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Showing posts with label GABA. Show all posts
Showing posts with label GABA. Show all posts

Friday 3 June 2016

Mefenamic acid (Ponstan) for some Autism


Caution:-

Ponstan (Mefenamic Acid) contains a warning:-
Caution should be exercised when treating patients suffering from epilepsy.

At lower doses Ponstan is antiepileptic, but at high doses it can have the opposite effect.  This effect depends on the biological origin of the seizures.
In an earlier post I wrote about a paper by Knut Wittkowski who applied statistics to interpret the existing genetic data on autism. 


“Autism treatments proposed by clinical studies and human genetics are complementary” & the NSAID Ponstan as a Novel AutismTherapy




His analysis suggested the early use of Fenamate drugs could potentially reduce the neurological anomalies that develop in autism as the brain develops.  The natural question arose in the comments was to whether it is too late to use Fenamates in later life.

Knut was particularly looking at a handful of commonly affected genes (ANO 2/4/7 & KCNMA1) where defects should partially be remedied by use of fenamates.

I recently received a comment from a South African reader who finds that his children’s autism improves when he gives them Ponstan and he wondered why.  Ponstan (Mefenamic Acid) is a fenamate drug often used in many countries as a pain killer, particularly in young children.

Ponstan is a cheap NSAID-type drug very widely used in some countries and very rarely used in other countries like the US.  It is available without prescription in some English-speaking countries (try a pharmacy in New Zealand, who sell online) and, as Petra has pointed out, it is widely available in Greece.

I did some more digging and was surprised what other potentially very relevant effects Ponstan has.  Ponstan affects GABAA receptors, where it is a positive allosteric modulator (PAM).  This may be very relevant to many people with autism because we have seen that fine-tuning the response of the sub-units that comprise GABAA receptors you can potentially improve cognition and also modulate anxiety. 

Anxiety seems to be a core issue in Asperger’s, whereas in Classic Autism, or Strict Definition Autism (SDA) the core issue is often actually cognitive function rather than “autism” as such.

In this post I will bring together the science showing why Ponstan should indeed be helpful in some types of autism.

Professor Ritvo from UCLA read Knut’s paper and also the bumetanide research and suggested that babies could be treated with Ponstan and then, later on, with  Bumetanide.

Autism treatments proposed by clinical studies and human genetics are complementary



I do not think the professor or Knut are aware of Ponstan’s effect on GABA.

The benefits from Ponstan may very well be greater if given to babies at risk of autism, but there does seem to be potential benefit for older children and adults, depending on their type of autism.

Professor Ritvo points out that that Ponstan is safely used in 6 month old babies, so trialing it in children and adults with autism should not be troubling.

Being an NSAID, long term use at high doses may well cause GI side effects.  An open question is the dosage at which Ponstan modulates the calcium activated ion channels that are implicated in some autism and also what dosage affects GABAA receptors.  It might well be lower than that required for Ponstan’s known ant-inflammatory effects.


Ponstan vs Ibuprofen

Ibuprofen is quite widely used in autism.  Ibuprofen is an NSAID but also a PPAR gamma agonist.  Ponstan is an NSAID but has no effect on PPAR gamma.

Research shows that some types of autism respond to PPAR gamma agonists.

So it is worth trying both Ponstan and Ibuprofen, but for somewhat different reasons.

They are both interesting to deal with autism flare-ups, which seem common.

Other drugs that people use short term, but are used long term in asthma therapy,  are Singulair (Montelukast) and an interesting Japanese drug called Ibudilast.  Singulair is a Western drug for maintenance therapy in asthma.  Ibudilast is widely used in Japan as maintenance therapy in Asthma, but works in a different way.  Ibudilast is being used in clinical trials in the US to treat Multiple Sclerosis.  Singulair is cheap and widely available, Ibudilast is more expensive and available mainly in Japan.


Pre-vaccination Immunomodulation

In spite of there being no publicly acknowledged link between vaccinations and autism secondary to mitochondrial disease (AMD), I read that short term immunomodulation is used prior to vaccination at Johns Hopkins, for some babies.

Singulair is used, as is apparently ibuprofen.  Ponstan and Ibudilast would also likely be protective.   Ponstan might well be the best choice; it lowers fevers better than ibuprofen.

For those open minded people, here is what a former head of the US National Institutes of Health, Bernadine Healy, had to say about the safe vaccination.  Not surprisingly she was another Johns Hopkins trained doctor, as is Hannah Poling’s Neurologist father.

The Vaccines-Autism War: Détente Needed

“Finally, are certain groups of people especially susceptible to side effects from vaccines, and can we identify them? Youngsters like Hannah Poling, for example, who has an underlying mitochondrial disorder and developed a sudden and dramatic case of regressive autism after receiving nine immunizations, later determined to be the precipitating factor. Other children may have a genetic predisposition to autism, a pre-existing neurological condition worsened by vaccines, or an immune system that is sent into overdrive by too many vaccines, and thus they might deserve special care. This approach challenges the notion that every child must be vaccinated for every pathogen on the government's schedule with almost no exception, a policy that means some will be sacrificed so the vast majority benefit.”


So if I was an American running the FDA/CDC I would suggest giving parents the option of paying a couple of dollars for 10 days of Ponstan prior to these megadose vaccinations and a few days afterwards.  No harm or good done in 99.9% of cases, but maybe some good done for the remainder.

The fact the fact that nobody paid any attention to the late Dr Healy on this subject tells you a lot.



Fenamates (ANO 2/4/7 & KCNMA1)

Here Knut is trying to target the ion channels expressed by the genes ANO 2/4/7 & KCNMA1. 

·        ANO 2/4/7 are calcium activated chloride channels. (CACCs)


·        KCNMA1 is a calcium activated potassium channel.  KCNMA1encodes the ion channel KCa1.1, otherwise known as BK (big potassium).  This was the subject of post that I never got round to publishing.
  
Fenamates are an important group of clinically used non-steroidal anti-inflammatory drugs (NSAIDs), but they have other effects beyond being anti-inflammatory.  They act as CaCC inhibitors and also stimulate BKCa channel activity.


But fenamates also have a potent effect on what seems to be the most dysfunctional receptor in classic autism, the GABAA receptor.




The fenamate NSAID, mefenamic acid (MFA) prevents convulsions and protects rats from seizure-induced forebrain damage evoked by pilocarpine (Ikonomidou-Turski et al., 1988) and is anti-epileptogenic against pentylenetetrazol (PTZ)-induced seizure activity, but at high doses induces seizures (Wallenstein, 1991). In humans, MFA overdose can lead to convulsions and coma (Balali-Mood et al, 1981; Young et al., 1979; Smolinske et al., 1990). More recent data by Chen and colleagues (1998) have shown that the fenamates, flufenamic, meclofenamic and mefenamic acid, protect chick embryo retinal neurons against ischaemic and excitotoxic (kainate and NMDA) induced neuronal cell death in vitro (Chen et al., 1998a; 1998b). MFA has also been reported to reduce neuronal damage induced by intraventricular amyloid beta peptide (Aβ1-42) and improve learning in rats treated with Aβ1-42 (Joo et al., 2006). The mechanisms underlying these anti-epileptic and neuroprotective effects are not well understood but together suggest that fenamates may influence neuronal excitability through modulation of ligand and/or voltage-gated ion channels. In the present study, therefore, we have investigated this hypothesis by determining the actions of five representative fenamate NSAIDs at the major excitatory and inhibitory ligand-gated ion channels in cultured hippocampal neurons


This study demonstrates for the first time that mefenamic acid and 4 other representatives of the fenamate NSAIDs are highly effective and potent modulators of native hippocampal neuron GABAA receptors. MFA was the most potent and at concentrations equal to or greater than 10 μM was also able to directly activate the GABAA gated chloride channel. A previous study from this laboratory reported that mefenamic acid potentiated recombinant GABAA receptors expressed in HEK-293 cells and in Xenopus laevis oocytes (Halliwell et al., 1999). Together these studies lead to the conclusion that fenamate NSAIDs should now also be considered a robust class of GABAA receptor modulators.


