protein (Liu et al,
    1994).  These anaesthetics also inhibit muscarinic receptors
    (Minami et al, 1997). Keifer et al (1996) have suggested that a
    cholinergic mechanism generates the key midbrain pedunculopontine
    and lateral dorsal tegmental nuclei in EEG sleep spindles.
    Anaesthetic agents also block choline uptake into synaptosomes,
    which would inhibit cholinergic functions (Griffiths et al, 1994).
    Experiments using brain microdialysis volatile anaesthetics have
    shown that these anaesthetics suppress ACh release, while nitrous
    oxide has the opposite effect (Shichino et al, 1998). Some cases of
    hepatic coma have a cholinergic element, which, as with the coma
    produced by atropine, can be relieved by intravenous physostigmine
    (Kabatnik et al, 1999).

    (ii) Both halothane and ketamine inhibit Ca2+-activated K channels
    and their phospholipase A2-arachidonic acid signal transduction
    pathways (Denson et al, 1996). Both ketamine and halothane also
    inhibit K currents through Kv2-1 channels, but do so by different
    mechanisms (Kulkarni et al, 1996).

    (iii) Volatile anaesthetics selectively inhibit plasma membrane
    Ca2+-transport APTase by binding to lipophilic sites and changing
    conformation (Lopez & Kosk-Kosicka 1995).

    (iv) Halothane inhibits Na+-mediated glutamate release (Ratnakumari
    & Hemmings, 1998). Liachenko et al (1998) report that the
    anaesthetic cyclobutane derivative F3 inhibits K+-evoked glutamate
    and GABA release, whereas the non-anaesthetic cyclobutane
    derivative F6 suppresses evoked glutamate release but has no effect
    on evoked GABA release. They conclude that the suppression of
    excitatory neurotransmitter release might not be the mode of action
    of general anaesthetics. The suppression of GABA release is
    somewhat paradoxical.

    (v) In the molecular layer of the cerebellum NMDAr stimulation
    activates the nitric oxide-guanyl cyclase signaling pathway. This
    effect is inhibited by halothane and isoflurane (Zou et al, 1996).

    (vi) Halothane increases the probability of opening of the
    glycine-activated channel in rat central neurons (Wakamori et al,
    1998).

    (vii) Isofluorane inhibits histamine metabolism in the hypothalamus
    (Hashimoto et al, 1998), and affects polyamine amine metabolism in
    the brain (Mills et al, 1997).

    (viii) Isoflurane disrupts synchronized neural oscillations at a
    frequency of <10 Hz (rather than ~40 Hz) (Tennigkeit et al, 1997).

    (ix) Low doses of anaesthetics induce hypethesis, i.e., disruption
    of memory formation without loss of awareness. On this basis,
    Andrade (1996) suggests that frontal lobe function is particularly
    sensitive to anaesthetics.

    (x) With regard to systems, Flohr (1995) suggests that
    consciousness involves the NMDAr mediated activation of large
    neuronal assemblies, and that anaesthetics and brain stem lesions
    have a common denominator for inducing coma, namely inhibition of
    the formation of these assemblies.

7. In conclusion, it appears that one important role for volatile
anaesthetics is to potentiate GABA-mediated inhibition at the receptor
level and to inhibit NMDAr-mediated conduction at the level of the
post-synaptic signaling cascade (Zou et al, 1996). Ketamine-like agents
inhibit glutamate-mediated conduction at the receptor level, while
nitrous oxide affects both systems. There is also evidence that other
systems, particularly the cholinergic, may be important.

III. SOME METABOLIC COMAS.

8. Some comas of metabolic origin also involve the glutamate and/or
GABA systems. For example, hepatic coma is associated with raised blood
levels of GABA and ammonia, which inhibits metabotropic glutamate
receptors (Albrecht, 1998). Abnormal sugar metabolism in comas suggests
that other systems are involved. Diabetic coma has been linked to a
reduction in brain cell volume caused by changes in brain osmolarity
(Fink et al, 1994). However, in this instance there was also an
increase of GABA release in the cortex. Hypoglycaemic coma may be
related to increased tyrosine phosphorylation of mitogen-activated
protein kinase (Kurihara and Wielock, 1994). Concussion has been linked
to the excessive release of glutamate (Bullock et al, 1998), of oxygen
free radicals, in particular superoxide anions (Mori et al, 1998;
Yunoki et al, 1998), and to changes in brain osmolarity (Katayama et
al, 1998).

