Ginseng, as a GABAb Antagonist, as an "Add-on Therapy" for some Autism? Also Homotaurine and Acamprosate

Rather like negotiating with North Korea, today’s post does rather meander. It does in the end up with some interesting options for some people. 

Korea - the centre of Ginseng research

This post was prompted by research highlighted by our reader Ling, which suggested that bumetanide responders (i.e. people with high intracellular chloride) might benefit from a GABA_B antagonist. 

There has been quite a lot of coverage in this blog about agonists of GABA_B receptors, like Baclofen and Arbaclofen. Some people with an autism diagnosis do indeed seem to benefit, ranging from some with Fragile-X to others with Asperger’s. Russian-developed GABA_B agonists like Phenibut and Pantogam are widely used by adults self-treating their behavioural/emotional disturbances.

Some Aspies have commented in this blog that far from helping, Baclofen made them feel worse; perhaps the opposite therapy might help? (the Goldilocks scenario, from the previous post) 

The paper below shows how a GABA_B antagonist (the opposite of Baclofen) might benefit some with autism.

Neuronal Chloride Regulation via KCC2 Is Modulated through a GABA_B Receptor Protein Complex.

GABA_B receptors are G-protein-coupled receptors that mediate inhibitory synaptic actions through a series of downstream target proteins. It is increasingly appreciated that the GABA_B receptor forms part of larger signaling complexes, which enable the receptor to mediate multiple different effects within neurons. Here we report that GABA_B receptors can physically associate with the potassium-chloride cotransporter protein, KCC2, which sets the driving force for the chloride-permeable ionotropic GABA_A receptor in mature neurons. Using biochemical, molecular, and functional studies in rodent hippocampus, we show that activation of GABA_B receptors results in a decrease in KCC2 function, which is associated with a reduction in the protein at the cell surface. These findings reveal a novel "crosstalk" between the GABA receptor systems, which can be recruited under conditions of high GABA release and which could be important for the regulation of inhibitory synaptic transmission.

SIGNIFICANCE STATEMENT Synaptic inhibition in the brain is mediated by ionotropic GABA_A receptors (GABA_ARs) and metabotropic GABA_B receptors (GABA_BRs). To fully appreciate the function and regulation of these neurotransmitter receptors, we must understand their interactions with other proteins. We describe a novel association between the GABA_BR and the potassium-chloride cotransporter protein, KCC2. This association is significant because KCC2 sets the intracellular chloride concentration found in mature neurons and thereby establishes the driving force for the chloride-permeable GABA_AR. We demonstrate that GABA_BR activation can regulate KCC2 at the cell surface in a manner that alters intracellular chloride and the reversal potential for the GABA_AR. Our data therefore support an additional mechanism by which GABA_BRs are able to modulate fast synaptic inhibition.

In bumetanide-responsive autism, neurons remain immature because the “GABA switch“ never flipped and so NKCC1 is overexpressed and KCC2 is underexpressed, chloride levels remain high and the neurotransmitter GABA works backwards (excitatory, rather than inhibitory).

Bumetanide partially counters the over-abundance of NKCC1 transporters that carry chloride into neurons, but is a partial solution.

The above research suggests that blocking GABA_B receptors might increase the flow of chloride ions exiting through KCC2.

All very complicated sounding, but in effect it means that a GABA_B antagonist might boost the effect of bumetanide.

Which GABA_B antagonist?

This was Ling’s question.

Saclofen is a competitive antagonist for the GABA_B receptor. This drug is an analogue of the GABA_B agonist baclofen.

Phaclofen/phosphonobaclofen, is a selective antagonist for the GABA_B receptor.

Since these “–aclofens” are not accessible we are left with a choice of homotaurine (developed to treat Alzheimer’s) or Ginsenosides from Korean/Panax ginseng.

Both homotaurine and Ginsenosides have various other effects beyond GABA_B.

Since Ling is in Scandinavia, homotaurine is an option. It seems to be banned in the US, though it is approved in Canada and sold in Europe.