Also demonstrated for the first time here is the direct activation of neuronal GABAA receptors by mefenamic acid. Other allosteric potentiators, including the neuroactive steroids and the depressant barbiturates share this property, with MFA at least equipotent to neurosteroids and significantly more potent than the barbiturates. The mechanism(s) of the direct gating of GABAA receptor chloride channels by MFA requires further investigation using ultra-fast perfusion techniques but may be distinct from that reported for neurosteroids (see, Hosie et al., 2006). Mefenamic acid induced a leftward shift in the GABA dose-response curve consistent with an increase in receptor affinity for the agonist. This is an action observed with other positive allosteric GABAA receptor modulators, including the benzodiazepine agonist, diazepam, the neuroactive steroid, allopregnanolone, and the intravenous anesthetics, pentobarbitone and propofol (e.g. Johnston, 2005). To our knowledge, a unique property of MFA was that it was significantly (F = 10.35; p≤ 0.001) more effective potentiating GABA currents at hyperpolarized holding potentials (especially greater than −60mV). Further experiments are required however to determine the underlying mechanism(s).

The highly effective modulation of GABAA receptors in cultured hippocampal neurons suggests the fenamates may have central actions. Consistent with this hypothesis, mefenamic acid concentrations are 40–80μM in plasma with therapeutic doses (Cryer & Feldman, 1998); fenamates can also cross the blood brain barrier (Houin et al., 1983; Bannwarth et al., 1989) Coyne et al. Page 5 Neurochem Int. Author manuscript; available in PMC 2008 November 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript and in overdose in humans are associated with coma and convulsions (Smolinske et al., 1990). In animal studies, mefenamic acid is anticonvulsant and neuroprotective against seizureinduced forebrain damage in rodents (Ikonomidou-Turski et al., 1988). The present study would suggest that the anticonvulsant effects of fenamates may be related, in part, to their efficacy to potentiate native GABAA receptors in the brain, although a recent study has suggested that activation of M-type K+ channels may contribute to this action (Peretz et al., 2005) Finally, Joo and co-workers (2006) have recently reported that mefenamic acid provided neuroprotection against β-amyloid (Aβ1-42) induced neurodegeneration and attenuated cognitive impairments in this animal model of Alzheimer’s disease. The authors proposed that neuroprotection may have resulted from inhibition of cytochrome c release from mitochondria and reduced caspase-3 activation by mefenamic acid. Clearly it would also be of interest to evaluate the role of GABA receptor modulation in this in vivo model of Alzheimer’s disease. Moreover, considerable evidence has emerged in the last few years indicating that GABA receptor subtypes are involved in distinct neuronal functions and subtype modulators may provide novel pharmacological therapies (Rudolf & Mohler, 2006). Our present data showing that fenamates are highly effective modulators of native GABAA receptors and that mefenamic acid is highly subtype-selective (Halliwell et al., 1999) suggests that further studies of its cognitive and behavioral effects would be of value.

  

Note in the above paper that NSAIDs other than mefenamic acid also modulate GABAA receptors.

Just a couple of months ago a rather complicated paper was published, again showing that NSAIDs modulate GABAA receptors and showing that this is achieved via the same calcium activated chloride channels (CaCC) referred to by Knut.

NSAIDs modulate GABA-activated currents via Ca2+-activated Cl channels in rat dorsal root ganglion neurons






"Schematic displaying the effects of CaCCs on GABA-activated inward currents and depolarization. GABA activates the GABAA receptor to open the Cl  channel and the Cl efflux induces the depolarization response (inward current) of the membrane of dorsal root ganglion (DRG) neurons. Then, voltage dependent L-type Ca2+ channels are activated by the depolarization, and give rise to an increase in intracellular Ca2+. CaCCs are activated by an increase in intracellular Ca2+ concentration which, in turn, increases the driving force for Cl efflux. Finally, the synergistic action of the chloride ion efflux through GABAA receptors and NFA-sensitive CaCCs causes GABA-activated currents or depolarization response in rat DRG neurons."


Note in the complex explanation above the L-type calcium channels, which are already being targeted by Verapamil, in the PolyPill.



Mefenamic Acid and Potassium Channels

We know that Mefenamic acid also affects Kv7.1 (KvLQT1).

A closely related substance called meclofenamic acid is known to act as novel KCNQ2/Q3 channel openers and is seen as having potential for the treatment of neuronal hyper-excitability including epilepsy, migraine, or neuropathic pain.



The voltage-dependent M-type potassium current (M-current) plays a major role in controlling brain excitability by stabilizing the membrane potential and acting as a brake for neuronal firing. The KCNQ2/Q3 heteromeric channel complex was identified as the molecular correlate of the M-current. Furthermore, the KCNQ2 and KCNQ3 channel  subunits are mutated in families with benign familial neonatal convulsions, a neonatal form of epilepsy. Enhancement of KCNQ2/Q3 potassium currents may provide an important target for antiepileptic drug development. Here, we show that meclofenamic acid (meclofenamate) and diclofenac, two related molecules previously used as anti-inflammatory drugs, act as novel KCNQ2/Q3 channel openers. Extracellular application of meclofenamate (EC50  25 M) and diclofenac (EC50  2.6 M) resulted in the activation of KCNQ2/Q3 K currents, heterologously expressed in Chinese hamster ovary cells. Both openers activated KCNQ2/Q3 channels by causing a hyperpolarizing shift of the voltage activation curve (23 and 15 mV, respectively) and by markedly slowing the deactivation kinetics. The effects of the drugs were stronger on KCNQ2 than on KCNQ3 channel  subunits. In contrast, they did not enhance KCNQ1 K currents. Both openers increased KCNQ2/Q3 current amplitude at physiologically relevant potentials and led to hyperpolarization of the resting membrane potential. In cultured cortical neurons, meclofenamate and diclofenac enhanced the M-current and reduced evoked and spontaneous action potentials, whereas in vivo diclofenac exhibited an anticonvulsant activity (ED50  43 mg/kg). These compounds potentially constitute novel drug templates for the treatment of neuronal hyperexcitability including epilepsy, migraine, or neuropathic pain. Volt




BK channel

KCNMA1encodes the ion channel KCa1.1, otherwise known as BK (big potassium). BK channels are implicated not only by Knut’s statistics, but numerous studies ranging from schizophrenia to Fragile X. 

Usually it is a case of too little BK channel activity.

The BK channel is implicated in some epilepsy.

  

Pharmacology

BK channels are pharmacological targets for the treatment of several medical disorders including stroke and overactive bladder. Although pharmaceutical companies have attempted to develop synthetic molecules targeting BK channels, their efforts have proved largely ineffective. For instance, BMS-204352, a molecule developed by Bristol-Myers Squibb, failed to improve clinical outcome in stroke patients compared to placebo. However, BKCa channels are reduced in patients suffering from the Fragile X syndrome and the agonist, BMS-204352, corrects some of the deficits observed in Fmr1 knockout mice, a model of Fragile X syndrome.
BK channels have also been found to be activated by exogenous pollutants and endogenous gasotransmitters carbon monoxide and hydrogen sulphide.
BK channels can be readily inhibited by a range of compounds including tetraethylammonium (TEA), paxilline and iberiotoxin.



