Drinking Baking Soda for Vagal Nerve Stimulation?

 The easy to read article: -

Drinking baking soda could be an inexpensive, safe way to combat autoimmune disease

 The original paper:-

Oral NaHCO3 Activates a Splenic Anti-Inflammatory Pathway: Evidence That Cholinergic Signals Are Transmitted via Mesothelial Cells

There are several posts in this blog about Vagal Nerve Stimulation, which may look like science fiction, but does have potent anti-inflammatory effects.  What if you could achieve some of those benefits in a much simpler, non-invasive way? And all in the name of actual science.

Many people currently take baking soda (sodium bicarbonate) for all kinds of different reasons. It is the bicarbonate (HCO3) that is the interesting part.

Bicarbonate is alkaline and it plays a key role in how the body regulates pH; above 7 is alkaline and below 7 is acidic. The other important factors are carbonic acid, carbon dioxide and water. The body is constantly having to maintain its pH in a narrow range. Sometimes this is not possible, as we saw in the case of long distance runners, the mitochondria in their muscles run out of enough oxygen and then lactic acidosis occurs. This drop in pH causes some side effects which in theory can be reduced if you increase bicarbonate in your bloodstream by consuming baking soda.

Many products for heartburn and indigestion contain baking soda to provide a short-term reduction in acidity. Some people just mix regular baking soda with a glass of water.

Many people seem to use baking soda to treat gout, which is caused by high levels of uric acid, but many do seem to worry about elevating their blood pressure. This is because of the sodium in baking soda.

Some people take baking soda long term to improve their sleep. This actually appears to be a DAN therapy.

Some people with autism are taking baking soda for all kinds of reasons other than poor sleep, including for allergy.

Kidney Disease and Baking Soda

I was surprised to see that baking soda has been shown in numerous studies to be beneficial for those with kidney disease; until very recently nobody really knew why it helps. Baking soda will reduce the pH of urine and some bicarbonate supplements even include pH measuring strips.

A recent study set out to investigate why baking soda has this positive effect and it came up with some very interesting conclusions. The bicarbonate is producing an anti-inflammatory effect very similar to that produced by vagal nerve stimulation (VNS). As we have seen in previous posts, by stimulating the vagus nerve you activate the cholinergic anti-inflammatory pathway. The problem with VNS is that you need a device connected to your vagus nerve to deliver electrical pulses to it. This exists today and special versions are being developed to treat arthritis. Half a teaspoon of baking soda in a glass of water is a much simpler therapy.

“We speculate that the anti-inflammatory effects of oral NaHCO3 ingestion are mediated by activation of the cholinergic ant-inflammatory pathway. The cholinergic anti-inflammatory pathway has been reported to be the efferent arm of the anti-inflammatory reflex, which acts via vagal efferents to promote M2 macrophage polarization in the spleen and limit activation of the innate immune system, thereby preventing damage caused by excessive cytokine production. Inflammatory macrophages and excessive TNF-a production have been implicated in the pathology of a broad range of disease states, including rheumatoid arthritis, cardiovascular disease, atherosclerosis, irritable bowel disease, type 2 diabetes, and neurodegenerative diseases as well as others. Conversely, FOXP3+ Tregs have been shown to be beneficial in a wide range of pathologies. FOXP3+ Tregs act to suppress activation of the immune system and induce immune tolerance. Evidence suggests that expansion of Tregs may be beneficial in a wide variety of disease states that involve pathological activation of the immune system, including allergy, asthma, multiple sclerosis (29), graft versus host disease, diabetes, and hypertension as well as many others. Given its therapeutic potential against inflammatory disease, there is currently much interest in methods to activate the cholinergic anti-inflammatory pathway.”

Macrophage polarization

This section is a cut and paste from the site below:

https://www.drthrasher.org/macrophages

Chronic inflammation is currently linked to a variety of diseases. The disease processes include the central nervous system through Rheumatoid Arthritis. The macrophages of the brain (microglia) and the peripheral innate immune system become chronically activated and release inflammatory cytokines. These cytokines cause tissue damage and cell death.

Macrophages function as control switches of the immune system, providing a balance between pro- and anti-inflammatory responses. To accomplish this, they develop into different subsets: classically (M1) or alternatively (M2) activated macrophages.

M1 macrophages display a cytotoxic, proinflammatory phenotype, much like the soldiers of The Dark Side of The Force in the Star Wars movies. M2 macrophages, like Jedi fighters, suppress immune and inflammatory responses and participate in wound repair and angiogenesis.

Critical to the actions of these divergent or polarized macrophage subpopulations is the regulated release of inflammatory mediators. When properly controlled, M1 macrophages effectively destroy invading pathogens, tumor cells and foreign materials. However, when M1 activation becomes excessive or uncontrolled, these cells can succumb to The Dark Side, releasing copious amounts of cytotoxic mediators that contribute to disease pathogenesis.

The activity of M1 macrophages is countered by The Force of alternatively activated M2 macrophages, which release anti-inflammatory cytokines, growth factors and mediators involved in extracellular matrix turnover and tissue repair.

It is the balance in the production of mediators by these two macrophage subpopulations that ultimately determines the outcome of the tissue response to chemical toxicants. 

Macrophages and Inflammatory Mediators in Chemical Toxicity: A Battle of Forces

 Baking Soda and Macrophage Polarization 

The recent research showed that oral bicarbonate reduced M1 macrophages and increased M2 macrophages, in a dose dependent fashion; so shifting away from the Dark Side towards the Jedi Order.

The above is for rats, but he same very likely applies to humans. 

So is baking soda a panacea for auto-immune disease?

The big drawback of baking soda is that very often causes irritation to your digestion, but this should also apply to those indigestion tablets containing baking soda.

These tablets do not just contain sodium bicarbonate, they often contain potassium bicarbonate. It has been reported that the effect of drinking sodium bicarbonate will affect your blood electrolytes as follows

·        Raise sodium (and hence potentially blood pressure)

·        Raise calcium

·        Lower potassium

·        Raise bicarbonate

·        Lower chloride

In another study below in the use of baking soda in humans (table III in the full paper) the level of potassium fell 10%, from 4.3 to 3.9 mmol/l. Sodium did not change much at all.

So adding potassium bicarbonate is quite clever. It will naturally increase potassium but it has a negative effect on sodium.

Some DAN-type doctors use Alka Seltzer Gold, which contains

Anhydrous citric acid 1000 mg
Potassium bicarbonate 344 mg
Sodium bicarbonate  1050 mg

Here is what Dr Sidney Baker writes on ARI’s website 

“A quarter of an Alka Seltzer Gold tablet for a toddler or two tablets for an adult, dissolved in a glass of water, is safe when given once or twice in a day to see its effect. In the context of sleep problems its first use is just to see if it does work. If it does—in, say, less than 35 minutes—then you’ve learned a lot and done some good. What you have learned is that there was something that didn’t agree with the person to whom it was administered. The good you have done is to find a temporary solution to the problem and take steps based on what you have learned.” 