Ginseng is very widely used, indeed it is the most widely consumed herbal nutritional product in the world, with sales of $400 million in 2012.

I was surprised that there actually is research in both humans and animal models using Ginseng in autism and indeed ADHD.

N-Acetyl homotaurine,  a derivative of homotaurine, is a registered drug called Acamprostate. It is used to treat alcohol dependence. It affects both NMDA and GABA receptors. Acamprostate has been shown to benefit Fragile-X, as has bumetanide. A drug that affects GABA_B will inevitably also affect NMDA receptors.

This was covered in this post from 2015.

NMDAR hypo-function causing E/I imbalance in Autism and Schizophrenia – Baclofen, Sodium benzoate and Cinnamon (again)

which highlighted this paper:

GABA_B-mediated rescue of altered excitatory–inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR-hypofunction

Homotaurine 

Homotaurine has been reported as a GABA antagonist as well as a GABA agonist. In vitro studies have found that homotaurine is a GABA_A partial agonist as well as a GABA_B receptor partial agonist with low efficacy, becoming an antagonist and a displacing full agonist of GABA or baclofen at this receptor.^[15] In a study in rats, homotaurine reversed the catatonia induced by baclofen (the prototypical GABA_B agonist),^[16] and was able to produce analgesia via the GABA_B receptor, an effect that was abolished when CGP 35348, a GABA_B receptor antagonist was applied.^[17][18] 

One study suggests Homotaurine increases dopamine levels.^[19]

One study in rats showed that homotaurine suppressed ethanol-stimulated dopamine release, as well as ethanol intake and preference in rats in a way similar to the N-acetyl derivative of homotaurine, acamprosate.^[20] Acamprosate was approved by the FDA in 2004 to treat alcohol dependence.^[3]

Impact of acamprosate on behavior and brain-derived neurotrophic factor: an open-label study in youth with fragile X syndrome.

Fragile X syndrome (FXS) is an inherited form of developmental disability and a single gene cause of autism. As a disorder with increasingly understood pathophysiology, FXS is a model form of developmental disability for targeted drug development efforts. Preclinical animal model findings have focused targeted drug treatment development in FXS on an imbalance between excessive glutamate and deficient gamma-aminobutyric acid (GABA) neurotransmission.

Acamprosate was generally safe and well tolerated and was associated with a significant improvement in social behavior and a reduction in inattention/hyperactivity. The increase in BDNF that occurred with treatment may be a useful pharmacodynamic marker in future acamprosate studies. Given these findings, a double-blind, placebo-controlled study of acamprosate in youth with FXS is warranted.

Back to Ginseng

 Supplementation of Korean Red Ginseng improves behavior deviations in animal models of autism

Background

Autism spectrum disorder (ASD) is heterogeneous neurodevelopmental disorders that primarily display social and communication impairments and restricted/repetitive behaviors. ASD prevalence has increased in recent years, yet very limited therapeutic targets and treatments are available to counteract the incapacitating disorder. Korean Red Ginseng (KRG) is a popular herbal plant in South Korea known for its wide range of therapeutic effects and nutritional benefits and has recently been gaining great scientific attention, particularly for its positive effects in the central nervous system.

Objectives

Thus, in this study, we investigated the therapeutic potential of KRG in alleviating the neurobehavioral deficits found in the valproic acid (VPA)-exposed mice models of ASD.

Design

Starting at 21 days old, VPA-exposed mice were given daily oral administrations of KRG solution (100 or 200 mg/kg) until the termination of all experiments. From P28, mice behaviors were assessed in terms of social interaction capacity, locomotor activity, repetitive behaviors, short-term spatial working memory, motor coordination, and seizure susceptibility.

Results

VPA-exposed mice showed sociability and social novelty preference deficits, hyperactivity, increased repetitive behavior, impaired spatial working memory, slightly affected motor coordination, and high seizure susceptibility. Remarkably, long-term KRG treatment in both dosages normalized all the ASD-related behaviors in VPA-exposed mice, except motor coordination ability.

Conclusion

As a food and herbal supplement with various known benefits, KRG demonstrated its therapeutic potential in rescuing abnormal behaviors related to autism caused by prenatal environmental exposure to VPA.