IV. IRON-RELATED COMA.

9. In 1985, Blake et al reported that a combination of the standard
iron-chelator desferrioxamine (100 mg) plus prochlorperazine (25 mg)
produces a profound and prolonged coma lasting 48-72 hours in humans,
whereas given singly, these drugs had no such effect. A similar result
was obtained with rats. The effect was explained as follows.
Desferrioxamine is a hydrophilic iron chelator and prochlorperazine is
a lipophilic iron chelator. This drug combination was shown to act
synergistically in transferring iron across a layer of chloroform
between two water compartments. The researchers suggested that this
combination produces a rapid flux of iron (and copper) out of neurons,
which in turn produces a disturbance of the plasma membrane, leading to
interference with serotoninergic and noradrenergic function, and coma.

10. During the period of coma the CSF levels of total non-haem iron
were significantly raised but levels of chelatable iron were
depressed.  It may be the loss of intraneuronal iron rather than the
transmembrane flux which is important, as iron deficiency in rats leads
to a greater susceptibility to this coma (Blake et al, 1985). As we
have seen, general anaesthesia and coma are usually associated with
disturbances of the glutamate/GABA systems rather than with
disturbances of serotonin and noradrenergic systems, which are more
related to mood disturbances, cognitive effects and REM sleep. There
is, however, some evidence that general anaesthetics affect
serotoninergic systems.  Halothane reduces 5-HT-induced currents at the
5HT2A receptor (Minami et al, 1997) and general anaesthetics potentiate
5-HT3 receptors (Franks & Lieb, 1997).

11. Thus the coma induced by the combination of desferrioxamine and
prochlorperazine may be due to inhibition of the glutamate synapse due
to low levels of iron in the post-synaptic neuron, although additional
actions at serotoninergic and perhaps other receptors cannot be ruled
out. In addition, the side chain of prochlorperazine somewhat resembles
a polyamine such as spermidine, and there is a polyamine modulatory
site on the NMDAr. This indicates that a possible antagonist effect of
prochlorperazine at the polyamine site on the NMDAr may be involved.

12. Since desferrioxamine is hydrophilic, it may be asked how it could
cross the cell membrane in the manner required by this hypothesis.
Ollinger and Brunk (1995) report that desferrioxamine is taken up by
endocytosis in the case of hepatocytes, and is then transported to the
acidic endosome. Furthermore, desferrioxamine inhibits the peroxidation
and lysis of lysosomal membranes by chelating intralysomal iron. The
next sections (paragraphs 9-16) address current knowledge of the
biochemical mechanisms that may support the operations of the brain
related to consciousness.

13. The general statement that glutamatergic mechanisms may be relevant
requires investigation. The details of this system include:

    (i) the redox balance at the glutamate synapse and inside neurons,

    (ii) the role of interactions between the dopamine system and the
    glutamate synapse,

    (iii) the possible role of iron-dopamine complexes,

    (iv) the possible role of endocytosis of receptors and their
    subsequent processing inside the postsynaptic neuron.

14. The first question to be addressed is how intraneuronal levels of
iron could affect glutamate synaptic function and so lead to a loss of
consciousness. I would suggest the following hypothesis: There is
considerable evidence to suggest than one important factor determining
the plasticity of the glutamate synapse (growth and diminution of
existing synapses, and the supply of new synapses and removal of old
ones) is the redox balance at the synapse, both in the synaptic cleft
and inside the dendritic spine, which I have reviewed elsewhere
(Smythies, 1997a). There is a constantly shifting balance at the
glutamate synapse between neurotoxic reactive oxygen species (ROS) and
reactive nitrogen species (RNS) on the one hand, and protective
antioxidants on the other. The ROS include the superoxide anion, the
hydroxyl radical and hydrogen peroxide. These are produced by enzymes
in the post-synaptic cascade, such as prostaglandin H synthase and
nitric oxide synthase, as well as by mitochondria. RNS include the
nitric oxide radical and peroxynitrite. ROS and RNS would tend to lead
to spine deletion if produced in excess. However, ROS do not just
function as neurotoxins. They may do this when produced in excess, or
when antioxidant defenses are inadequate, but normally ROS are
important signaling molecules in their own right. There is evidence to
suggest that they play such a role in many cellular process such as
cell growth, chemotaxis, apoptosis, transcription factor activation,
gene expression and others (Suzuki et al, 1997; Kamata and Hirata,
1998). An interesting link between iron, ROS and antioxidants has been
provided by Fuchs (1997), who showed that the level of expression of
the gene for the important antioxidant enzyme glutathione peroxidase is
modulated by intracellular chelatable iron levels, as well as by the
level of oxidative stress.