Achieving a better understanding of BK channel function is important not only for furthering our knowledge of the involvement of these channels in physiological processes, but also for pathophysiological conditions, as has been demonstrated by recent discoveries implicating these channels in neurological disorders. One such disorder is schizophrenia where BK channels are hypothesized to play a role in the etiology of the disease due to the effects of commonly used antipsychotic drugs on enhancing K+ conductance [101]. Furthermore, this same study found that the mRNA expression levels of the BK channel were significantly lower in the prefrontal cortex of the schizophrenic group than in the control group [101]. Similarly, autism and mental retardation have been linked to haploinsufficiency of the Slo1 gene and decreased BK channel expression [102].
Two mutations in BK channel genes have been associated with epilepsy. One mutation has been identified on the accessory β3 subunit, which results in an early truncation of the protein and has been significantly correlated in patients with idiopathic generalized epilepsy [103]. The other mutation is located on the Slo1gene, and was identified through genetic screening of a family with generalized epilepsy and paroxysmal dyskinesia [104]. The biophysical properties of this Slo1 mutation indicates enhanced sensitivity to Ca2+ and an increased average time that the channel remains open [104107]. This increased Ca2+ sensitivity is dependent on the specific type of β subunit associating with the BK channel [106, 107]. In association with the β3 subunit, the mutation does not alter the Ca2+-dependent properties of the channel, but with the β4 subunit the mutation increases the Ca2+ sensitivity [105107]. This is significant considering the relatively high abundance of the β4 subunit compared to the weak distribution of the β3 subunit in the brain [12, 13,15, 106, 107]. It has been proposed that a gain of BK channel function may result in increases in the firing frequency due to rapid repolarization of APs, which allows a quick recovery of Na+ channels from inactivation, thereby facilitating the firing of subsequent APs [104]. Supporting this hypothesis, mice null for the β4 subunit showed enhanced Ca2+ sensitivity of BK channels, resulting in temporal lobe epilepsy, which was likely due to a shortened duration and increased frequency of APs [108]. An interesting relevance to the mechanisms of BK channel activation as discussed above, the Slo1 mutation associated with epilepsy only alters Ca2+ dependent activation originated from the Ca2+ binding site in RCK1, but not from the Ca2+bowl, by altering the coupling mechanism between Ca2+ binding and gate opening [100]. Since Ca2+dependent activation originated from the Ca2+ binding site in RCK1 is enhanced by membrane depolarization, at the peak of an action potential the binding of Ca2+ to the site in RCK1 contributes much more than binding to the Ca2+ bowl to activating the channel [84, 109].
Although these associations between specific mutations in BK channel subunits and various neurological disorders have been demonstrated by numerous studies, it is also important to point out certain caveats with these studies, such as genetic linkage between BK channels and different diseases do not necessary show causation as these studies were performed based on correlation between changes in the protein/genetic marker and overall phenotype. Furthermore, studies performed using a mouse model also can fail to indicate what may happen in higher-order species, and this is especially true for BK channels, where certain β subunits are only primate specific [110].


  

Possible role of potassium channel, big K in etiology of schizophrenia.

Schizophrenia (SZ), a common severe mental disorder, affecting about 1% of the world population. However, the etiology of SZ is still largely unknown. It is believed that molecules that are in an association with the etiology and pathology of SZ are neurotransmitters including dopamine, 5-HT and gamma-aminobutyric acid (GABA). But several lines of evidences indicate that potassium large conductance calcium-activated channel, known as BK channel, is likely to be included. BK channel belongs to a group of ion channels that plays an important role in regulating neuronal excitability and transmitter releasing. Its involvement in SZ emerges as a great interest. For example, commonly used neuroleptics, in clinical therapeutic concentrations, alter calcium-activated potassium conductance in central neurons. Diazoxide, a potassium channel opener/activator, showed a significant superiority over haloperidol alone in the treatment of positive and general psychopathology symptoms in SZ. Additionally, estrogen, which regulates the activity of BK channel, modulates dopaminergic D2 receptor and has an antipsychotic-like effect. Therefore, we hypothesize that BK channel may play a role in SZ and those agents, which can target either BK channel functions or its expression may contribute to the therapeutic actions of SZ treatment.




Conclusion

It appears that Ponstan and related substances have some interesting effects that are only now emerging in the research.

People with autism, and indeed schizophrenia, may potentially benefit from Ponstan and for a variety of different reasons.

I think it will take many decades for any conclusive research to be published on this subject, because this is an off-patent generic drug.

As with most NSAIDS, it is simple to trial Ponstan.

Thanks to Knut for the idea, Professor Ritvo for his endorsement of the idea and our reader from South Africa for sharing his positive experience with Ponstan. 







Monday 11 January 2016

The GABA Switch, Altered GABAa Receptor subunit expression in Autism and Basmisanil





In today’s post I intended to dig a little deeper into the GABA switch, which appears to underlie much autism, schizophrenia, epilepsy, even Down Syndrome and, not to forget, many mood disorders.  

Once you start digging, it is rather hard to stop.

There is literature on the subject, but very little (almost none, really) looks at the big picture of what is going on.  It is the big picture that matters.





The GABA switch(es); but how many are there?


The post starts out relatively simple, but then it does get complicated, because I discovered a lot interesting avenues exist, that seem to have been completed ignored by autism research.  It seems Down Syndrome researchers are better informed.  

So if you make it to the end of this post, you will have done well.

It seems that there are tens, if not hundreds, of possible ways to repair the faulty GABA switches.  It would very much become a case for personalized medicine, correcting the precise dysfunctions, without disturbing anything else.  Each case will be slightly different.

Back to the simple part.

During very early development certain changes in the brain are expected to occur, that control how the GABA neurotransmitter functions.  If they do not all occur, some of the following may occur:-

·        Autism
·        Epilepsy
·        Mental retardation / Intellectual Disability; including the MR/ID in Down Syndrome
·        Mood disorders (anxiety, depression etc)

If later in life, after brain maturation, these same changes occur the following may occur:-

·        Schizophrenia
·        Bipolar
·        Other mood disorders (anxiety, depression etc)
·        Epilepsy

There are at least two distinct processes involved, both can be considered as part of the GABA switch. It is likely that two further process are involved, but they have not yet been adequately researched.


1.     The lowering of intracellular chloride levels

This has been very deeply documented already in this blog.  If the GABA switch has not been “flipped”, we have overexpression of a cotransporter NKCC1, which overwhelms the effect of another called KCC2 and this results in elevated intracellular chloride levels.  This then prevents GABA signaling switching from excitatory to inhibitory.  This creates an excitatory/inhibitory imbalance and neurons fire when they should not.  This disables cognitive function and creates a tendency towards pre-epilepsy and then epilepsy.

The neurons never reach their expected mature state.


2.     Change in GABAA-receptor subunit expression

We have already seen in this blog, that drugs that positively modulate the α2 and α3 subunits of GABAA receptors, like low-dose clonazepam, rescue some aspects of autism.  This is Professor Catterall’s research.  As far as I can see, he did not really explain the big picture behind this.

We saw in early posts that the composition of the GABA receptor vary over time. Remember there are 5 sub-units in each receptor.  These changes in composition of the receptor directly affect people’s mood and behavior.





Five subunits can combine in different ways to form GABAA channels. The minimal requirement to produce a GABA-gated ion channel is the inclusion of both α and β subunits, but the most common type in the brain is a pentamer comprising two α's, two β's, and a γ (α1β2γ2)
In humans, the units are as follows:
·         six types of α subunits (GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRA6)
·         three βs (GABRB1, GABRB2, GABRB3)
·         three γs (GABRG1, GABRG2, GABRG3)
·         as well as a δ (GABRD), an ε (GABRE), a π (GABRP), and a θ (GABRQ)


What appears likely to me is that this variation in subunit expression both over time and throughout different parts of the brain is really just another part of the GABA switch.