In the trial below the dose appears very low at 250mg. In the more encouraging study in ADHD the dose was 1000mg twice a day.

First preliminary results of an observation of Panax ginseng treatment in patients with autistic disorder.

Autism is a pervasive developmental disorder, with impairments in reciprocal social interaction and verbal and nonverbal communication. There is often the need of psychopharmacological intervention in addition to psychobehavioral therapies, but benefits are limited by adverse side effects. For that reason, Panax ginseng, which is comparable with Piracetam, a substance effective in the treatment of autism, was investigated for possible improvement of autistic symptoms. There was some improvement, which suggests some benefits of Panax ginseng, at least as an add-on therapy.

Three male outpatients (age range 18.4–22.2 years; mean=21.3 years; SD =4.1 years) meeting ICD-10 criteria for autistic disorder participated in our observation. IQs ranged from 54 to 82 (68 +/− 14), which were obtained from the Wechsler Intelligence Scale. At least two child and adolescent psychiatrists independently diagnosed the subjects for autistic disorder. All subjects had no additional medical or neurological illnesses. They had been treated with either methylphenidate, or neuroleptics before entry into the study, without any positive effect (nonresponder). One patient’s language consisted of monosyllabic utterances, second patient’s language consisted of single words(10-word vocabulary),and the third patient spoke in sentences. Parents and mentors’ (i.e., the person who takes care of the patient in daily life, and supports the patient’s educational efforts) rated instruments included weekly ratings by means of the Aberrant Behavior and Symptom Checklist. Clinician ratings consisted of the Global Assessment Scale, Psychiatric Rating Scale (CPRS), and Clinical Global Improvement. Panax ginseng (oral administration of tablets containing 250-mg alcoholic Panax ginseng berry extract, pure encapsulations) was administered for 4 weeks (dosage: 250 mg daily). Patients were free of medication for at least 4 weeks before the beginning of the study. During that time, there were no changes in the symptoms of the patients. Subjects continued to receive educational and behavioral interventions, which were not altered substantially in any of the patients during their participation in the study. The means of parent and mentor ratings were averaged over the 4-week treatment period. Clinician and mentor ratings were made at the beginning of the treatment period and then weekly up to the end of the treatment. Ratings were compared by paired t-test.

RESULTS

Panax ginseng slightly improved the ratings on the ABC factors: irritability (before treatment, 13.2 +/− 5.9; after treatment, 11.3 +/− 6.2; p =.41), hyperactivity (before treatment, 20.6+/−12.4;     after,18.4+/− 9.4; p = .33), inadequate eye contact (before treatment, 8.6 +/− 5.4; after, 7.5 +/− 3.2; p .35), and inappropriate speech (before treatment, 6.1+/−2.2;after, 4.3 +/− 3.6; p = .41). The symptom checklist scores revealed a slight increase in drowsiness (before treatment, 1.6 +/− 2.2; after, 2.9+/−4.2; p =.31) and decreased activity (before treatment, 2.5 +/− 3.3; after, 4.4 +/− 3.1; p = 0.40). None of the clinician ratings showed significant improvement. This may result from different impressions of clinical visits and daily life observations of caregivers. Panax ginseng has some moderate sedative effect with effects especially on daily life, a fact that also makes it effective in the treatment of attention deficit/hyperactive disorders. None of the subjects appeared to have headaches or stomach aches, although report of such side effects was limited by the expressive language and social skills of these subjects. Medication was continued after the observation period. We did not see any significant changes in symptoms.

DISCUSSION 

Although this was a very small study (n = 3), which revealed very modest therapeutic effect of Panax ginseng in the management of autistic patients in some of the subjects (which might be due to the small sample size), it may be mentioned that its role in the management of these symptoms in patients with autistic disorder may be limited, especially because of its risk for estrogen-associated problems in females (Papapetropoulos, 07). Since there does not seem to be any significant improvement caused by Panaxginseng, its effect as an add-on therapy remains completely open and requires further investigation. Before knowing its efficacy for adults, Panax ginseng should not be recommended for treating children suffering from autism.