Potassium bicarbonate is an approved food additive often used in making wine.  Club soda usually contains potassium bicarbonate. In the European Union, it is identified by the E number E501.

Some people with high blood pressure self-treat with potassium bicarbonate.

You will struggle to find Alka Seltzer Gold outside North America, but you can easily make your own.

It looks like the researchers at Augusta University have put some science behind Dr Baker’s therapy. If an adult keeps taking two of these Aka Seltzer Gold tablets a day, he will tamp down his immune system. If he has any kind of autoimmune condition, this would appear as an improvement in the symptoms he was accustomed to.

Most people with autism do seem to have auto-immune comorbidities of one kind or another which would be expected to make their autism symptoms worse.

So it is pretty clear what one of those North American autism researchers needs to do. Find the bicarbonate product that causes the least GI problems and test it on people with autism and an auto-immune comorbidity (asthma, allergy, irritable bowel syndrome etc). The results would be interesting. 

If you read the full text of the paper you will see that the researcher’s still do not fully understand what is going on.

It is currently fashionable to talk about alkalinity and how it is good for you, but there is much more to it than this idea. Baking soda does reduce acidity, but so do drugs called Proton Pump Inhibitors (e.g. Nexium) and H2 antihistamines (Zantac), these drugs do not activate the cholinergic ant-inflammatory pathway. Worse still the researchers showed that by taking a Proton Pump Inhibitor (e.g. esomeprazole, below), you blocked the clever anti-inflammatory effect of baking soda. You need acid (H+) to be present.

In the chart below we want as much M2 as possible and as little M1. This is only achieved by bicarbonate alone.

So, people with IBS will not benefit from this bicarbonate effect unless they stop taking their Nexium/Zantac/ …prazole.

Conclusion

A combination of sodium bicarbonate (baking soda) and potassium bicarbonate (a food additive) equal to about 2g dissolved in a water bottle and drunk either at once, or throughout the day, would be a good trial for those autism researchers to think about. They would have to make sure no drugs were being used to inhibit the production of gastric acids, so no H2 antihistamines or more modern PPIs allowed; they will stop the anti-inflammatory effect.

It also looks like marathon runners might benefit from taking Alka Seltzer Gold, unless it counts as a banned substance.

Drinking your baking soda slowly apparently reduces the incidence of GI problems. 

Long term use of Proton Pump Inhibitors, like Nexium, to lower stomach acidity, may have the unintended consequence of aggravating auto-immune disease.

=========

The original paper

Oral NaHCO3 Activates a SplenicAnti-Inflammatory Pathway: Evidence That Cholinergic Signals Are Transmitted via Mesothelial Cells

Some highlights:- 

Participants. To examine the effects of NaHCO3 on acute changes in parasympathetic activity, 12 healthy participants (six men, six women, age 27 6 2 y, body mass index [BMI] 25.3 6 1.2 kg/m2) were provided 2 g of NaHCO3 dissolved in 250 ml of bottled water (treatment [TXT] group).

An additional six participants (four men, two women, age 25 6 1, BMI 25.7 6 2.1 kg/m2) were recruited as controls and were provided 1.39 g of NaCl (equivalent molar load to 2 g of NaHCO3) dissolved in 250 ml of bottled water (CON group).

 Serum electrolytes. Blood samples were collected via an i.v. catheter (Nexiva; Becton Dickinson, Franklin Lakes, NJ) at baseline and at 60 min intervals posttreatment to examine changes in serum electrolyte balance (Na, K, and Cl2). Analytical flow cytometry (humans). In the NaHCO3 TXT group, 10 of 12 subjects had blood drawn at 3 h posttreatment. Blood was taken at all time points for all control subjects. No data were excluded from the analysis. Flow cytometric analysis of heparinized whole blood was performed as described previously (11–13). Briefly, cells were incubated with Abs for surface markers (15 min on ice in dark) before incubation with Abs against intracellular cytokines and factors (after permeabilization for 15 min using fix/Perm mixture; eBioscience), including CD11b, CD68, TNF-a (for M1 macrophages); CD11b, CD68, CD206 and IL-10 (for M2 macrophages) (purchased from BD BioSciences); and CD16 and TNF-a (for neutrophils; from eBioscience). 

To determine whether oral NaHCO3 had a similar antiinflammatory action in humans as we found in rats, we evaluated blood samples at baseline and 1, 2, and 3 h following ingestion of a single dose (2 g) of NaHCO3 (n = 11) or equimolar NaCl (n = 6), each dissolved in 250 ml of bottled water. Pre- and posttreatment values of serum electrolytes are presented in Table III. There was a significant group by time interaction for changes in serum potassium (p = 0.029, h2 P = 0.279). Specifically, serum potassium decreased with NaHCO3 treatment (p = 0.008), but there was no change with NaCl treatment (p = 0.381). BMI and C-reactive protein levels were not significantly different at baseline between either group, indicating a similar baseline inflammatory state (Table IV). No other significant differences were observed between TXT groups at baseline in any variables tested (Table IV). Baseline flow cytometry values of all subjects, before ingesting NaHCO3 or NaCl in solution, are presented in Table IV. Prior to any treatment, the percentages of blood leukocytes that were TNFa+ neutrophils, M1 macrophages, or M2 macrophages were all significantly higher in the NaHCO3 TXT group when compared with baseline values obtained in the NaCl TXT group (Table IV). There was a significant TREATMENT 3 TIME effect on both M1 macrophages (p = 0.0004) and TNF-a–positive neutrophils (p = 0.0146), with the levels of these inflammatory cells in the plasma being reduced to a significantly greater degree following ingestion of NaHCO3 when compared with NaCl (Fig. 3). The greatest decreases in blood inflammatory cells were observed at 2 and 3 h following NaHCO3 ingestion. Similar to our observations in rats, oral NaHCO3 ingestion increased the percentage of blood leukocytes identified by flow cytometry as M2 macrophages (p = 0.00165) (Fig. 3). Decreases in inflammatory TNFa+ neutrophils and M1 macrophages in the NaHCO3 TXT group did not appear to be related to the differing baseline levels observed between TXT groups. When comparing individual responses between subjects of different groups, subjects with similar baseline levels of blood leukocytes responded differently if they received NaHCO3 compared with NaCl (Supplemental Fig. 1). Splenic involvement. In the current study, we found that, prior to beginning NaHCO3 or vehicle treatment, either complete removal of the spleen or simple manipulation of the spleen to midline during sterile surgical laparotomy completely abolished the effect of NaHCO3 to promote M1 to M2 polarization in the kidney of Dahl SS rats fed an HS diet for 2 wk (Fig. 4). Furthermore, both of these maneuvers resulted in a significant decrease in renal M2 macrophages when compared with sham laparotomy only (p = 0.02 and 0.0002, comparing laparotomy only to sham splenectomy and splenectomy for vehicle- and bicarbonate-treated groups, respectively; Fig. 4). We confirmed a functional antiinflammatory response using the MLR.  