It appears that in autism the α3 subunit is under-expressed.  In its place it appears we have the α5 subunit over-expressed.  This lowers your IQ.

This under-expression of the α3 subunit has been compensated for by Catterall by using a positive allosteric modulator.

In effect the α5 subunit is dominating, where it should not be and this presents itself as autism.

There is research showing how numerous influences can affect the sub unit expression.  Some are transitory and reversible, but others can be permanent.  It is proposed that, when permanent, the underlying change is epigenetic, when “tags” are placed on perfectly functional genes that either turn them on or off.  One example resulting in permanent change is stress. 

Repeated neonatal handling with maternal separation permanently alters hippocampal GABAA receptors and behavioral stress responses



We will focus on the reversible changes, since that is the purpose of this blog.

After some digging, I did find a nice graphic that does illustrate the first two elements of the GABA switch.  It is based on mouse research and shows the changes that should happen in the first 20 days of life. 

In the first few days NKCC1 is highly expressed, while KCC2 is weakly expressed. This results high levels of intracellular chloride.  As NKCC1 drops, less chloride enters and so intracellular chloride falls.  So depolarizing GABA (excitatory) becomes hyperpolarizing GABA (inhibitory).

In the twenty days while this is happening, the sub unit structure of the GABAA receptors is also changing.

In some autism neurons remain in their immature state, with NKCC1 highly expressed and so high levels of intracellular chloride and depolarizing GABA (excitatory).

My suggestion is that the programmed changes in sub unit expression may also fail to occur.









3.     Density of GABA A receptors ?

In the literature, when they talk about density of GABA A receptors, they are talking about either an increased or reduce number of receptors at a given location.

The density can vary over time and within different parts of the brain.










Source:  http://www.nature.com/articles/srep16347/figures/1


It appears that in autism, there is a reduced number of GABAA receptor, in other words lower density.

GABAA receptor downregulation in brains of subjects with autism


I am suggesting that the under-expression of GABAA receptor, which appears likely to be linked to disturbed calcium channel signaling, could be considered as the third element of the GABA switch.

In the science jargonVGCC (Voltage Gated Calcium Channel) activation is involved in GABA-induced GABAAR down-regulation”. Which begs the question, what is the effect on GABAA receptor density in humans of blocking VGCCs?

Nifedipine is specifically suggested.  My Polypill already includes verapamil, another blocker of VGCCs.




4.     GABAB subunits ?

There probably is a fourth element of the GABA switch.  If there is, it possibly relates to the GABAB receptor.  GABAB receptors are made up of just two subunits, GABAB1 and  GABAB2. This is much less well researched than GABAA, but from what little there is, it is clear that GABAB1 and GABAB2 receptor subunits expression is disturbed in some epilepsy and after TBI (traumatic brain injury).  TBI is interesting because, in many ways, that is what autism is; just it was not a physical trauma, it was a genetic/environmental trauma.

The expression of GABA(B1) and GABA(B2) receptor subunits in the cNS differs from that in peripheral tissues.

Modification of GABA(B1) and GABA(B2) receptor subunits in the somatosensory cerebral cortex and thalamus of rats with absence seizures (GAERS)





The Implication?  Repair the GABA Switch(es)

Before getting involved in the complexities of the research, we can already draw a nice simple conclusion.

Many types of autism are likely associated with a faulty GABA switch.  So if you want to treat someone’s autism, start by repairing the GABA switch(es).  Just realize there is more than one and so therapies will have to vary.

Now to the science, for those who like to go into details:-
                                                         
This is a good paper that was highlighted earlier by Tyler, a reader of this blog:-



The GABAergic neurons of the thalamic reticular nucleus (nRt) provide the primary source of inhibition within the thalamus. Using physiology, pharmacology and immunohistochemistry in mice we characterized post-synaptic developmental changes in these inhibitory projection neurons. First, at postnatal day 3-5 (P3-5), inhibitory postsynaptic currents (IPSCs) decayed very slowly, followed by a biphasic developmental progression, becoming faster at P6-8, then slower again at P9-11 before stabilizing in a mature form around P12. Second, the pharmacological profile of GABAAR mediated IPSCs differed between neonatal and mature nRt neurons and this was accompanied by reciprocal changes in α3 (late) and α5 (early) subunit expression in nRt. Zolpidem, selective for α1- and α3-containing GABAARs, augmented only mature IPSCs, while clonazepam enhanced IPSCs at all stages. This effect was blocked by the α5-specific inverse agonist L-655,708 but only in immature neurons. In α3H126R mice in which α3 subunits were mutated to become BZ insensitive, IPSCs were enhanced compared to wild type animals in early development. Third, tonic GABAAR activation in nRt is age-dependent, and more prominent in immature neurons, which correlates with early expression of α5 containing GABAARs. Thus neonatal nRt neurons show relatively high expression of α5 subunits which contributes to both slow synaptic and tonic extrasynaptic inhibition. The postnatal switch in GABAAR subunits from α5 to α3 could facilitate spontaneous network activity in nRt that occurs at this developmental time point and which is proposed to play a role in early circuit development.



The following paper really covers the first GABA switch very well:-






The switch from excitatory to inhibitory GABAAR-related effects is closely related to the lowering of [Cl−]i during the course of the development. This latter mainly relies on the differential ontogenic expression of the Na+/K+/2Cl− cotransporter isoform 1 (NKCC1), which uptakes chloride ions [76–78], and the neuronal K+/Cl−cotransporter type 2 (KCC2) [79], which extrudes chloride ions [49, 80]. However, other exchangers can control the chloride gradient as the anion (Cl−–HCO3 −) exchangers, either Na+- independent (AE) or Na+-driven (NDCBE also called NDAE) [81] (NCBE) [82]. AE mediates influx of Cl− while exporting HCO3 −, these exchanges being triggered by intracellular alkalinisation. NDCBE, known as an acid extruder (extrudes H+), moves Cl− out in exchange of HCO3 −, driven by the Na+ gradient [83, 84]. NCBE also lowers [Cl−]i (and [H+]i) while importing Na+ and HCO3 − [82, 85].


This is one of Professor Catterall’s papers we looked at previously





Moreover, autistic-like behavioral impairments can be treated effectively in both BTBR and Scn1a+/− mice by enhancement of inhibitory neurotransmission with low doses of subunit-selective positive allosteric modulators of GABAA receptors containing α2 and/or α3 subunits. Together, our results support the hypothesis that reduced GABAergic inhibitory neurotransmission contributes to autism-associated behavioral and cognitive deficits and suggest that enhancement of GABAergic neurotransmission with next-generation subunit-specific pharmacological agents may be beneficial.


Subsynaptic GABAAreceptor subtypes are composed of two α, two β, and one γ subunit (Fritschy and Mohler, 1995). The action of GABA at these ionotropic receptors is increased through positive allosteric modulation by benzodiazepines, which are used to treat anxiety, insomnia, and epilepsy (Rudolph and Knoflach, 2011). In order to determine whether treatment with a benzodiazepine reverses the constitutively decreased GABAergic inhibitory signaling, we treated C57BL/6J and BTBR hippocampal slices with 0.5 μM clonazepam, a broad-acting, traditional benzodiazepine. These recordings revealed increased spontaneous IPSC amplitude (Figures 1E and 1F) and frequency (Figure S1C) in BTBR slices. In contrast, a significant increase of spontaneous IPSC amplitude (Figure S1I), but no change in IPSC frequency (Figure S1J), was observed in C57BL/6J slices. The increased GABAergic signaling after treatment with clonazepam led to a decrease in frequency of spontaneous EPSCs (Figures 1G and 1H), without change in amplitude in BTBR hippocampal slices (Figure S1D). Interestingly, the frequency of spontaneous EPSC was also decreased by clonazepam (Figure S1K), without change in amplitude (Figure S1L) in C57BL/6J slices. These data support the idea that low-dose clonazepam can reverse the underlying deficit in spontaneous GABAergic inhibitory neurotransmission in BTBR mice.