Ginseng for ADHD? 

Effects of Korean Red Ginseng Extract on Behavior in Children with Symptoms of Inattention and Hyperactivity/Impulsivity: A Double-Blind Randomized Placebo-Controlled Trial

Objective: There is evidence that Korean red ginseng (KRG) can reduce the production of the adrenal corticosteroids, cortisol, and dehydroepiandrosterone (DHEA), and thus may be a viable treatment for attention-deficit/hyperactivity disorder (ADHD). The present randomized double-blind placebo-controlled clinical trial tested the effect of KRG on children with ADHD symptoms.

Methods: Subjects 6–15 years, who satisfied the inclusion criteria and had ADHD symptoms, were randomized into a KRG group (n=33) or a control group (n=37). The KRG group received one pouch of KRG (1g KRG extract/pouch) twice a day, and the control group received one pouch of placebo twice a day. At the 8 week point, the primary outcomes were the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria for inattention and hyperactivity scale scores, which were measured at baseline and 8 weeks after starting treatment. Secondary outcomes were quantitative electroencephalography theta/beta ratio (QEEG TBR) (measured at baseline and week 8) and salivary cortisol and DHEA levels (measured at baseline and at 4 and 8 weeks).

Results: The baseline characteristics of the KRG and control groups were not statistically different. The mean ages of the KRG and control groups were 10.94±2.26 and 10.86±2.41, respectively. The KRG group had significantly decreased inattention/hyperactivity scores compared with the control group at week 8 (least squared means of the differences in inattention adjusted for baseline scores: −2.25 vs. −1.24, p=0.048; hyperactivity: −1.53 vs. −0.61, p=0.047). The KRG group had significantly decreased QEEG TBR compared with the control group (least squared means of the differences: −0.94 vs. −0.14, p=0.001). However, neither the KRG group nor the control group exhibited significant differences in salivary cortisol or DHEA levels at week 8 compared with the baseline levels. No serious adverse events were reported in either group.

Conclusions: These results suggest that KRG extract may be an effective and safe alternative treatment for children with inattention and hyperactivity/ impulsivity symptoms. Further studies to investigate the efficacy and safety of KRG are warranted. 

Although medications to treat psychiatric disorders for children and adolescents have been widely researched and several are on the market, natural products may also be effective in these patients while inducing fewer significant adverse effects. The present randomized controlled trial was performed to assess whether KRG, a well-known traditional medicine plant that is used particularly frequently in Eastern Asia, can improve the adrenal function and inattention/hyperactivity symptoms of chronically stressed children with ADHD symptoms. KRG extract significantly improved the inattention and hyperactivity of the subjects and had a good safety profile. However, the KRG extract did not have significant effects on cortisol or DHEA levels

Clinical Significance

To our knowledge, this is the first randomized controlled trial to investigate the efficacy and safety of Korean red ginseng extract for children with ADHD. The stimulant medications for ADHD have demonstrated not only clinical efficacy, but also significant adverse events such as poor growth, tics, and psychosis. Although KRG extract did not affect the salivary cortisol or DHEA, it significantly improved ADHD symptoms and QEEG TBR. And the safety profile of KRG extract was good. The results imply that KRG extract is a possible effective alternative medication for ADHD children.

Effect of the herbal extract combination Panax quinquefolium and Ginkgo biloba on attention-deficit hyperactivity disorder: a pilot study.

OBJECTIVE:

A combination herbal product containing American ginseng extract, Panax quinquefolium, (200 mg) and Ginkgo biloba extract (50 mg) (AD-FX; CV Technologies, Edmonton, Alta.) was tested for its ability to improve the symptoms of attention-deficit hyperactivity disorder (ADHD). 

DESIGN:

Open study. 

PATIENTS:

36 children ranging in age from 3 to 17 years who fit the diagnostic criteria for ADHD. 

INTERVENTIONS:

AD-FX capsules were taken twice a day on an empty stomach for 4 weeks. Patients were instructed not to change any other medications during the study. 

