Glutamate as a mediating transmitter for auditory hallucinations in schizophrenia – an opportunity to target NO?

Glutamate as a mediating transmitter for auditory hallucinations in schizophrenia: A 1H MRS study

This is a (1)H MR spectroscopy (MRS) study of glutamate (Glu), measured as Glx, levels in temporal and frontal lobe regions in patients with schizophrenia compared with a healthy control group with the objective of revealing aspects of the underlying neurochemistry of auditory hallucinations. We further compared and correlated Glu(Glx) levels for the patients-only against frequency and severity of auditory hallucinations and the sum of Positive symptoms, and also for frequency and severity of emotional withdrawal, and sum of Negative symptoms. The sample included 23 patients with an ICD-10 and DSM-IV diagnosis of schizophrenia, and 26 healthy control subjects without any known psychiatric or neurological disorders. Symptom scores were obtained from the Positive and Negative Syndrome Scale (PANSS). (1)H MRS data were acquired on a 3T MR scanner from two temporal and two frontal voxels, using standard sequences and analysis parameters. The results showed that schizophrenia patients as a group had reduced Glu(Glx) levels in the voxels of interest compared to the healthy control subjects, while increased levels were found for patients with frequent and severe auditory hallucinations, relative to patients with less frequent and severe hallucination. We further found significant positive correlations between frequency and severity of auditory hallucinations, and for sum Positive symptoms, and Glu(Glx) levels in all regions, not seen when the analysis was done for negative symptoms. It is concluded that the results show for the first time that glutamate may be a mediating factor in auditory hallucinations in schizophrenia.

“It is therefore not unreasonable to suggest that Glu levels actually may be increased in patients with frequent and severe auditory hallucinations, despite that schizophrenia patients in general have been found to show decreased Glu levels. It would similarly not be unreasonable to suggest that such a relationship between Glu and auditory hallucinations might extend to other positive symptoms, considering that the hallucination symptom is highly correlated with other positive symptoms, but not with negative symptoms.

…[results] point towards an alternative hypothesis; that glutamatergic hyper-activity is not kept in balance by corresponding increased GABA release to inhibit excessive Glu release in frontal and temporal areas, because of a specific glutamate-GABA deficit that is underlying auditory hallucinations. Thus, auditory hallucinations may be the result not only of striatal dopamine excess at D2-receptors, as the classic model predicts, but also of glutamate over-activation in cortical regions. Such a hypothesis has the advantage of being parsimonious and closer to the neuroanatomical substrates of auditory hallucinations”

Are auditory hallucinations distinct aspects of schizophrenia that require a different treatment approach?

From: Drug development in schizophrenia: are glutamatergic targets still worth aiming at?

“…efforts to enhance interneuron firing via GABAergic agents have largely been unsuccessful. [a second] approach follows from the observation of increased glutamate release following ketamine administration. Excessive glutamate concentrations may be disruptive to network synchrony and, at high concentrations, can be neurotoxic. Preliminary evidence has suggested that elevation of glutamate in hippocampus may precede gray matter loss early in the illness. Both lamotrigine and mGluR2/3 agonists reduce excessive glutamate release and each has shown promise in attenuating behavioral effects of ketamine in humans. Each produced promising results in early trials that were not replicated in subsequent multicenter trials, although a meta-analysis suggested that lamotrigine may be effective when added to clozapine. Given the evidence that excessive glutamate transmission may be present only in early stage illness, this approach also should be studied in first episode or prodromal patients.”

Targeting NO as a therapeutic intervention?

“A future direction for drug development involves targeting intracellular pathways downstream of NMDA receptors. Following NMDA-gated channel opening, calcium influx activates the calmodulin, nitric oxide synthase, and adenylate cyclase pathway, which results in generation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) and phosphorylation of cAMP response element-binding protein which regulates synaptic plasticity and neurogenesis. The nitric oxide synthase pathway has been strongly linked to schizophrenia in genetic association studies and offers multiple potential targets. A recent preliminary study of the nitric oxide donor, nitroprusside, reported a marked improvement of positive and negative symptoms in acutely ill patients following a single intravenous administration. Phosphodiesterase (PDE) inhibitors may also enhance the production of cAMP and cGMP and are in development for cognitive enhancement. Results with PDE5 inhibitors in schizophrenia, which increase cGMP and improve memory in animals, have been mixed; these results are difficult to interpret in light of uncertainty about PDE5 levels in human brain. Inhibitors of PDE4 and PDE10 are currently in development for schizophrenia and several classes of PDE inhibitors have shown promise for cognitive enhancement”

Other interventions targeting NO?