In humans, efforts to stimulate the cholinergic anti-inflammatory pathway chronically by implanting stimulating electrodes on the vagal nerves have shown promise in patients with rheumatoid arthritis

Consistent with activation of the cholinergic anti-inflammatory pathway, in rats, removal of the spleen or treatment with the a7 nicotinic Ach receptor antagonist MLA abolished the anti-inflammatory effect of oral NaHCO3 intake. Our data indicate that oral NaHCO3 loading may provide a cheap, relatively safe, effective, and easily accessible and/or non-invasive method to activate cholinergic anti-inflammatory pathways in humans, which may be of benefit to patients suffering from a multitude of inflammatory disease states. As such, our findings could potentially have significant clinical application to the treatment of human disease. Future studies testing the efficacy of oral NaHCO3 to limit injury in models of inflammatory disease will be required to determine the therapeutic potential of this stimuli. 

We speculate that the anti-inflammatory effects of oral NaHCO3 ingestion are mediated by activation of the cholinergic antiinflammatory pathway. The cholinergic anti-inflammatory pathway has been reported to be the efferent arm of the anti-inflammatory reflex (15), which acts via vagal efferents to promote M2 macrophage polarization in the spleen and limit activation of the innate immune system, thereby preventing damage caused by excessive cytokine production (4, 16). Inflammatory macrophages and excessive TNF-a production have been implicated in the pathology of a broad range of disease states, including rheumatoid arthritis (17), cardiovascular disease (18), atherosclerosis (19, 20), irritable bowel disease (21), type 2 diabetes (22), and neurodegenerative diseases as well as others (23–26). Conversely, FOXP3+ Tregs have been shown to be beneficial in a wide range of pathologies. FOXP3+ Tregs act to suppress activation of the immune system and induce immune tolerance (27). Evidence suggests that expansion of Tregs may be beneficial in a wide variety of disease states that involve pathological activation of the immune system, including allergy (28), asthma (28), multiple sclerosis (29), graft versus host disease (30), diabetes (31), and hypertension (32, 33) as well as many others. Given its therapeutic potential against inflammatory disease, there is currently much interest in methods to activate the cholinergic anti-inflammatory.

Interestingly, we found that inhibition of gastric proton pumps prevented oral NaHCO3 from activating an anti-inflammatory response, suggesting that gastric H+ secretion is required. This finding may be particularly relevant to CKD, as long-term use of proton pump inhibitors has been associated with increased risk of developing CKD (36). 

Our data indicating that oral ingestion of NaHCO3 promotes an anti-inflammatory response, which is inhibited by an antagonist of the gastric proton pump, raises the possibility that the effect of vagal stimulation or denervation to promote or inhibit the anti-inflammatory response, respectively, is secondary to the common denominator between these stimuli: the stimulation of acid secretion in the stomach (53–55). This hypothesis is consistent with findings that Ghrelin, which also stimulates acid secretion, can activate the anti-inflammatory pathway (56). Our finding that mesothelial cells are required to mediate this anti-inflammatory response provides a potential sensory mechanism for this alternative hypothesis, whereby stomach acid secretion alters some factor within the peritoneal milieu, such as pH, that is sensed by the mesothelium that lines this compartment. Such a mechanism may be of physiological importance in deciphering whether Ags absorbed by the gut are inert (coming after a meal) or represent a potential infection of the peritoneum with ensuing acid production by invading bacteria and providing the appropriate response, either tolerance or inflammatory immune response, respectively. This alternative hypothesis challenges our current understanding of how vagal nerve stimulation promotes the cholinergic anti-inflammatory response in the spleen, suggesting for the first time that there may be no direct interface between the nervous and immune systems. In light of our data, further studies are warranted to determine whether promotion of an anti-inflammatory effect following stimulation of vagal nerves (classical activation of the cholinergic anti-inflammatory response) occurs independent of a requirement to stimulate stomach acid secretion. 

In summary, we report that oral NaHCO3 activates splenic anti-inflammatory pathways in both rats and humans. Our novel finding provides a potentially practical and/or cost-effective and relatively safe method to activate splenic anti-inflammatory pathways in humans and therefore may have significant therapeutic potential for inflammatory disease. We provide both functional (flow cytometry) and anatomical and histological evidence that the signals that mediate this response are transmitted to the spleen via a novel neuronal-like function of mesothelial cells. To our knowledge, this is the first evidence that mesothelial cells may have a role in transmitting cholinergic signals to distal sites and, combined with evidence that gastric acid secretion is required to promote an anti-inflammatory response to NaHCO3, raises the possibility that there may be no direct interface between the nervous and immune systems. Future studies testing the efficacy of oral NaHCO3 to limit injury in models of inflammatory disease will be required to determine the therapeutic potential of this stimuli.

Increased blood pH but not performance with sodium bicarbonate supplementation in elite rugby union players.

PURPOSE:

This study investigated the effect of ingesting 0.3 g/kg body weight (BW) of sodium bicarbonate (NaHCO₃) on physiological responses, gastrointestinal (GI) tolerability, and sprint performance in elite rugby union players.

CONCLUSIONS: 

NaHCO₃ supplementation increased blood HCO₃⁻ concentration and attenuated the decline in blood pH compared with placebo during high-intensity exercise in well-trained rugby players but did not significantly improve exercise performance. The higher incidence and greater severity of GI symptoms after ingestion of NaHCO₃ may negatively affect physical performance, and the authors strongly recommend testing this supplement during training before use in competitive situations. 

Some ideas from Dr Baker over at ARI

https://www.autism.com/sleeplessness 

Alka Seltzer Gold and activated charcoal (in sequence—not mixed together!)
A dose of Alka Seltzer Gold followed in at least 20 minutes by a dose of activated charcoal provides information gained from seeing it work that is worth almost as much as the relief it provides. The equivalent of “Alka-Gold” comes in the form of tri-salts —sodium, magnesium, and potassium bicarbonate powder and capsules—from various nutritional supplement suppliers and compounding pharmacies. Alka Seltzer Gold (not Cold) contains only sodium and potassium bicarbonate. Not to be taken immediately after a large meal, it is safe and makes just about everything better. It is absorbed from the intestine quickly into the bloodstream and results in a slight, transient adjustment (called an alkaline tide) of the acidity that is associated with just about everything that goes wrong with us acutely and chronically when we are sick.