Rescue by α23-Specific Positive Allosteric Modulators of GABAA Receptors

Diversity of GABA receptor function is conferred by more than 20 different subunits, and receptors with different α subunits play distinct roles in the physiological and pharmacological actions of GABA and benzodiazepines (Fritschy and Mohler, 1995, Harmar et al., 2009, Rudolph and Knoflach, 2011,Rudolph and Möhler, 2004, Smith and Olsen, 1995). We tested the effects of subunit-selective positive allosteric modulators of GABAA receptors on social behavior in BTBR mice and C57BL/6J mice. A low dose of the α2,3-subunit-selective positive allosteric modulator L-838,417 (Löw et al., 2000, Mathiasen et al., 2008) increased social interactions in BTBR mice, with maximal effective dose of 0.05 mg/kg, and the beneficial effect was lost when the dose increased (Figures 4I and S4E). In contrast, L-838,417 did not change the social interaction behavior of C57BL/6J mice (Figure S4I). Moreover, the α1-subunit-selective positive GABAA modulator zolpidem (Mathiasen et al., 2008, Sieghart, 1995) failed to show beneficial effects in BTBR mice and actually aggravated their social interaction deficit at high doses (Figures 4J and S4F). Interestingly, a high dose of zolpidem also impaired social behavior in C57BL/6J mice (Figure S4J). Total movement tended to increase at high doses of L-838,417 (Figure S4G; not significant) but significantly decreased at 0.5 mg/kg zolpidem (Figure S4H). These results indicate that different subtypes of GABAA receptors may have opposite roles in social behavior, with activation of GABAA receptors containing α2,3 subunits favoring and activation of GABAA receptors with α1 subunits reducing social interaction, respectively.
Subunit-selective GABAA receptor modulators may also have an important effect on cognitive behaviors. In the context-dependent fear conditioning test, treatment with 0.05 mg/kg L-838,417 improved short-term (30 min) and long-term (24 hr) spatial memory in BTBR mice (Figure 4K), whereas 0.05 mg/kg zolpidem enhanced short-term memory but not long-term memory (Figure 4L). These data show that α2,3-subunit-containing GABAA receptors may also be important for cognitive behaviors in BTBR mice. The bell-shaped dose-response curves observed for both L-838,417 and clonazepam may explain why high-dose benzodiazepine treatment for prevention of anxiety and seizures has not been reported to improve autistic traits in ASD patients. As illustrated in Figures 4N and 4O, treatment with low doses of L-838,417 also improves social interactions in the Scn1a+/− mice, a model of Dravet syndrome with severe autistic-like behaviors (Han et al., 2012), within a narrow dose range. In contrast, similar treatment with zolpidem is not effective. Altogether, these experiments show that treatment with an α2,3-selective positive allosteric modulator of GABAA receptors is sufficient to rescue autistic-like behaviors and cognitive deficit in both a monogenic model of autism-spectrum disorder and the BTBR mouse model of idiopathic autism.

The following paper highlights disrupted subunit expression in one type of epilepsy:-

Altered thalamic GABAA-receptor subunit expression in the stargazer mouse model of absence epilepsy.


Abstract

PURPOSE:

Absence seizures, also known as petit mal seizures, arise from disruptions within the cortico-thalamocortical network. Interconnected circuits within the thalamus consisting of inhibitory neurons of the reticular thalamic nucleus (RTN) and excitatory relay neurons of the ventral posterior (VP) complex, generate normal intrathalamic oscillatory activity. The degree of synchrony in this network determines whether normal (spindle) or pathologic (spike wave) oscillations occur; however, the cellular and molecular mechanisms underlying absence seizures are complex and multifactorial and currently are not fully understood. Recent experimental evidence from rodent models suggests that regional alterations in γ-aminobutyric acid (GABA)ergic inhibition may underlie hypersynchronous oscillations featured in absence seizures. The aim of the current study was to investigate whether region-specific differences in GABAA receptor (GABAAR) subunit expression occur in the VP and RTN thalamic regions in the stargazer mouse model of absence epilepsy where the primary deficit is in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression.

METHODS:

Immunofluorescence confocal microscopy and semiquantitative Western blot analysis were used to investigate region-specific changes in GABAAR subunits in the thalamus of the stargazer mouse model of absence epilepsy to determine whether changes in GABAergic inhibition could contribute to the mechanisms underlying seizures in this model of absence epilepsy.

KEY FINDINGS:

Immunofluorescence confocal microscopy revealed that GABAAR α1 and β2 subunits are predominantly expressed in the VP, whereas α3 and β3 subunits are localized primarily in the RTN. Semiquantitative Western blot analysis of VP and RTN samples from epileptic stargazers and their nonepileptic littermates showed that GABAAR α1 and β2 subunit expression levels in the VP were significantly increased (α1: 33%, β2: 96%) in epileptic stargazers, whereas α3 and β3 subunits in the RTN were unchanged in the epileptic mice compared to nonepileptic control littermates.

SIGNIFICANCE:

These findings suggest that region-specific differences in GABAAR subunits in the thalamus of epileptic mice, specifically up-regulation of GABAARs in the thalamic relay neurons of the VP, may contribute to generation of hypersynchronous thalamocortical activity in absence seizures. Understanding region-specific differences in GABAAR subunit expression could help elucidate some of the cellular and molecular mechanisms underlying absence seizures and thereby identify targets by which drugs can modulate the frequency and severity of epileptic seizures. Ultimately, this information could be crucial for the development of more specific and effective therapeutic drugs for treatment of this form of epilepsy

This is another good paper, this time looking at how fragile X mental retardation protein (FMRP) may disrupt sub-unit expression and how this appears not only in those with Fragile-X but also in schizophrenia, mood disorders, and autism.  mGluR5 is involved not surprisingly and you may recall that mGluR5 is surprisingly also involved in GERD/GORD/reflux, which affects many people with autism.

GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism.



Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in the brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), the absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems.


The logical next step - Regulation of GABAA Receptor Subunit Expression by Pharmacological Agents


The first thing to note is that, if you are using low dose clonazepam, you are already compensating for the lack of α3 subunits. Clonazepam is a so-called positive allosteric modulator of α3.

Are there further options?  Yes there are very many, but are they safe for long term use?  Most fail this test.

For example, ethanol will down-regulate α5 expression and it appears possible that the right benzodiazepine at the right dose might also achieve this, but much mouse research is contradictory.  We are very lucky that the clonazepam dose is so tiny, it appears to have no side effects whatsoever.  At conventional doses, benzodiazepines do have problems.

Initially I looked at research that sets out very generally to look at what I am interested in.  This yielded some interesting studies, some of which we looked at already when looking at Professor Catterall and Clonazepam.

Then I decided to look at very specific things I want to modulate and I found a great deal more.  It looks like we will be able to borrow a drug being developed for Down Syndrome (Basmisanil / RG-1662).

Numerous substances do affect GABAA receptor subunit expression, but the ones available today are generally non-specific.  They will change things, but will it be for the better?

So there are two approaches:-


·        Roll the dice

Some simple substances are known to affect GABAA receptor subunit expression and some of these substances have already been associated with autism.  It is conceivable that a lit bit, more or less, of one of this might just stir things up so that the end result might be better.