Role of nitric oxide and related molecules in schizophrenia pathogenesis: biochemical, genetic and clinical aspects

agmatine NO
l-Arginine is converted to NO in the postsynaptic neuron. The NO that is produced diffuses back to the presynaptic neuron, where it enhances the release of glutamate via guanylate cyclase (GC) and cGMP. Glutamate that is released from the presynaptic terminal activates NMDA receptors, and Ca2+ enters and, via calmodulin (CaM), activates the NOS again

“Recent evidence suggests a link between NO and dopamine in psychiatric disorders like schizophrenia. Genetic studies examining a large population of schizophrenic patients revealed that polymorphisms in the neuronal NOS gene may confer increased susceptibility to schizophrenia. NO is increased in plasma of schizophrenic patients and NOS inhibitors like L-NAME or L-NNA could block several symptoms in animal models of schizophrenia like apomorphine induced climbing, PPI and amphetamine, ketamine or phencyclidine induced hyperlocomotion etc., comparable to that of conventional antipsychotics like haloperidol and clozapine. Moreover, potent antipsychotics are reported to decrease NO synthesis in brain. Subsequent studies have shown an inverse relation between NO levels and release of prolactin. In fact NOS inhibitors increase the prolactin secretion in rodents.”

glu no da
Effects of nitric oxide (NO) on dopamine (DA) transmission. (1) Glutamate (Glu) activates N-methyl-d-aspartate receptors (NMDAr) in NO neurons, opening calcium channels and leading to Ca2+ influx. (2) Increased intracellular Ca2+ activates neuronal nitric oxide synthase (nNOS), generating NO from l-arginine (L-arg). (3) Nitric oxide diffuses to the extracellular space, inhibiting the dopamine transporter (DAT), which reduces DA reuptake and increases extracellular DA. (4) Dopamine activates postsynaptic D2 receptors. (5) Nitric oxide also directly facilitates D2 activation or postsynaptic transduction mechanisms, increasing DA transmission. (6) Nitric oxide also increases Glu release, which exacerbates its effect on NO production. Apomorphine acts at postsynaptic D2 receptors, and amphetamine stimulates presynaptic DA release and decreases DA reuptake. 7-NI blocks NO synthesis, reducing the facilitatory effect of NO on DA transmission. Solid line: activation; dashed line: inhibition. [source]

“Nitric oxide (NO) can positively modulate dopaminergic neurotransmission, since it appears to block dopamine reuptake and to facilitate postsynaptic activation, and drugs that inhibit NO may have potential antipsychotic effects. 7-Nitroindazole (7-NI) and NG-nitro-l-arginine (L-NOARG) are specific and nonspecific NO synthase inhibitors, respectively, and attenuate the disruption of prepulse inhibition induced by methylphenidate and phencyclidine, proposed models of antipsychotic action. Furthermore, in reserpine-treated mice, L-NOARG and 7-NI attenuated the increase in locomotor activity induced by D1 and D2 agonists. Locomotor hyperactivity induced by dopamine agonists is another animal model for the study of antipsychotic-like action. Clinical studies found changes in NO synthase (NOS)-containing interneurons in frontal and limbic cortices in schizophrenia patients”


Agmatine: α2-adrenergic receptor activation, imidazoline receptor activation, NMDA receptor blockage and NOS inhibition or NO-NMDA cascade inhibition.

See for a full overview

“Agmatine is an endogenous polyamine intermediary derived from the biosynthesis of the proteinogenic amino acid l-arginine through the enzyme arginine decarboxylase (ADC) and inactivated by agmatinase. Highly expressed in brain, especially in the hippocampus and cortex, synthesis of agmatine occurs primarily in glial astrocytes but also in microglia. Agmatine is also dietary derived, readily crossing the blood brain barrier where endogenous levels in the hippocampus can increase several folds. Synthesised or exogenously derived, astrocyte agmatine is packaged and stored in synaptic vesicles where reports suggest that it may be co-localised with glutamine for release by depolarisation into the extracellular space and uptake by neurons where glutamine is converted to glutamate.

The neuronal glutamate released is cleared from the extracellular space along with agmatine through uptake by astrocytes and microglia, where glutamate is converted back to glutamine completing the highly regulated glial and neuronal cell compartmentation of glutamate metabolism through this critical glutamine–glutamate cycling that maintains glutamatergic homeostasis. Tightly regulated itself (agmatine homeostasis), agmatine plays a key role along with d-serine in these plasticity-related processes of glutamatergic homeostasis that keeps the brain far from excitation. In delightful reciprocity, glutamatergic homeostasis contributes to the regulation of agmatine synthesis and homeostasis.