A quarter of an Alka Seltzer Gold tablet for a toddler or two tablets for an adult, dissolved in a glass of water, is safe when given once or twice in a day to see its effect. In the context of sleep problems its first use is just to see if it does work. If it does—in, say, less than 35 minutes—then you’ve learned a lot and done some good. What you have learned is that there was something that didn’t agree with the person to whom it was administered. The good you have done is to find a temporary solution to the problem and take steps based on what you have learned.


Unless the Alka Seltzer Gold is an instant success by itself, the next step in the sequence comes with the administration of activated charcoal. It comes as tablets (crushable) or encapsulated in doses of 100 to 560 mg. For individuals who cannot swallow capsules, the powder can be taken carefully from the capsules to avoid getting the powder on your clothing. It is, however, washable. If administered as a powder it must first be mixed in water. (Grape juice frozen concentrate— undiluted or minimally diluted—is a vehicle for children needing a strong disguise of taste and color, provided they can tolerate an exceptional bit of sugar.) A recipient who is likely to chew a capsule should be given the charcoal as a liquid suspension (water or juice) to avoid the risk of inhaling the fine black powder.

Many parents and individuals with problems discover from the use of charcoal for die-off reactions that it works—as just described—under circumstances that include just having a “bad day” or reactions to stresses such as allergenic foods, too much sugar, or alcohol, not enough sleep, or even just being hungry and irritable. Similar to Alka Seltzer Gold or its generic equivalent, activated charcoal works as a kind of panacea.

The risk that activated charcoal will absorb important nutrients is minimized by using it only for short-term diagnostic and treatment purposes and keeping it at least an hour away from foods and other medications. 

Here we have another DAN doctor using Alka Seltzer:-

https://www.ochec.com/2de646a5-4b92-4b42-becf-b2c39226e6d1

4. FOOD ALLERGIES

Inflammation is a common result of the histamine release that takes place because of food allergies. Dr. Lendon Smith says children tend to crave what they are allergic to—dairy and wheat, for example. Many parents have seen dramatic changes when they not only reduce sugar and simple carbohydrates but when they start an allergy elimination diet, beginning with dairy items (not even one teaspoonful!). In a few weeks, they may also eliminate gluten products. For encouraging parent testimonials, go to www.gfcfdiet.com and www.blockcenter.com, and read Dr. Mary Ann Block’s success with elimination diets. Histamine is such a factor in behavior that Dr. Block recommends the use of Alka-Seltzer Gold (no medicine, just sodium and potassium), to help a child calm down from a tantrum or anxiety. A histamine reaction floods acid into the system, and this product serves to neutralize that acid, calming the body. A mother named Amy recently e-mailed me and thanked me for the suggestion of Alka-Seltzer Gold for her son Michael, age 10. When a meltdown or anxiety begins to occur (after all, no one can follow a diet perfectly), she goes “plop, plop, fizz, fizz,” and finds what a relief it is—for his nervous system.

For those of you interested to see what happens to your blood/urine when you take sodium/potassium bicarbonate, there is plenty of data in the full version of the paper below 

Potassium bicarbonate, but not sodium bicarbonate, reduces urinary calcium excretion and improves calcium balance in healthy men.

Previous studies demonstrated that the administration of NaHCO3 or sodium citrate had either only a small effect to reduce urinary Ca excretion or no effect, but that potassium citrate significantly reduced urinary Ca excretion. In order to further evaluate and compare the effects of NaHCO3 and of KHCO3, we performed ten metabolic balances in healthy men during 18 control days, 12 days of NaHCO3, 60 mmol/day and 12 days of KHCO3, 60 mmol/day. Six subjects were fed a low Ca diet (5.2 +/- 0.7 SD mmol/day) and three of these were also given calcitriol (0.5 microgram 6-hourly). Four subjects ate a normal Ca diet (19.5 +/- 1.3 mmol/day). For all 10 subjects, KHCO3 administration reduced urinary Ca excretion from control by -0.9 +/- 0.7 mmol/day, P less than 0.001. Net intestinal Ca absorption did not change detectably so that Ca balances became less negative by a +0.9 +/- 0.9 mmol/day; P = 0.01. KHCO3 administration was also accompanied by more positive PO4 and Mg balances. NaHCO3 administration had no significant effect on urinary Ca excretion or Ca balance. NaHCO3 and KHCO3 administration were accompanied by equivalently more positive Na or K balances, respectively and equivalently more negative acid balances (HCO3 retention). Neither NaHCO3 or KHCO3 altered fasting serum HCO3 concentrations, blood pH, serum 1,25-(OH)2-D or PTH concentrations. We conclude that KHCO3 promotes more positive Ca balances by either enhancing renal Ca retention or skeletal Ca retention or both.

GABA A Receptors in Autism – How and Why to Modulate Them

This post will get complicated, since it will look at many aspects of the GABA _A receptor, rather than just a small fraction that usually appear in the individual pieces of the scientific literature. 

It was prompted by comments I have received from regular readers, regarding Bumetanide, Clonazepam, epilepsy and whether there might be alternatives with the same effect.  So it is really intended to answer some complex issues. 

There are some new interesting facts/observations that may be of wider interest, just skip the parts that too involved.

Regarding today’s picture, most readers of this blog are female and by the way, while the US is the most common location by far,  a surprisingly high number of page views come from France, Hong Kong, South Africa and Poland.

GABA

We have seen that GABA is one of the brain's most important neurotransmitters and we know that various forms of GABA dysfunction are associated with autism, epilepsy and indeed schizophrenia.

One recurring aspect in the research is the so-called excitatory-inhibitory balance of GABA.

The way the brain is understood to function assumes that GABA should be inhibitory and NMDA should be excitatory.

What makes GABA inhibitory is the level of the electrolyte chloride within the cells.  If the level is “wrong”, then GABA may be excitatory and the fine balance required with the NMDA receptor is lost.  The brain then cannot function as intended.

Source: Sage Therapeutics, a company that is developing new drugs that target GABA_A and NMDA receptors

To understand what is going wrong in autism and how to treat it we need to take a detailed look at the GABA_A receptor and all the anion transport mechanism associated with it.  Most research looks at either the receptor OR the transporters and exchangers.

Anion Transport Mechanisms of the GABA_A receptor

You will either need to be a doctor, scientist or very committed to keep reading here.

We know that level of chloride within the cells is critical to whether GABA behaves as excitatory or inhibitory.  This has all been established in the laboratory.

The usual target in autism is the NKCC1 transporter that lets chloride INTO cells, but as you can see in the two figures below, there are other ways to affect the concentration of chloride.  

·        The KCC2 transporter lets chloride out of the cells

·        The sodium dependent anion exchanger (NDAE) lets chloride out of the cells

·        The sodium independent anion exchanger 3 (AE3), lets chloride in.  It extrudes intracellular HCO₃^- in exchange for extracellular Cl^-.