OUTCOME MEASURES:

At the beginning of the study, after 2 weeks, and then at the end of the 4-week trial, parents completed the Conners' Parent Rating Scale--revised, long version, a questionnaire that assesses a broad range of problem behaviours (and was used as an indication of ADHD symptom severity). 

RESULTS: 

After 2 weeks of treatment, the proportion of the subjects exhibiting improvement (i.e., decrease in T-score of at least 5 points) ranged from 31% for the anxious-shy attribute to 67% for the psychosomatic attribute. After 4 weeks of treatment, the proportion of subjects exhibiting improvement ranged from 44% for the social problems attribute to 74% for the Conners' ADHD index and the DSM-IV hyperactive-impulsive attribute. Five (14%) of 36 subjects reported adverse events, only 2 of which were considered related to the study medication. 

CONCLUSIONS:

These preliminary results suggest AD-FX treatment may improve symptoms of ADHD and should encourage further research on the use of ginseng and Ginkgo biloba extracts to treat ADHD symptoms.

Interactions of ginsenosides with ligand-bindings of GABA(A) and GABA(B) receptors.

Abstract

1. Total saponin fraction decreased the affinity of specific [3H]muscimol binding without changes in Bmax. Ginsenoside Rb1 Rb2, Rc, Re, Rf and Rg1 inhibited the specific [3H]muscimol binding to the high-affinity site. 2. Total saponin fraction increased the affinity of specific [3H]flunitrazepam binding. Ginsenoside Re and Rf enhanced specific [3H]flunitrazepam binding.

3. Total saponin fraction decreased the affinity of specific [35S]TBPS binding without changes in Bmax. Ginsenosides did not affect specific or non-specific [35S]TBPS binding.

4. Total saponin fraction decreased the affinity of specific [3H]baclofen binding without changes in Bmax. Ginsenoside Rc inhibited specific [3H]baclofen binding.

Emerging signals modulating potential of ginseng and its active compounds focusing on neurodegenerative diseases

very detailed paper

Also (Ling) note that there is an effect on ERbeta

A ginseng-derived oestrogen receptor beta (ERbeta) agonist, Rb1 ginsenoside, attenuates capillary morphogenesis.

 Ginseng extracts contain a variety of active ingredients and have been shown to promote or inhibit angiogenesis, depending on the presence of different ginsenosides that exert opposing effects on blood vessel growth. Leung et al. in this issue of the British Journal of Pharmacology report that Rb1, a ginsenoside that constitutes only 0.37–0.5% of ginseng extracts (depending on manufacturing and processing methods), blocks tube-like network formation by endothelial cells in vitro. At the molecular level, Rb1 binds to the oestrogen receptors and stimulates the transcription of pigment epithelium-derived factor that, in turn, inhibits matrix-driven capillary morphogenesis.

Ginseng, the root of Panax ginseng and related species, has been a key component of traditional medicine in the Far East for over a thousand years. The genus name Panax means ‘cure all' in Greek; it, thus, comes as no surprise that ginseng has been described as beneficial in many different ailments (Huang, 1999; Kiefer and Pantuso, 2003; Ng, 2006). Perhaps the most studied biological actions of ginseng extracts and constituents are those relating to its inhibitory effects on solid tumour growth (Yun, 2001). The main active ingredients in ginseng-based herbal preparations are thought to be the ginsenosides, comprising 3–6% of ginseng extracts (Huang, 1999). 

Effects and mechanisms of ginseng and ginsenosides on cognition. 