·        Clever approach

The clever approach would make sure changes only affect the specific subunits that need to be modulated.  The chance of success is then very high.  The problem is that this requires waiting for a new drug, currently in phase 2 trials, to complete its approval process 


Rolling the Dice

In the following papers there numerous ideas and some of these that have already appeared in previous posts.  Those ideas include:-

·        Calcium channel blockers, like Nifedipine

They may increase GABA receptor density.

·        Zinc

We saw in research from Taiwan, that it appears there is a problem with zinc in autism and schizophrenia.  It is not a lack of zinc, rather it is in the “wrong” place.  They have a drug, Clioquinol, that can move it to the “right” place, but this drug is not regarded as safe in many countries.

Altered Homeostasis in Autism: Cl-, K+, Ca2+, and quite possibly Zn2+


It claimed that immature neurons are more sensitive to zinc than adult neurons.  Even in adults, autistic neurons remain immature.


·        BDNF

We have already seen that growth factors, including BDNF are disrupted in autism.  Some people with autism have too much BDNF and some too little.
BNDF seems to affect GABA receptor density.


·        Progesterone/Pregnenolone

We know that transdermal Progesterone/Pregnenolone helps many people with autism + anxiety.  We know, from Hardan at Stanford, that high dose of oral Pregnenolone seem to help adults with autism or schizophrenia.

We saw in earlier posts that allopregnanolone possesses biphasic, U-shaped actions at the GABAA receptor, meaning that a tiny dose can have the same effect as a large dose. Giving large doses of a female hormone to young boys does not seem a clever idea, presumably this is why the Stanford trial was on adults.

I did previously suggest small doses of oral Pregnenolone might be worthy of a clinical trial. 

Progesterone/Pregnenolone will change GABAA sub unit expression.


·        We have another mushroom-derived substance called muscimol

“The GABAAergic agonist muscimol increases KCC2 mRNA in male neurons, via activation of voltage sensitive calcium channels and calcium signaling [79, 80]; in contrast, muscimol decreases KCC2 mRNA in female SNR neurons”


So mushrooms just for the boys.

Muscimol is a selective agonist for the GABAA receptors that seems to do some clever things.


For those of you who like “natural” substance, here is a paper for you:-



It does actually mention something very interesting.  We know that ethanol would down regulate α5, but clearly you cannot “treat” a child with alcohol.  It appears that the fragrant components of whiskey, wine, sake, brandy may have the same effect as ethanol, but require only tiny concentrations.  So there may be scope for alcohol as a therapy after all.  Note that propolis is usually sold as a solution in alcohol; this widely given to children.

“Volatile components of alcoholic drinks, such as whiskey, wine, sake, brandy, and shochu potentiate GABA responses to varying degrees (Hossain et al., 2002a). Although these fragrant components are present in alcoholic drinks at low concentrations (extremely small quantities compared with ethanol), they may also modulate the mood or consciousness through the potentiation of GABAA responses after absorption into the brain, because these hydrophobic fragrant compounds are easily absorbed into the brain through the blood–brain barrier and are several thousand times as potent as ethanol in the potentiation of GABAA receptor‐mediated responses (Hossain et al., 2002a).”


The following paper is very extensive, but completely omits some extremely important possibilities I later came across.  It should be a must read for those interested in the science.

Regulation of GABAA Receptor Subunit Expression by Pharmacological Agents





F. Mechanisms Regulating GABAA Receptor Subunit Expression

The large number of GABAA receptor genes and the various types of neurons and glial cells in the brain with different patterns of subunit expression suggest a complex system regulating their transcription

Activity-dependent signaling pathways modulate the function of both transcriptional activators and repressors (West et al., 2002). Calcium is a crucial second messenger in the transduction of synaptic activity into gene expression (Carafoli et al., 2001), and it is involved in the mechanisms of GABAA receptor up- and down-regulation
 It was recently shown that the activation of protein kinase C in primary rat neocortical cultures increases transcription of α1 mRNA via phosphorylation of CREB that is bound to the GABRA1 promoter (Hu et al., 2008). In contrast, activation of protein kinase A (PKA) represses α1 mRNA transcription via inducible cAMP early repressor (ICER) that forms inactive heterodimers with CREB (Hu et al., 2008). Brain-derived neurotrophic factor (BDNF) decreases α1 transcription via activation of the Janus kinase/signal transducer and activator of transcription (STAT) pathway (Lund et al., 2008). BDNF-dependent phosphorylation of STAT3 induces the synthesis of ICER that binds with phosphorylated CREB at the GABRA1 promoter CRE site, thereby repressing transcription (Lund et al., 2008).

In cultured CGCs, BDNF induces α6 mRNA expression and enhances the expression of α1 and γ2 mRNA (Bulleit and Hsieh, 2000). These enhancements are mediated via mitogen-activated protein kinase pathway (Bulleit and Hsieh, 2000). In contrast, in cultured hippocampal pyramidal cells, BDNF reduced cell surface expression of α2, β2/3, and γ2 subunits (Brünig et al., 2001). The results suggest that BDNF affects GABAAreceptor expression in a brain region- and cell-specific manner.


Regulation of GABAA Receptor Expression By Pharmacological Agents

A. Benzodiazepines


Read the full paper!


B. Neurosteroids

5. α5 Subunit.

The expression of α5 mRNA was down-regulated by CE in the cerebral cortex (Mhatre and Ticku, 1992), whereas no effect was found by Devaud et al. (1995)(Table 14). CE did not affect cerebral cortical α5 polypeptide expression (Charlton et al., 1997). Withdrawal from CE down-regulated cortical α5 mRNA expression (Mhatre and Ticku, 1992). CE down-regulated α5 polypeptide expression in the cerebellum (Charlton et al., 1997). In the hippocampus, CE up-regulated α5 mRNA expression, although it had no effect on α5 polypeptide expression (Charlton et al., 1997). Withdrawal from CE did not affect hippocampal α5 mRNA expression (Mahmoudi et al., 1997; Petrie et al., 2001). Long-term ethanol treatment or withdrawal from it did not affect α5 mRNA expression in cultured rat hippocampal neurons (Sanna et al., 2003). The results of studies on the CE effect on α5 subunit suggest brain region-specific modulation of the expression.

Here we look at the down/up regulation of the number of receptors, rather than their substructure.  This was shown to vary in nice chart with green spots earlier in this post. In other words this about modulating receptor density.





Changes in GABA receptor (GABAAR) gene expression are detected in animal models of epilepsy, anxiety and in post-mortem schizophrenic brain, suggesting a role for GABAAR regulation in neurological disorders. Persistent (48 h) exposure of brain neurons in culture to GABA results in down-regulation of GABAAR number and uncoupling of GABA and benzodiazepine (BZD) binding sites. Given the central role of GABAARs in fast inhibitory synaptic transmission, GABAAR down-regulation and uncoupling are potentially important mechanisms of regulating neuronal excitability, yet the molecular mechanisms remain unknown. In this report we show that treatment of brain neurons in culture with tetrodotoxin, glutamate receptor antagonists, or depolarization with 25 mm K+ fails to alter GABAAR number or coupling. Changes in neuronal activity or membrane potential are therefore not sufficient to induce either GABAAR down-regulation or uncoupling. Nifedipine, a voltage-gated Ca2+ channel (VGCC) blocker, inhibits both GABA-induced increases in [Ca2+]i and GABAAR down-regulation, suggesting that VGCC activation is required for GABAAR down-regulation. Depolarization with 25 mm K+ produces a sustained increase in intracellular [Ca2+] without causing GABAAR down-regulation, suggesting that activation of VGCCs is not sufficient to produce GABAAR down-regulation. In contrast to GABAAR down-regulation, nifedipine and 25 mm K+ fail to inhibit GABA-induced uncoupling, demonstrating that GABA-induced GABAAR down-regulation and uncoupling are mediated by independent molecular events. Therefore, GABAAR activation initiates at least two distinct signal transduction pathways, one of which involves elevation of intracellular [Ca2+] through VGCCs.