All this does actually matter since we will be able to link it back to a known genetic dysfunction and it would suggest alternative therapeutic avenues.  We can also see how epilepsy fits into the picture.

Source: Two types of chloride transporters are required for GABA_A receptor-mediated inhibition in C. elegans

 Source:- InhibitoryRegulation of Excitatory Neurotransmission

NKCC1 in Autism

Without doubt, the transporter that controls the flow of chloride into the brain is the expert field of Ben Ari.

His recent summary paper is below:-

The GABA excitatory /inhibitory developmental sequence: a personal journey

He showed that by reducing the level of chloride in the autistic brain using the common diuretic Bumetanide, a marked improvement in many peoples’ autism could be achieved

This post is really about expanding more on what he does not tell us.

KCC2 in Autism

In typical people, very early in life the KCC2 transporter develops and as a result level of chloride falls inside the cells, since the purpose of the transporter is to extrude chloride.

It appears that in autism this mechanism has been disrupted.  The existing science can show us what has gone wrong.

The following study shows that KCC2 is itself regulated by neuroligin-2 (NL2), a cell adhesion molecule specifically localized at GABAergic synapses.

It gets more interesting because the scientists looking for genetic causes of autism have already identified the gene that encodes NL2, which they call NLGN2 (neuroligin 2) as being associated with autism and schizophrenia (adult onset autism).

GeneCards – the human gene compendium

  

An unexpected role of neuroligin-2 in regulating KCC2 and GABA functional switch

Abstract

Background

GABA_A receptors are ligand-gated Cl^- channels, and the intracellular Cl^- concentration governs whether GABA function is excitatory or inhibitory. During early brain development, GABA undergoes functional switch from excitation to inhibition: GABA depolarizes immature neurons but hyperpolarizes mature neurons due to a developmental decrease of intracellular Cl^- concentration. This GABA functional switch is mainly mediated by the up-regulation of KCC2, a potassium-chloride cotransporter that pumps Cl^- outside neurons. However, the upstream factor that regulates KCC2 expression is unclear.

Results

We report here that KCC2 is unexpectedly regulated by neuroligin-2 (NL2), a cell adhesion molecule specifically localized at GABAergic synapses. The expression of NL2 precedes that of KCC2 in early postnatal development. Upon knockdown of NL2, the expression level of KCC2 is significantly decreased, and GABA functional switch is significantly delayed during early development. Overexpression of shRNA-proof NL2 rescues both KCC2 reduction and delayed GABA functional switch induced by NL2 shRNAs. Moreover, NL2 appears to be required to maintain GABA inhibitory function even in mature neurons, because knockdown NL2 reverses GABA action to excitatory. Gramicidin-perforated patch clamp recordings confirm that NL2 directly regulates the GABA equilibrium potential. We further demonstrate that knockdown of NL2 decreases dendritic spines through down-regulating KCC2.

Conclusions

Our data suggest that in addition to its conventional role as a cell adhesion molecule to regulate GABAergic synaptogenesis, NL2 also regulates KCC2 to modulate GABA functional switch and even glutamatergic synapses. Therefore, NL2 may serve as a master regulator in balancing excitation and inhibition in the brain.

KCC2 in Peripheral nerve injury (PNI)

Autism is not the only diagnosis associated with reduced function of the KCC2 transporter; Peripheral nerve injury (PNI) is another.

In this condition researchers sought to counter the failure of KCC2 to remove chloride from within the cell by increasing the flow chloride through the Cl^-/HCO₃^- anion exchanger known as AE3.

Inhibition of carbonic anhydrase augments GABAA receptor-mediated analgesia via a spinal mechanism of action.

Abstract

Peripheral nerve injury (PNI) negatively influences spinal gamma-aminobutyric acid (GABA)ergic networks via a reduction in the neuron-specific potassium-chloride (K(+)-Cl(-)) cotransporter (KCC2). This process has been linked to the emergence of neuropathic allodynia. In vivo pharmacologic and modeling studies show that a loss of KCC2 function results in a decrease in the efficacy of GABAA-mediated spinal inhibition. One potential strategy to mitigate this effect entails inhibition of carbonic anhydrase activity to reduce HCO3(-)-dependent depolarization via GABAA receptors when KCC2 function is compromised. We have tested this hypothesis here. Our results show that, similarly to when KCC2 is pharmacologically blocked, PNI causes a loss of analgesic effect for neurosteroid GABAA allosteric modulators at maximally effective doses in naïve mice in the tail-flick test. Remarkably, inhibition of carbonic anhydrase activity with intrathecal acetazolamide rapidly restores an analgesic effect for these compounds, suggesting an important role of carbonic anhydrase activity in regulating GABAA-mediated analgesia after PNI. Moreover, spinal acetazolamide administration leads to a profound reduction in the mouse formalin pain test, indicating that spinal carbonic anhydrase inhibition produces analgesia when primary afferent activity is driven by chemical mediators. Finally, we demonstrate that systemic administration of acetazolamide to rats with PNI produces an antiallodynic effect by itself and an enhancement of the peak analgesic effect with a change in the shape of the dose-response curve of the α1-sparing benzodiazepine L-838,417. Thus, carbonic anhydrase inhibition mitigates the negative effects of loss of KCC2 function after nerve injury in multiple species and through multiple administration routes resulting in an enhancement of analgesic effects for several GABAA allosteric modulators. We suggest that carbonic anhydrase inhibitors, many of which are clinically available, might be advantageously employed for the treatment of pathologic pain states.

PERSPECTIVE:

Using behavioral pharmacology techniques, we show that spinal GABAA-mediated analgesia can be augmented, especially following nerve injury, via inhibition of carbonic anhydrases. Carbonic anhydrase inhibition alone also produces analgesia, suggesting these enzymes might be targeted for the treatment of pain

Acetazolamide and midazolam act synergistically to inhibit neuropathic pain

Treatment of neuropathic pain is a major clinical challenge that has been met with minimal success. After peripheral nerve injury, a decrease in the expression of the K–Cl cotransporter KCC2, a major neuronal Cl⁻ extruder, leads to pathologic alterations in GABA_A and glycine receptor function in the spinal cord. The down-regulation of KCC2 is expected to cause a reduction in Cl⁻ extrusion capacity in dorsal horn neurons, which, together with the depolarizing efflux of HCO−3 anions via GABA_A channels, would result in a decrease in the efficacy of GABA_A-mediated inhibition. Carbonic anhydrases (CA) facilitate intracellular HCO−3 generation and hence, we hypothesized that inhibition of CAs would enhance the efficacy of GABAergic inhibition in the context of neuropathic pain. Despite the decrease in KCC2 expression, spinal administration of benzodiazepines has been shown to be anti-allodynic in neuropathic conditions. Thus, we also hypothesized that spinal inhibition of CAs might enhance the anti-allodynic effects of spinally administered benzodiazepines. Here, we show that inhibition of spinal CA activity with acetazolamide (ACT) reduces neuropathic allodynia. Moreover, we demonstrate that spinal co-administration of ACT and midazolam (MZL) act synergistically to reduce neuropathic allodynia after peripheral nerve injury. These findings indicate that the combined use of CA inhibitors and benzodiazepines may be effective in the clinical management of neuropathic pain in humans.