15. Protective antioxidants available at the glutamate synapse include
ascorbic acid (vitamin C), the dipeptide carnosine, and glutathione.
The effect of positive reinforcement on this system may be mediated by
the release of dopamine from its boutons-en-passage, which are attached
to the sides of many glutamate synapses (Kotter, 1994). Dopamine exerts
powerful anti-oxidant effects through three separate mechanisms:  (a)
the direct scavenging of ROS and redox cycling between dopamine and
dopamine quinone, in conjunction with an antioxidant such as ascorbate
or glutathione (Liu and Mori 1994); (b) the activation of D 2 dopamine
receptors, which induces the synthesis of an antioxidant enzyme,
probably catalase (Sawada et al, 1998) in the post-synaptic neuron; and
(c) forming dopamine-iron complexes which act as powerful scavengers of
superoxide anions by redox cycling between ferrous and ferric iron and
between dopamine and dopamine quinone (Zhao et al, 1998). This system
effectively transmutes 2 molecules of superoxide into oxygen and 3
molecules of superoxide into the much less toxic hydrogen peroxide. How
do iron and dopamine molecules come into contact, in order that this
third mechanism may occur? Since free iron is extremely reactive,
almost all the iron in the body is located inside shielding proteins,
mainly the iron storage protein ferritin and the iron transport
proteins transferrin and lactoferrin. One answer is suggested by the
following facts.

16. A paradigm change has recently occurred in cell biology relating to
the mechanism by which receptors for neurotransmitters and
neuromodulators function. It used to be thought that when a receptor
located in the membrane bound a molecule of the transmitter/modulator
it underwent a conformational change. In some cases this opened an
ionic channel, while in others it activated a second molecule (such as
a G-protein) which started a post-synaptic cascade. The receptor itself
was supposed to eject that molecule of transmitter/modulator and then
wait in the membrane for the next, when the whole process would be
repeated. Although it was recognized that the receptor molecule was
eventually replaced, possibly because of accumulated oxidative damage,
the general picture of the plasma membrane was that seen under the
microscope, i.e., a motionless structure.

17. It is now known that much of the entire membrane, proteins and
lipids alike (Kobayashi et al, 1998), is in a constant state of flux,
being continually endocytosed, processed by the endosome system and
then recycled back to the plasma membrane. This process involves
G-protein linked and other similar receptors (Koenig & Edwardson, 1997;
Mukherjee et al, 1997). When one of these binds with a molecule from
the transmitter/modulator (e.g., a neuropeptide, catecholamine or
acetylcholine), the receptor-ligand complex is rapidly endocytosed
inside a clathrin-lined pit which converts to a vesicle inside the
post-synaptic neuron. This is transported (~10 minutes) to the
tubulovesicular endosome system. Here, the vesicle membrane fuses with
the membrane of the early endosome and delivers the receptor-ligand
complex into the lumen of the endosome, where the acidic environment
leads to the dissociation of the ligand from the receptor protein. The
ligand is then transported to the late endosome, where the receptor
protein is subjected by the endosome/lysosome system to a triage
process. This includes those receptor proteins that have been
down-regulated by phosphorylation.