Reviewed here is the existing evidence for the effects of ginseng extracts and isolated ginsenosides relevant to cognition in humans. Clinical studies in healthy volunteers and in patients with neurological disease or deficit, evidence from preclinical models of cognition, and pharmacokinetic data are considered. Conditions under which disease modification may indirectly benefit cognition but may not translate to cognitive benefits in healthy subjects are discussed. The number of chronic studies of ginseng effects in healthy individuals is limited, and the results from acute studies are inconsistent, making overall assessment of ginseng's efficacy as a cognitive enhancer premature. However, mechanistic results are encouraging; in particular, the ginsenosides Rg3 , Rh1 , Rh2 , Rb1 , Rd, Rg2 , and Rb3 , along with the aglycones protopanaxadiol and protopanaxatriol, warrant further attention. Compound K has a promising pharmacokinetic profile and can affect neurotransmission and neuroprotection. Properly conducted trials using standardized tests in healthy individuals reflecting the target population for ginseng supplementation are required to address inconsistencies in results from acute studies. The evidence summarized here suggests ginseng has potential, but unproven, benefits on cognition.

Ginseng is the most widely consumed herbal nutritional product in the world. According to the most recent data available, ginseng had a total world export value in 2010 of over US$350 million, which was expected to rise to more than US$400 million in 2012

A survey of Korean medicine doctors’ clinical practice patterns for autism spectrum disorder: preliminary research for clinical practice guidelines

The survey had 54 respondents and 4 (8.5%) used Ginseng.

Conclusion

There is a long list of substances shown to have a benefit in some autism. Today we can add the Asian type of Ginseng and also Acamprosate (at least for Fragile-X).

It would be interesting to see the effect of Phaclofen and Saclofen which may be more selective for GABA_B receptors.

Ginseng has so many effects there is no way to know which is the one that benefited autism and ADHD in today’s highlighted posts.

We also have the problem with natural substances that there is natural variation and that supplement companies are known to cheat with ingredients. Ginseng roots are not cheap and apparently ginseng is known to get adulterated.  Drug companies are usually much more reliable.

If anyone tries out homotaurine or ginseng, let us all know the result.

Homotaurine was originally developed as an Alzheimer’s drug, but did not work well enough, its developer then tried to sell it as a supplement called Vivimind, but it was rejected by the FDA. It is sold in Canada and Europe. 

For our Aspie readers, here is a link for them:-

https://www.reddit.com/r/phenibut/comments/5ii692/homotaurine_gabab_antagonist_tolerance_reversal/

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 GABA_A-receptor subunit expression

We have already seen in this blog, that drugs that positively modulate the α₂ and α₃ subunits of GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A receptor, in other words lower density.

GABA_A receptor downregulation in brains of subjects with autism

I am suggesting that the under-expression of GABA_A 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 jargon “VGCC (Voltage Gated Calcium Channel) activation is involved in GABA-induced GABA_AR down-regulation”. Which begs the question, what is the effect on GABA_A receptor density in humans of blocking VGCCs?

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

4.     GABA_B subunits ?

There probably is a fourth element of the GABA switch.  If there is, it possibly relates to the GABA_B receptor.  GABA_B receptors are made up of just two subunits, GABA_B1 and  GABA_B2. This is much less well researched than GABA_A, but from what little there is, it is clear that GABA_B1 and GABA_B2 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)

Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury

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:-

Early Postnatal Switch in GABA_AR α Subunits in the Reticular Thalamic Nucleus

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 GABA_AR 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 GABA_ARs, 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 GABA_AR activation in nRt is age-dependent, and more prominent in immature neurons, which correlates with early expression of α5 containing GABA_ARs. 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 GABA_AR 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:-

Maturation of the GABAergic Transmission in Normal and Pathologic Motoneurons

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

Enhancement of Inhibitory Neurotransmission by GABA_A Receptors Having α_2,3-Subunits Ameliorates Behavioral Deficits in a Mouse Model of Autism

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 GABA_A 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 GABA_Areceptor 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 α₂,α₃-Specific Positive Allosteric Modulators of GABA_A 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 GABA_A receptors on social behavior in BTBR mice and C57BL/6J mice. A low dose of the α_2,3-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 GABA_A 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 GABA_A receptors may have opposite roles in social behavior, with activation of GABA_A receptors containing α_2,3 subunits favoring and activation of GABA_A receptors with α₁ subunits reducing social interaction, respectively.

Subunit-selective GABA_A 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 α_2,3-subunit-containing GABA_A 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 α_2,3-selective positive allosteric modulator of GABA_A 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 GABA_A-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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A sub unit expression.