In conclusion, the major finding of the present work is that ACT and MZL act synergistically to inhibit neuropathic allodynia. In light of the available in vitro data reviewed above, a parsimonious way to explain this synergism is that CA inhibition blocks an HCO−3 -dependent positive shift in the E_r of GABA and/or glycine-mediated currents and the consequent tonic excitatory drive mediated by extrasynaptic GABA_A receptors, while preserving shunting inhibition that is augmented by benzodiazepine actions at postsynaptic GABA_A receptors. Obviously, further work is needed at the in vitro level in order to directly examine the cellular and synaptic basis of the ACT-MZL synergism and clinical studies are required to determine the safety of intrathecally applied CA inhibitors in humans. Since MZL and ACT, as well as several other inhibitors of CA [37], are clinically approved, we propose that their use in combination opens up a novel approach for the treatment of chronic neuropathic pain

Midazolam and Acetazolamide

The therapeutic as well as adverse effects of midazolam are due to its effects on the GABA_A receptors; midazolam does not activate GABA_A receptors directly but, as with other benzodiazepines, it enhances the effect of the neurotransmitter GABA on the GABA_A receptors (↑ frequency of Cl− channel opening) resulting in neural inhibition. Almost all of the properties can be explained by the actions of benzodiazepines on GABA_A receptors. This results in the following pharmacological properties being produced: sedation, hypnotic, anxiolytic, anterograde amnesia, muscle relaxation and anti-convulsant.

Acetazolamide, usually sold under the trade name Diamox in some countries.  Acetazolamide is a diuretic, and it is available as a (cheap) generic drug.

It is a carbonic anhydrase inhibitor that is used for the medical treatment of glaucoma, epileptic seizure, idiopathic intracranial hypertension, altitude sickness, cystinuria, periodic paralysis, central sleep apnea, and dural ectasia.

In epilepsy, the main use of acetazolamide is in menstrual-related epilepsy and as an adjunct in refractory epilepsy.

Acetazolamide is not an immediate cure for acute mountain sickness; rather, it speeds up part of the acclimatization process which in turn helps to relieve symptoms.  I am pretty sure, many years ago, it was Diamox that I took with me when crossing the Himalayas from Nepal into Tibet.  We did not have any problems with mountain sickness.

If periodic paralysis above rings some bells it should.  Two forms already mentioned in this blog are Hypokalemic periodic paralysis and Andersen Tawil syndrome.  We even referred to a paper suggesting the use of Bumetanide.

Bumetanide prevents transient decreases in muscle force in murine hypokalemic periodic paralysis.

Acetazolamide is a carbonic anhydrase inhibitor, hence causing the accumulation of carbonic acid Carbonic anhydrase is an enzyme found in red blood cells that catalyses the following reaction:

hence lowering blood pH, by means of the following reaction that carbonic acid undergoes

Anion exchanger 3 (AE3) in autism

Anion exchange protein 3 is a membrane transport protein that in humans is encoded by the SLC4A3 gene. It exchanges chloride for bicarbonate ions.  It increases chloride concentration within the cell.  AE3 is an anion exchanger that is primarily expressed in the brain and heart

Its activity is sensitive to pH. AE3 mutations have been linked to seizures

Characterization of anEpilepsy-associated variant of the AE3 Cl-/HCO3-exchanger

Bicarbonate (HCO₃^-) transport mechanisms are the principal regulators of pH in animal cells. Such transport also plays a vital role in acid-base movements in the stomach, pancreas, intestine, kidney, reproductive organs and the central nervous system.

Two types of chloride transporters are required for GABA_A receptor-mediated inhibition in C. elegans

Abstract

Chloride influx through GABA-gated Cl⁻ channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl⁻ gradient established in part by K⁺–Cl⁻ cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na⁺-driven Cl⁻–HCO₃⁻ exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K⁺–Cl⁻ cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl⁻ extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl⁻ gradient such that Cl⁻ flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl⁻ gradient. Our results show that Na⁺-driven Cl⁻–HCO₃⁻ exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain.

NKCC1 and AE3 Appear to Accumulate Chloride in Embryonic Motoneurons

Abstract

During early development, γ-aminobutyric acid (GABA) depolarizes and excites neurons, contrary to its typical function in the mature nervous system. As a result, developing networks are hyperexcitable and experience a spontaneous network activity that is important for several aspects of development. GABA is depolarizing because chloride is accumulated beyond its passive distribution in these developing cells. Identifying all of the transporters that accumulate chloride in immature neurons has been elusive and it is unknown whether chloride levels are different at synaptic and extrasynaptic locations. We have therefore assessed intracellular chloride levels specifically at synaptic locations in embryonic motoneurons by measuring the GABAergic reversal potential (E_GABA) for GABA_A miniature postsynaptic currents. When whole cell patch solutions contained 17–52 mM chloride, we found that synaptic E_GABA was around −30 mV. Because of the low HCO₃⁻ permeability of the GABA_A receptor, this value of E_GABA corresponds to approximately 50 mM intracellular chloride. It is likely that synaptic chloride is maintained at levels higher than the patch solution by chloride accumulators. We show that the Na⁺-K⁺-2Cl⁻ cotransporter, NKCC1, is clearly involved in the accumulation of chloride in motoneurons because blocking this transporter hyperpolarized E_GABA and reduced nerve potentials evoked by local application of a GABA_A agonist. However, chloride accumulation following NKCC1 block was still clearly present. We find physiological evidence of chloride accumulation that is dependent on HCO₃⁻ and sensitive to an anion exchanger blocker. These results suggest that the anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons.

Sodium dependent anion exchanger (NDAE)

Not much has been written about these exchangers, outside of very technical literature.

Anion transport and GABA signaling

Sodium-coupled anion exchange is activated by intracellular acidification (Schwiening and Boron, 1994), suggesting that regulation of the chloride gradient by NDAEs may be closely linked to the regulation of cellular pH. As prolonged neuronal activity can cause neuronal acidification by efflux of bicarbonate through GABA_A receptors (Kaila and Voipio, 1987), sodium-coupled anion exchange may help to maintain a hyperpolarizing chloride reversal potential and thus promote the inhibitory action of GABA. Thus activation of sodium-coupled anion exchange by acidosis may also contribute to seizure termination by promoting a more negative chloride reversal potential and thus promoting the inhibitory effects of GABA.