18. Because the phosphate groups cannot be removed in situ, the protein
is endocytosed to allow their removal, and the newly sensitized
receptor is returned to the external membrane (Ferguson & Caron,
1998).  Other proteins subject to the triage process may include those
damaged by oxidative attack. These have to be broken down into their
constituent amino acids. The oxidized amino acids are metabolized and
excreted, while the undamaged ones are recycled. The same process may
apply to membrane lipids which are also under constant oxidative
attack. Dephosphorylated and reduced receptors are recycled back to the
cell membrane. Normal proteins and lipids are recycled by the
appropriate mechanisms, and endosome membrane is also recycled back to
the surface. The whole cycle in some cases takes around 30 minutes
(e.g., for NGF receptors (Zapf-Colby and Olefsky,1998)). It seems
unlikely that the function of the endocytotic mechanism is only to
resensitize receptors, since much of the membrane itself is
endocytosed.

19. If the ligand is a polypeptide it is transmitted to the cell
nucleus, where it plays an essential role in gene expression (Jans and
Hassan, 1998). As Koenig and Edwardson (1997) note in the case of
polypeptide transmitters and receptors "the purpose of endocytosis is
to capture the ligand for consequent use by the cell". For our present
purposes it is of particular interest that dopamine G-protein related
receptors are also rapidly and robustly endocytosed following
transmitter binding (Dumartin et al, 1998). The D1 receptor is
endocytosed by clathrin-lined vesicles, which also transport the iron
transporter transferrin, whereas the non-clathrin lined vesicles that
endocytose the D2 receptor do not perform this additional function
(Vickery et al, 1998). Koenig and Edwardson (1997) state that low
affinity agonists (like muscarine or dopamine) are unlikely to be
internalized in sufficient quantity to "cause significant receptor
activation in endosomes". However, the relationship between
internalization and the intrinsic activity of the ligand is non-linear,
and so very weak partial agonists can produce significant receptor
internalization (Szekeres et al, 1998). Moreover, the role of
intracellular dopamine may not be receptor activation but something
quite different. This prompts the question of what, if anything, could
be the function of dopamine inside the post-synaptic neuron?

20. In the post-dopamine D1 receptor endosome system, transferrin and
the D1 receptor-dopamine complex co-localize in the same endosome,
which enables chelatable iron and dopamine to come into close contact.
The path of iron from the late endosome to its target - the
iron-containing enzymes being synthesized in the neuron (including
enzymes like tyrosine and tryptophan hydroxylase and certain
mitochondrial enzymes) - is not clear. There have been suggestions that
a small molecule acts as the carrier (Jacobs 1977; Bradbury 1997).
However, when Vyoral and Petrk (1998) used gel electrophoresis, they
could not find any evidence for these. They suggested that the endosome
is physically in contact with all the structures that use iron
(mitochondria, ribosomes, etc.), and that the iron is transported from
one to the others by means of an extensive system of channels and
tubes.

21. Breuer et al (1997) found that the catalytic potential of iron was
highest while in transit between the endosomes and cytosolic ligands.
They also found that iron is released from endosomes and enters a
cytoplasmic pool at a concentration of 0.3-0.5 mM (Breuer et al,1995).
The mean transit time through the chelatable pool is 1-2 hours. Moos
and Morgan (1998) have recently presented experimental evidence for the
existence of low-molecular weight transporters for iron in the brain
and in cerebrospinal fluid, and suggest that citrate or ascorbate might
act in this way. If the postulated iron-dopamine complex is carried
attached to some protein, this may perhaps explain Vyoral and Petrk's
results. The function of dopamine inside the post-synaptic neuron, in
the form of an iron-dopamine complex, may be similar to what I have
suggested is its role in the synapse; that is, as an antioxidant
protection for the spine and dendrite. This would have the added
advantage of the safe transport of iron to its cytoplasmic
destinations.

22. As noted earlier, free iron is far too toxic to be loose in the
cytoplasm in anything more than minute quantities. The dopamine
complex-mediated iron transporter could be transported to the sites of
iron usage through the cytoplasm, or within extensions of the
endoplasmic tubules, as proposed by Vyoral and Petrk. In the first case
the superoxide anions would encounter the dopamine-iron complexes in
the cytoplasm, and in the second case superoxide anions could enter the
endosomes from their main sites of production i.e., the mitochondria,
to which the endoplasmic tubes would give direct access. Qian et al
(1997) have reviewed the whole question of how iron is transported from
inside the endosome to its cytosolic targets. Their conclusion is that
little is known about this subject, and that there may be multiple
carriers (e.g., p97, integrin, H+-ATPase and the transferrin
receptor).