·        We have another mushroom-derived substance called muscimol

“The GABA_Aergic 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 GABA_A receptors that seems to do some clever things.

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

Modulation of Ionotropic GABA Receptors by Natural Products of Plant Origin

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 GABA_A Receptor Subunit Expression by Pharmacological Agents

F. Mechanisms Regulating GABA_A Receptor Subunit Expression

The large number of GABA_A 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 GABA_A 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 GABA_Areceptor expression in a brain region- and cell-specific manner.

Regulation of GABA_A 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.

Distinct signal transduction pathways for GABA-induced GABA_A receptor down-regulation and uncoupling in neuronal culture: a role for voltage-gated calcium channels

Changes in GABA receptor (GABA_AR) gene expression are detected in animal models of epilepsy, anxiety and in post-mortem schizophrenic brain, suggesting a role for GABA_AR regulation in neurological disorders. Persistent (48 h) exposure of brain neurons in culture to GABA results in down-regulation of GABA_AR number and uncoupling of GABA and benzodiazepine (BZD) binding sites. Given the central role of GABA_ARs in fast inhibitory synaptic transmission, GABA_AR 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 GABA_AR number or coupling. Changes in neuronal activity or membrane potential are therefore not sufficient to induce either GABA_AR down-regulation or uncoupling. Nifedipine, a voltage-gated Ca²⁺ channel (VGCC) blocker, inhibits both GABA-induced increases in [Ca²⁺]i and GABA_AR down-regulation, suggesting that VGCC activation is required for GABA_AR down-regulation. Depolarization with 25 mm K⁺ produces a sustained increase in intracellular [Ca²⁺] without causing GABA_AR down-regulation, suggesting that activation of VGCCs is not sufficient to produce GABA_AR down-regulation. In contrast to GABA_AR down-regulation, nifedipine and 25 mm K⁺ fail to inhibit GABA-induced uncoupling, demonstrating that GABA-induced GABA_AR down-regulation and uncoupling are mediated by independent molecular events. Therefore, GABA_AR 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 GABA_AR down-regulation and uncoupling are mediated by independent molecular events, indicating that GABA_AR activation leads to initiation of at least two distinct signal transduction pathways. We present evidence that VGCC activation is involved in GABA-induced GABA_AR down-regulation. Understanding molecular mechanisms of GABA_AR 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.

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

Developmental Changes in GABA_A Receptor Structure and Pharmacology

Most studies describing developmental changes in GABA_Aergic 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, GABA_A 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 / GABA_A 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 GABA_Aergic influence within the outer neocortical layers inhibits proliferation [8,105, 177], enhances migration [20], and may therefore permit further neuronal differentiation. GABA_Aergic signaling is also important for neuronal survival at this stage [128]. In further support of the importance of GABA_Aergic signaling for brain development, in utero exposure to GABA_A 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 GABA_Aergic 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 GABA_A 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 GABA_Aergic 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 GABA_A receptor mediated inhibition is more efficient.

There is though regional specificity of the evolution of these changes [56]. Sex differences in GABA_Areceptor 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, GABA_A 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 GABA_A receptor subunit expression, at times with a lasting effect, suggesting that epigenetic influences may be as important in shaping the GABA_Areceptor related differentiation and communication patterns [122,170, 202, 238].

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

Fig. (4)

Differential regulation of KCC2 in neurons with depolarizing or hyperpolarizing GABA_Aergic signaling.GABA_A receptor activation and BDNF increase KCC2 in immature neurons with depolarizing GABA_A 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 GABA_Aergic 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 GABA_Aergic responses [79]. These indicate that the maturational state of a neuron, as it relates to the mode of GABA_Aergic 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 GABA_A 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 escape from 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 GAB_AA 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 α5GABA_ARs 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 GABA_A α₅

·         α₅IA

·         Basmisanil (RG-1662, RO5186582): derivative of Ro4938581, negative allosteric modulator at GABA_A 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]

A Study of RG1662 (Basmisanil) in Adults and Adolescents With Down Syndrome (CLEMATIS)

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