Given that calcium is a ubiquitous signaling molecule, it seems reasonable that increased Ca2+ alone is not sufficient to mediate the effects of signal transduction pathways initiated by activation of a specific receptor. Studies of hippocampal neurons demonstrate that increases in [Ca2+]i by NMDA receptors or VGCCs initiate distinct signal transduction pathways (Bading et al. 1993; Xia et al. 1996). The route of Ca2+influx appears to influence which signal transduction pathway is stimulated. Compartmentalization of molecules involved in second messenger pathways may also account for the observation that Ca2+ influx in dendrites initiates signal transduction cascades distinct from those triggered in the soma (Ghosh and Greenberg 1995). Recent evidence shows that increases in [Ca2+]i initiate different signaling mechanisms depending on whether the Ca2+ increase occurs in the cytoplasm or in the nucleus (Hardingham et al. 1997).
Our results demonstrate that GABA-induced GABAAR down-regulation and uncoupling are mediated by independent molecular events, indicating that GABAAR activation leads to initiation of at least two distinct signal transduction pathways. We present evidence that VGCC activation is involved in GABA-induced GABAAR down-regulation. Understanding molecular mechanisms of GABAAR down-regulation will clarify the role of GABA-induced changes in gene expression in both normal nervous system function and in neurological disease.


This is another paper on epilepsy.  It is a very good one again talks about zinc.  It also talks about using AE3 as well as NKCC1 for therapeutic intervention.

GABAA Receptors in Normal Development and Seizures: Friends or Foes?







Developmental Changes in GABAA Receptor Structure and Pharmacology

Most studies describing developmental changes in GABAAergic signaling have been done in rats. To better understand how might these reflect changes in humans, it is generally thought that brain development in a postnatal day 8-10 (PN8-10) rat is almost equivalent to a newborn human baby. The infantile stage in rats spans from PN7-21 and is followed by the juvenile stage. Puberty onset in rats occurs at approximately P32-37, whereas adulthood is reached at 2 months [230, 342, 343]. GABA is present in the embryonic neural system from the very early days [105, 162]. In the embryonic rat neocortex, GABA is detected diffusely as early as embryonic day 10 (E10) but after E14 its presence is limited to the subplate, cortical plate, marginal and intermediate zones [105]. In parallel, GABAA receptors are expressed, even before the establishment of GABAergic synapses, to permit the autocrine and paracrine actions of GABA on brain development [164,183, 278]. Regional differences in subunit expression have been reported in rats, with α4, β1, γ1 detected in the premigratory neuroblasts of the ventricular zone [164, 183] and α2, α3, β3, γ2 at the cortical or subcortical plate [164, 183, 190]. The spatiotemporal developmental patterns of GABA / GABAA receptor expression are thought to be important in the orchestration of the normal GABA-related regulation of proliferation and migration or neural and glial progenitors [105]. The high levels of GABA in the early stages of development promote the proliferation of ventricular zone progenitors [105], whereas the subsequent decline and restriction of GABAAergic influence within the outer neocortical layers inhibits proliferation [8,105, 177], enhances migration [20], and may therefore permit further neuronal differentiation. GABAAergic signaling is also important for neuronal survival at this stage [128]. In further support of the importance of GABAAergic signaling for brain development, in utero exposure to GABAA receptor inhibitors decreases the number of parvalbumin-immuno-reactive GABAergic neurons in the striatum, by impairing the survival or differentiation of these neurons [182]. Moreover, focal application of GABAAergic agonists in the cortex of newborn rats may induce abnormal migration and heterotopias [107].
Age-related, species, and region-specific changes, gradual or transient, continue through postnatal development, adulthood and ageing for GABAA receptor subunits like α1, α2, α3, α4, α5, γ1, γ2 [138, 171,214, 255, 260, 340]. Fritschy et al. have proposed that during the early postnatal life, a gradual parallel decrease in α2 / α3 and increase in α1 expression occurs in rat brain [74, 120] (Fig. 11). Similar developmental switch from α2 / α3 to α1 subunit predominance has been observed in mouse superior colliculus [111] and visual cortex [37, 109]. Functionally, the postnatal increase in α1 has been linked to increased sensitivity to neurosteroids [214], zolpidem [111] and benzodiazepines [140], and acquisition of mature type postsynaptic IPSCs with shorter duration [29]. The latter may be important for a brain that learns to respond appropriately to novel patterns of neuronal activation. Using α1 knockout mice, Bosman et al. have elegantly shown that lack of α1 subunits leads to preservation of juvenile, long duration IPSCs and impairs spatiotemporal excitation patterns to local high frequency stimulation in the visual cortex [28, 29]. In the dentate granule cells of the rat hippocampus, the developmental switch from α5 to α1, α4, and γ2 subunits correlates with decreasing sensitivity to zinc and increase in the affinity for benzodiazepines [34, 140]. Sensitivity to zinc is important in the functional regulation of GABAAergic transmission, particularly in immature neurons. Large amounts of zinc can be stored in synaptic vesicles of nerve terminals, as in the hippocampal mossy fibers of the immature hippocampus. Stimulation-dependent zinc release in this system may therefore be useful to keep under control the excessive depolarizing effects of GABA, in a subunit-specific pattern [16, 53, 166, 285,331]. This may be less important in adult neurons, which lose their sensitivity to zinc, as GABAA receptor mediated inhibition is more efficient.
There is though regional specificity of the evolution of these changes [56]. Sex differences in GABAAreceptor subunit expression further increase the diversity. These include increased expression of α1 subunit in the female substantia nigra of infantile and juvenile rats [255] and increased γ1 expression in the male rat juvenile medial preoptic area [219]. At the cellular level, GABAA receptor trafficking also evolves. Early in development and before synaptic integration occurs, receptor complexes can be diffusely expressed at the cell membrane and can be tonically activated in the presence of GABA [61, 172, 177, 236, 311]. As the establishment and differentiation of GABAergic synapses begins, they initially occupy both extrasynaptic and synaptic sites; finally targeting and clustering at synaptic sites and dendritic processes increases with maturation and spontaneous IPSCs can be detected [1, 236, 253].
The temporal, regional, sex, and species specific variability in the expression of these subunits in the brain emphasizes that generalization across brain regions, species, genders, and ages is not possible, but one needs to specifically study each structure, age, and condition independently. To further complicate these studies, handling, caloric restriction, and even swim stress regulate GABAA receptor subunit expression, at times with a lasting effect, suggesting that epigenetic influences may be as important in shaping the GABAAreceptor related differentiation and communication patterns [122,170, 202, 238].

Repeated neonatal handling with maternal separation permanently alters hippocampal GABAA receptors and behavioral stress responses



Differential regulation of KCC2 in neurons with depolarizing or hyperpolarizing GABAAergic signaling.GABAA receptor activation and BDNF increase KCC2 in immature neurons with depolarizing GABAA ergic responses, but decrease it in neurons with hyperpolarizing ...


In addition, the intracellular concentrations of Cl-and HCO3- are regulated by anion exchangers (AE). The sodium independent electroneutral AEs exchange HCO3- for extracellular Cl-, lowering intracellular pH and increasing Cl- [112, 300, 336]. Sodium Dependent Anion (Cl- / HCO3-) Exchangers (NDAE), also called sodium-dependent Cl-/HCO3- exchangers (NDCBE or NCBE) function in the opposite direction increasing intracellular pH and lowering intracellular Cl-[87, 92, 151, 287, 288, 315, 321]. The expression of NCBE precedes KCC2 in the embryonic mouse brain and, unlike KCC2, NCBE is expressed in the peripheral nervous system and epithelial non-neuronal tissues [125].