The GABA_A receptor  (background is cut and paste from Wikipedia)

In order for GABA_A receptors to be sensitive to the action of benzodiazepines they need to contain an α and a γ subunit, between which the benzodiazepine binds. Once bound, the benzodiazepine locks the GABA_A receptor into a conformation where the neurotransmitter GABA has much higher affinity for the GABA_A receptor, increasing the frequency of opening of the associated chloride ion channel and hyperpolarizing the membrane. This potentiates the inhibitory effect of the available GABA leading to sedative and anxiolytic effects.

Structure and function

Schematic diagram of a GABA_A receptor protein ((α1)₂(β2)₂(γ2)) which illustrates the five combined subunits that form the protein, the chloride (Cl^-) ion channel pore, the two GABA active binding sites at the α1 and β2 interfaces, and the benzodiazepine (BDZ) allosteric binding site

The receptor is a pentameric transmembrane receptor that consists of five subunits arranged around a central pore. Each subunit comprises four transmembrane domains with both the N- and C-terminus located extracellularly. The receptor sits in the membrane of its neuron, usually localized at a synapse, postsynaptically. However, some isoforms may be found extrasynaptically. The ligand GABA is the endogenous compound that causes this receptor to open; once bound to GABA, the protein receptor changes conformation within the membrane, opening the pore in order to allow chloride anions (Cl⁻) to pass down an electrochemical gradient. Because the reversal potential for chloride in most neurons is close to or more negative than the resting membrane potential, activation of GABA_A receptors tends to stabilize or hyperpolarise the resting potential, and can make it more difficult for excitatory neurotransmitters to depolarize the neuron and generate an action potential. The net effect is typically inhibitory, reducing the activity of the neuron. The GABA_A channel opens quickly and thus contributes to the early part of the inhibitory post-synaptic potential (IPSP).

Subunits

GABA_A receptors are members of the large "Cys-loop" super-family of evolutionarily related and structurally similar ligand-gated ion channels that also includes nicotinic acetylcholine receptors, glycine receptors, and the 5HT₃ receptor. There are numerous subunit isoforms for the GABA_A receptor, which determine the receptor's agonist affinity, chance of opening, conductance, and other properties.
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)

There are three ρ units (GABRR1, GABRR2, GABRR3), however these do not coassemble with the classical GABA_A units listed above,^[18] but rather homooligomerize to form GABA_A-ρ receptors (formerly classified as GABA_C receptors but now this nomenclature has been deprecated^[19] ).
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 (α₂β₂γ)
The receptor binds two GABA molecules, at the interface between an α and a β subunit



The important subunits for this post are:-

GABRA2,

Very little is written about this subunit.

GABRA3

While the effect of editing on protein function is unknown, the developmental increase in editing does correspond to changes in function of the GABA_A receptor. GABA binding leads to chloride channel activation, resulting in rapid increase in concentration of the ion. Initially, the receptor is an excitatory receptor, mediating depolarisation (efflux of Cl^- ions) in immature neurons before changing to an inhibitory receptor, mediating hyperpolarization(influx of Cl^- ions) later on. GABA_A converts to an inhibitory receptor from an excitatory receptor by the upregulation of KCC2 cotransporter. This decreases the concentration of Cl^- ion within cells. Therefore, the GABA_A subunits are involved in determining the nature of the receptor in response to GABA ligand. These changes suggest that editing of the subunit is important in the developing brain by regulating the Cl^- permeability of the channel during development. The unedited receptor is activated faster and deactivates slower than the edited receptor.

Editing modifies the GABAA receptor subunit α3

Editing of the I/M site is developmentally regulated

A switch in the GABA response from excitatory to inhibitory post-synaptic potentials occurs during early development where an efflux of chloride ions takes place in immature neurons, while there is an influx of chloride ions in mature neurons (Ben-Ari 2002). GABA switches from being excitatory to inhibitory by an up-regulation of the cotransporter KCC2 that decreases the chloride concentration in the cell. However, if GABA itself promotes the expression of KCC2 is still under debate (Ganguly et al. 2001; Ludwig et al. 2003; Titz et al. 2003). Further, the α subunits are critical elements in determining the nature of the GABA_A receptor response to GABA (Böhme et al. 2004). The α3 mRNA (Gabra-3) is present at high levels in several forebrain regions at birth with a major decline after post-natal day 12 (P12), when the expression of α1 is going up (Laurie et al. 1992). The change from α3 to α1 may cause the switch in GABA behavior from excitatory to inhibitory post-synaptic potentials during development.

GABA_A receptors respond to anxiolytic drugs such as benzodiazepines and are thus important drug targets. The benzodiazepine binding site is located at the interface of the α and γ2 subunits (Cromer et al. 2002). Antagonists that bind to this site enhance the effect of GABA by increasing the frequency of GABA-induced channel opening events. Post-transcriptional modifications of the α3 subunit, such as the I/M editing described here, could be important in determining the mechanistic features that are responsible for the diversity of GABA_A receptors and the variability in sensitivity to drugs

Ligands

A number of ligands have been found to bind to various sites on the GABA_A receptor complex and modulate it besides GABA itself.

Types

• Agonists: bind to the main receptor site (the site where GABA normally binds, also referred to as the "active" or "orthosteric" site) and activate it, resulting in increased Cl⁻ conductance.
• Antagonists: bind to the main receptor site but do not activate it. Though they have no effect on their own, antagonists compete with GABA for binding and thereby inhibit its action, resulting in decreased Cl⁻ conductance.
• Positive allosteric modulators: bind to allosteric sites on the receptor complex and affect it in a positive manner, causing increased efficiency of the main site and therefore an indirect increase in Cl⁻ conductance.
• Negative allosteric modulators: bind to an allosteric site on the receptor complex and affect it in a negative manner, causing decreased efficiency of the main site and therefore an indirect decrease in Cl⁻ conductance.
• Open channel blockers: prolong ligand-receptor occupancy, activation kinetics and Cl ion flux in a subunit configuration-dependent and sensitization-state dependent manner.
• Non-competitive channel blockers: bind to or near the central pore of the receptor complex and directly block Cl^- conductance through the ion channel.

The GABA_A receptor include a site where benzodiazepine can bind.  These are drugs that include like valium. Binding at this site increase the effect of GABA.  Since this receptor is meant to be inhibitory, giving valium should make it strong inhibitory, ie calming. 

It was noted that in autism the effect of valium was often the reversed, instead of calming it further increased anxiety.

The Valium is working just fine, it is magnifying the effect the effect of GABA, the problem is that the receptor is functioning as excitatory, the Valium is making it over-excitatory.  Now we come to the reason why.

We know that the excitatory-inhibitory balance is set by the chloride concentration within the cells.  We also know that exact mechanism that determines this level.