23. There may also be different transporters in different cells, and
the evidence suggests some type of carrier 'chaperone' protein rather
than an iron-conducting channel. If the dopamine-iron complex forms in
the endosome, it may be transmitted to the cytosol by such a carrier
mechanism. Alternatively, there may be separate transport mechanisms
for iron and dopamine out of the endosome and the complex forms only in
the cytosol. Qian et al (1998) state that iron is maintained in a
chelateable pool in the cytoplasm after leaving the endosome.
Catecholamine-iron complexes function as iron siderophores for the
bacterium Listeria monocytogenes, acquiring iron from the environment
and transporting it into the cell by means of a ferric reductase in the
membrane (Coulanges et al, 1997). Perhaps the endosome membrane has a
similar ferric reductase system. Mitochondria, descended from bacteria,
may act in the same way. It is also possible that the dismuting
dopamine-iron complex forms in the glutamatergic synaptic cleft
itself.  It is likely that free dopamine, and free iron in low
concentrations, are present in this location. Superoxide anions could
only be present if the neuronal membrane contains superoxide channels,
as the erythrocyte does. No information on this point is currently
available.

24. This system enables us to explain how the combination of
desferrioxamine and prochlorperazine induce coma. One factor may be
that in promoting iron transport across membranes, this combination
depletes intracellular iron by the synergistic effect of its
components. This prevents the formation of the dopamine-iron complex
inside the post-synaptic cell at the glutamate synapse, so that the
superoxide ions, produced by mitochondria and enzymes on the post NMDAr
cascade, cannot be scavenged. This effectively shuts down the glutamate
synapse and the functional effect is akin to the effect of ketamine,
although the details are different, as ketamine acts at the receptor
level, while the combination of desferrioxamine and prochlorperazine
may act at the post-synaptic level. A second factor, or contributory
mechanism, may be that prochlorperazine (the side chain of which has
structural similarities with spermine-type polyamines) blocks the
polyamine site on the NMDA receptor protein, and thus acts in a
synergistic manner, with lowered intracellular iron leading to down
regulation of the NMDA receptor.

25. Thus, it could be argued that consciousness depends on the general
level of activity of the whole cortex via a wide range of
cortico-thalamic relays - as determined by a proper balance between
glutamatergic activation and GABAergic inhibition, plus modulation by
other factors, such as local cholinergic neurotransmission - as much as
on the activity of strategically placed brain stem nuclei such as the
pedunculopontine nucleus and the intralaminar nuclei of the thalamus
(Smythies, 1997b). However, it is not clear why it is NMDAr antagonists
rather than AMPAr or mGlur antagonists that induce coma. This would
suggest a further field for study.

26. This hypothesis mirrors that reached by Angel (1991), that
anaesthesia is associated with a general impairment of communication
between the thalamus and cortex. Consciousness may be realized only
when some critical threshold of global neural activity is present.
Llins and Par (1991) have suggested that the content of consciousness
is provided by activity in cortico-thalamic loops which involves
specific thalamic nuclei, and that the 'binding' of these into a
'unitary experience' involves activity in the intralaminar thalamic
nuclei. However, another hypothesis is possible. As the molecular
targets of general anaesthetics (in either the classical or the
receptor-based theories) are widely distributed in the brain, it seems
likely that anaesthesia would affect all brain systems in a rather
unselective manner. What may be of greater significance is their
interaction with a subclass of key 'consciousness neurons' (should
these exist), rather than their global effect on the brain. Thus it can
be argued that general anaesthetics can tell us little or nothing about
the specific neural constituents of conscious phenomena. It may be
possible to clarify this point through the following experimental
approach:

    (i) prepare a number of rats with indwelling canulae suitable for
    local perfusion in a number of key locations, such as the medial
    thalamus, midbrain tegmental area, midbrain reticular formation,
    and basal forebrain,

    (ii) locally perfuse these awake animals with a series of general
    anaesthetic agents in these different anatomical loci and see if
    coma develops.