The GABAAergic agonist muscimol increases KCC2 mRNA in male neurons, via activation of voltage sensitive calcium channels and calcium signaling [79, 80]; in contrast, muscimol decreases KCC2 mRNA in female SNR neurons with hyperpolarizing GABAAergic responses [79]. These indicate that the maturational state of a neuron, as it relates to the mode of GABAAergic signaling, is critical in defining its reaction to stimuli that tend to disturb its GABA-related developmental pathway. 

Role of sex hormones in the sexually dimorphic expression of KCC2 in rat substantia nigra.


KCC2 is a neuronal-specific potassium chloride cotransporter. The level of KCC2 expression is a factor determining whether GABA(A) receptor agonists depolarize or hyperpolarize neurons. Substantia nigra reticulata (SNR) neurons of male postnatal day 15 (PN15) rats have low KCC2 mRNA expression and respond to GABA(A) receptor activation with depolarization and activation of calcium-regulated gene expression. Female PN15 SNR neurons have high KCC2 mRNA expression and GABA(A) receptor agonists cannot activate calcium-dependent signaling processes. We investigate whether sex hormones regulate KCC2 mRNA expression in PN15 rat SNR. Using in situ hybridization, we studied the effects of acute (4 h) or prolonged (52 h) subcutaneous (s.c.) administration of testosterone (100 microg), dihydrotestosterone (180 microg) or 17beta-estradiol benzoate (5 microg) on KCC2 mRNA expression in male and female PN15 rat SNR. Different doses of estradiol (1 and 10 microg s.c., 4 h) were also acutely administered in female PN15 rats. Controls received oil injections. Separate groups of PN15 male rats were pretreated with antagonists of L-type voltage-sensitive calcium channels (L-VSCCs) [nifedipine, 100 mg/kg s.c.] or GABA(A) receptors [bicuculline, 2 mg/kg intraperitoneally (i.p.)] or their vehicles, 30 min before estradiol (5 microg s.c., 4 h). Testosterone and dihydrotestosterone upregulated KCC2 mRNA in both sexes. Estradiol downregulated KCC2 mRNA in males but not in females. Both acute and prolonged hormonal administration had similar effects. In male PN15 SNR, nifedipine and bicuculline decreased KCC2 mRNA acutely and prevented further downregulation of KCC2 mRNA by estradiol. Estradiol therefore downregulates KCC2 mRNA in male PN15 SNR, by interacting with the GABA(A) receptor and L-VSCC signaling pathway.

Stimulation of prolactin and growth hormone secretion by muscimol, a gamma-aminobutyric acid agonist.





Conclusion


As we have seen, the normal function of GABAA receptor-mediated inhibition is governed by several factors, including subunit composition and density of the receptors and in by the appropriate ionic gradient of chloride (Cl-) and finally the release of GABA.

From a therapeutic perspective, the options are numerous and include:-


Modify the Chloride gradient

·        Reduce NKCC1 expression and increase KCC2, thereby making mature neurons

·        Block NKCC1 using bumetanide

·        Use a KCC2 agonist to stimulate KCC2

There are no KCC2 agonists currently available, but they are being developed for the treatment of neuropathic pain.



“KCC2 represents a fresh avenue to pain medication because stimulating KCC2 normalizes endogenous pain inhibition,” said De Koninck. “In normal neurons, Cl– levels are kept very low. Therefore, the effect of KCC2 enhancers will mainly touch on troubled neurons with elevated Cl– levels.”

Besides neuropathic pain, other neurological disorders with imbalances in Cl– homeostasis, like epilepsy, migraine or anxiety, could benefit from KCC2 stimulation. De Koninck plans to study KCC2-targeted compounds in an epilepsy model in which epilepsy-related neurological changes develop before they eventually trigger epileptic episodes. The model will provide insights on the effects of KCC2 agonists on seizure-evoking hyperexcited neurons and on changes in neuronal networks.


Recall the link between fibromyalia and autism?  I suggested this is what happened to females who nearly had autism.  Genetically down regulated KCC2 would be the link.

·        Modulate AE3 or NDAE to extrude  Cl 

This is possible using carbonic anhydrase inhibitors, such as Methazolamide and Acetazolamide  (Diamox)



Modulate subunit structure

·        Change the physical sub structure of GABA receptors to increase expression of α3 and perhaps α2

·        Upregulate the existing α3 receptors using a PAM (positive allosteric modulator) such as low dose clonazepam

·        Perhaps down regulate the overexpressed α5 receptors using a negative allosteric modulator

This might sound rather farfetched, but a chance would have it there are studies that show by down regulating α5 receptors you do indeed improve cognitive function.  We need a sub unit selective inverse agonist  or a Negative Allosteric Modulator.

Perhaps more interesting is that researchers trying to reverse cognitive deficit in Down Syndrome have already focused on down regulating α5 receptors.  They even have drugs in the approval pipeline.

But Down Syndrome is not autism, you are thinking.  But recall that in mouse models, bumetanide reverses cognitive dysfunction in Down Syndrome.

So perhaps the GABA switch is key to Down Syndrome, as well as Autism, Schizophrenia?

So keep your eyes out for news from Hoffmann-La Roche regarding RG1662 / Basmisanil currently in Phase 2 trials for Down Syndrome.


Allosteric Modulation of GABAA Receptor Subtypes: Effects on Visual Recognition and Visuospatial Working Memory in Rhesus Monkeys



 in mice and rats, investigational drugs that are negative allosteric modulators (NAMs) at α5GABAARs improved performance 

L-655,708 enhances cognition in rats but is not proconvulsant at a dose selective for alpha5-containing GABAA receptors.

http://www.ncbi.nlm.nih.gov/pubmed/17046030?dopt=Abstract&holding=npg
The in vitro and in vivo properties of L-655,708, a compound with higher affinity for GABA(A) receptors containing an alpha5 compared to an alpha1, alpha2 or alpha3 subunit have been examined further. This compound has weak partial inverse agonist efficacy at each of the four subtypes but, and consistent with the binding data, has higher functional affinity for the alpha5 subtype.
These data further support the potential of alpha5-containing GABA(A) receptors as a target for novel cognition enhancing drugs.



Another α5 NAM is MRK-016, it also demonstrates nootropic effects, but may be developed as an antidepressant.



Inverse agonists of GABAA α5

·         α5IA
·         Basmisanil (RG-1662, RO5186582): derivative of Ro4938581, negative allosteric modulator at GABAA α5, in human trials for treating cognitive deficit in Down syndrome.[3]
·         L-655,708
·         MRK-016
·         PWZ-029: moderate inverse agonist[4]
·         Pyridazines[5]
·         Ro4938581[6]
·         TB-21007[7][8]





Final thoughts


There really is a lot to digest in this post.  I really did not know where to stop; it could have just kept going. 

For example, the amount of GABA itself should start out high in very early life and then should rapidly fall; this may also have been disrupted in autism. This process appears linked to certain growth factors (bFGF/ FGF2) and perhaps physical growth itself, both of which we know are disrupted in some autism. Recently FGF2 was found to be an endogenous inhibitor of anxiety.  Anxiety is mediated through GABA subunit expression.  It turns out some Mexican doctor is injecting FGF2 into kids with autism.  Other recent research shows that FGF2 can promote remyelination.  I think that demyelination is a likely shared feature of severe autism and mitochondrial disease.  There will be a post on demyelination/ remyelination.

 FGF2 and FGFR1 signaling regulate functional recovery following cuprizone demyelination