Enhancement of Inhibitory Neurotransmission by GABAA ReceptorsHaving α2,3-Subunits Ameliorates Behavioral Deficits in a Mouse Modelof Autism

 Highlights

•BTBR mice have reduced spontaneous GABAergic inhibitory transmission

•Nonsedating doses of benzodiazepines improved autism-related deficits in BTBR mice

•Impairment of GABAergic transmission reduced social interaction in wild-type mice

•Behavioral rescue by low-dose benzodiazepine is GABA_A receptor α_2,3-subunit specific

Summary

Autism spectrum disorder (ASD) may arise from increased ratio of excitatory to inhibitory neurotransmission in the brain. Many pharmacological treatments have been tested in ASD, but only limited success has been achieved. Here we report that BTBR T⁺ Itpr3^tf/J (BTBR) mice, a model of idiopathic autism, have reduced spontaneous GABAergic neurotransmission. Treatment with low nonsedating/nonanxiolytic doses of benzodiazepines, which increase inhibitory neurotransmission through positive allosteric modulation of postsynaptic GABA_A receptors, improved deficits in social interaction, repetitive behavior, and spatial learning. Moreover, negative allosteric modulation of GABA_A receptors impaired social behavior in C57BL/6J and 129SvJ wild-type mice, suggesting that reduced inhibitory neurotransmission may contribute to social and cognitive deficits. The dramatic behavioral improvement after low-dose benzodiazepine treatment was subunit specific—the α_2,3-subunit-selective positive allosteric modulator L-838,417 was effective, but the α₁-subunit-selective drug zolpidem exacerbated social deficits. Impaired GABAergic neurotransmission may contribute to ASD, and α_2,3-subunit-selective positive GABA_A receptor modulation may be an effective treatment.

  

These results indicate that different subtypes of GABAA receptors may have opposite roles in social behavior, with activation of GABAA receptors containing α_2,3 subunits favoring and of GABAA receptors with α₁  subunits reducing social interaction, respectively.

Because of their broad availability and safety, benzodiazepines and other positive allosteric modulators of GABAA receptors administered at low nonsedating, nonanxiolytic doses that do not induce tolerance deserve consideration as a near-term strategy to improve the core social interaction deficits and repetitive behaviors in ASD.

These results are most consistent with the hypotheses that reduced inhibitory neurotransmission is sufficient to induce autistic-like behaviors in mice and that enhanced inhibitory neurotransmission can reverse autistic-like behaviors.

Epilepsy

I have received various comments about epilepsy.  Epilepsy has many variants, just like autism.  Epilepsy is often comorbid with autism.  GABA dysfunction is known to be closely involved in some types of autism and some types of epilepsy.

It is known that Bumetanide has very different effects in different types of epilepsy.

The question that naturally arises is whether you can give Bumetanide to someone who has autism and epilepsy and if you cannot, is there an alternative with the same desired effect?

Well it appears that any method that changes chloride levels is likely to affect epilepsy.  It appears that all three methods (NKCC1, KCC2 and AE3) would likely have the same impact on epilepsy.

But would it be a good effect or a bad effect?

Would it interact with any existing anti-epilepsy drugs?

I suspect that Bumetanide might be an effective anti-epileptic in people with autism and that other GABA related drugs might no longer be needed.  Quite likely the effect of Bumetanide and the anti-epileptic targeting GABA might be too much.  So the blog reader that pointed out that the bumetanide clinical trial excluded children with epilepsy has highlighted an important point.

While epilepsy is not fully understood and there are various variants, it would seem plausible that the epilepsy common in core classic autism and early regressive autism is the same type and that it is linked to the same excitatory/inhibitory dysfunction.

You may be wonder if other diuretics have anti-epileptic properties. Here is a paper by a Neurologist from Denver on the subject:-

Diuretics as anti-epileptic drugs – Should we go with the Flow

Why is there an excitatory/inhibitory dysfunction in Autism?

People are writing entire books on the GABA excitatory/inhibitory balance.  I was curious as to why this dysfunction exists at all in autism. 

We learnt from Ben-Ari in earlier posts all about this switch from excitatory to inhibitory that is supposed to occur very early on in life, we now have two reasons why this may fail to happen in autism:-

1.     Editing modifies the GABA_A receptor subunit α₃.  The change from α₃ to α₁ may cause the switch in GABA behavior from excitatory to inhibitory post-synaptic potentials during development.  This change appears not to occur in some types of autism.  We see from the Clonazepam research that α₃ and  α₁ have opposite effects in autism.   In autism, activation of GABA_A receptors containing α_2,3 subunits favours social interaction  and activation of α₁  subunits reduces social interaction.

And/Or

2.     The GABA functional switch is mainly mediated by the up-regulation of KCC2, a potassium-chloride cotransporter that pumps Cl^- outside neurons.  NL2 also regulates KCC2 to modulate GABA functional switch. Therefore, NL2 may serve as a master regulator in balancing excitation and inhibition in the brain.  The gene that encodes NL2 is called NLGN2 (neuroligin 2).  Dysfunction in gene NLGN2 is known to occur in both autism and schizophrenia (adult onset autism).

Conclusion

We came full circle back to Bumetanide and Clonazepam as most likely the safest and most effective therapy to adjust the E/I (excitatory/inhibitory) balance in autism. KCCI agonists do not seem to exist.  The bicarbonate exchanger agonist Acetazolamide/Diamox is another common diuretic and I see no reason why it would not also be effective, but we would then affect bicarbonate levels.  Since these ions play a role in controlling pH levels, I think we might risk seeing some unintended effects.  We know that Bumetanide is safe in long term use.  We know that all diuretics that change chloride level within the cell and will affect epilepsy; so it is a case of “better the devil you know”. 

I finally understood exactly why tiny dose of Clonazepam are effective and how this fits in with the changes the Bumetanide has produced.  Thankfully, such tiny doses are free of the typical side effects expected from benzodiazepines.  One tablet lasts 10 days.

It also answers somebody else’s question about starting with Clonazepam before the Bumetanide.  If you did that you might well make things much worse, you would magnify the unwanted excess brain cell firing.  Once you added bumetanide things would then reverse and brain cell firing would be inhibited.

I rather like the parallel with neuropathic pain, the other condition we looked at with reduced KCC2 transporter function, the researchers there proposed the combination of a diuretic (Acetazolamide) to lower cellular chloride (via exchanger AE3) and a benzodiazepine (Midazolam) as a positive allosteric modulator.  This is extremely similar to Ben Ari’s bumetanide (diuretic affecting transporter NKCC1) plus Catterall’s tiny doses of clonazepam (benzodiazepine) as a positive allosteric modulator.

As for epilepsy and bumetanide, we know that bumetanide has different effects on different types of autism. It seems plausible that people with autism might tend to have the same type of epilepsy.  In any case Monty, aged 11 with ASD, does not have epilepsy/seizures and I suspect taking bumetanide has decreased the chance he ever will.  Of course I cannot prove this, it is just conjecture.