27. Clearly, if a full coma (general anaesthesia) followed such a
perfusion of area A but not of areas B, C, and D, this would indicate
that the general anaesthetic effect of that agent was mediated by its
effects on area A, as a necessary and sufficient cause of the coma, and
not by any effects on areas B, C, or D, or on the cortex as a whole.
Similarly, if no such local effect could be found to support the
hypothesis, then general anaesthesia must depend on more widespread
effects. However, it could not be assumed from such a result alone that
a widespread cortical inhibition is required. Additional experiments
involving the local perfusion of combinations of these subcortical
areas would first be necessary. It would not seem technically possible
to perfuse the entire cortex without leakage to subcortical areas, or
to perfuse the reticular nucleus of the thalamus uniquely, owing to its
special anatomy. Gases such as halothane and nitrous oxide might
produce technical difficulties but agents such as anaesthetic
barbiturates, ketamine and the desferrioxamine/prochlorperazine mixture
should not. I can find no evidence, in spite of an intensive literature
search, that this experiment has ever been done.

28. In the past, the generally accepted position was that the key
neuronal functions to be considered for any aspect of brain function
(behavioral as well as pharmacological) were neurotransmitter
synthesis, release, activation of specific postsynaptic (and some
presynaptic) receptors, and a complex series of postsynaptic cascades
involving various nucleotides. These provided the framework for
investigating what were considered the important events at this level
of brain function, events that might relate, inter alia, to the
evolution of consciousness. Recent advances in cell biology (which I
mentioned in section 12, but with which many neuroscientists seem
unfamiliar) have added a new and highly significant dimension to this
research.

29. This new territory demonstrates that neurotransmitter receptors are
subject to a continuing and massive process of endocytosis into the
post-synaptic neuron, transfer to the endosome system (in some cases in
clathrin coated vesicles and in other cases in non-clathrin coated
vesicles), molecular processing in the endosome system, and a final
triage process, during which some of the receptor protein is broken
down by lysosomes but most is recycled back to the external membrane.
In many cases the bound neurotransmitter (or neuromodulator) is
endocytosed together with the receptor. This means that agents like
general anaesthetics, which appear to affect a number of receptors, may
be affecting the endocytotic mechanism common to these receptors. It
also entails the revolutionary concept that some neurotransmitters, or
neuromodulators, could play a key role inside the post-synaptic neuron.
In the case of neuropeptide neuromodulators, it is now known that they
are trafficked to the nucleus, where they play a role in transcription
processes. No such role has as yet been discovered for smaller
neurotransmitters. I presented an hypothesis based on this possibility
relating to dopamine earlier (paragraph 15). At present there is no
direct evidence linking the endocytotic mechanism to the maintenance of
consciousness or the induction of coma. However, this revolution in
neuronal biology is so important that it deserves inclusion in any
consideration of any higher neuronal activity.

V. NON-REM SLEEP.

30. Another instance of coma is familiar non-REM (NREM) sleep. During
both the awake state and REM sleep we are basically conscious, even
though the phenomenology of these states is very different. Recently
two good reviews of this topic have appeared (Kahn et al, 1997; Hobson
et al, 1998). The key feature of the awake state appears to be a
balanced activity in the cholinergic, noradrenergic and serotoninergic
systems permitting attentive behavior. During REM sleep, cholinergic
activity in the peribrachial pedunculopontine and lateral dorsal
tegmental cholinergic nuclei increases, and activity in the
noradrenergic and serotoninergic activity greatly decreases. In NREM
sleep, activity of all three of these neurotransmitters is low and
there is deactivation of almost all brain systems. This concurs with
the theory that general anaesthesia is associated with a widespread
down regulation of the cortex. In NREM sleep there is also a reduction
in the activity of some peribrachial cholinergic cells, which results
in a change from tonic to phasic firing of thalamic relay nuclei. This
is consistent with the specific effect of general anaesthetics in
reducing cholinergic activity as reviewed above (see section 4 (i)).
Kahn et al (1997) conclude that "conscious actions require activated
cortical networks".

NOTE: I have written about the problem of the nature of consciousness
elsewhere (Smythies 1994 a, 6).

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