Early findings of imidazolidine binding notwithstanding, it is now recognised that agmatine’s role in brain is as an inhibitory modulator of excitatory glutamatergic neurogliotransmitter events, the putative consequence of antagonist activity of NMDA receptors and their Ca2+ ion channels, (followed by) the essential inhibition of NOS, thus inhibiting the induction of the free radical proinflammatory mediator NO and oxidative stress . Agmatine is unique to date among endogenous biogenic amines, as selectively exhibiting antagonist activity at non-glycine β sites of NMDA receptors. Astrocyte derived d-serine is a co-agonist modulator of glutamate neurotransmission as an endogenous ligand for the glycine site of NMDA receptors [see more]. These agmatine induced inhibitory effects lead to the downstream inhibitory modulation of glutamate/Ca2+ and NO expression in glial/neuronal cells of the hippocampus, as well as the essential lowering of extracellular glutamate, the combined effects of which downregulates excitatory brain activity. This, thus, is the putative molecular basis of agmatine’s central neuroprotective and anti-inflammatory action that keeps the complex biological system of brain far from excitation, neuronoglial cyotoxicity, enhanced apoptotic signalling and cell death that characterise the gamut of neuropathic brain disorders that range from Multiple Sclerosis (MS), Amyotropic Lateral Sclerosis (ALS), Huntington’s and Parkinson’s disease to Alzheimer’s and clinical depression, among others.

In addition to its inhibition of both intracellular and extracellular glutamate and Ca2+-induced cytotoxicity, there exists an ever ripening body of data to suggest that agmatine’s more critical protection against cytotoxicity is related to its inhibition of NOS, with consequent downregulation in production of proinflammatory mediators, such as NO and oxidative stress, downregulating genes associated with microglial activation, recognised as a primary cause of neurological pathogenicity in the brain, oxidative cytotoxicity induces destabilisation of astrocyte lysosomes with leakage into the cytosol, decreased mitochondrial ability/capacity and damage with the release of mitochondrial cytochrome c. More ominous, oxidative toxicity may advantageously lead to protein misfolding and neurodegeneration. It is, thus, that agmatine’s impressive neuroprotective repertoire includes the essential safeguarding of neurons by stabilising astrocytes and mitochondria, achieved through stabilisation of lysosomes, peroxisosomes and ubiquitin–proteosome function in astrocyte mitochondria. While providing such abundant layers of neuroprotection, agmatine’s central inhibitory modulation of excitatory glutamatergic transmission participates in the brain plasticity-related modulation of synaptic plasticity and the Long term Potentiation (LTP) linked processes of learning and memory-cognition.” [1]

Nitric oxide synthase inhibitors have been demonstrated to normalize PCP-induced impairments, not only in PPI, but also in higher cognitive functions such as latent inhibition. Similarly, agmatine attenuates the disruptive effects of phencyclidine on prepulse inhibition [2], blocks conditioned avoidance responses; attenuates apomorphine induced climbing and diminishes amphetamine and ketamine-induced hyperlocomotor activity [3]

“…agmatine has an important role in regulating metabolic pathways of l-arginine. It inhibits all isoforms of enzyme, NOS which synthesize NO from l-arginine. …NOS inhibition by agmatine may be the basis for ability to antagonize ketamine induced hyperlocomotor activity as well as dopamine mediated behaviors relevant to schizophrenia. Alternately besides NMDA receptors, contribution of other receptor system like imidazoline, α2-adrenergic in agmatine induced behavior can not be completely ruled out.”

“…agmatine demonstrates an interesting profile when tested in the PCP model of schizophrenia, partially blocking a deficit in pre-attentive sensory information processing. However, this study suggests that agmatine does not alter the PPI response per se in mice. Further studies of the potential role of agmatine in psychiatric disease may answer important questions regarding pathophysiology and the role of arginine metabolism in brain function. Thus, the agmatine system may be of future interest both as a novel treatment target and as a part of the pathophysiological mechanisms underlying several brain disorders.”


6g/day of L-lysine added to risperidone has shown superiority over placebo [4] Large doses can deplete intra-cellular L-arginine stores, leading to a reduction in NO [more here].

Methylene Blue:

Methylene blue, which blocks NO-dependent soluble guanylate cyclase-mediated intracellular signalling, has been shown to exert therapeutic effects as an adjuvant to established antipsychotics in the treatment for schizophrenia. [5]

Myricitrin – a nitric oxide (NO) and protein kinase C (PKC) inhibitor [6]

Sigma-1 agonism may exert NMDA/nNOS-mediated neuroprotection [7] [full text]

See also

Effects of nitric oxide-related compounds in the acute ketamine animal model of schizophrenia.

Sodium nitroprusside – a rapid acting antipsychotic

Effect of l-theanine on glutamatergic function in patients with schizophrenia (2015)


Sodium nitroprusside – a rapid acting antipsychotic


Sodium nitroprusside:

“…a placebo controlled clinical trial by Hallak et al. has reported that has for the first time demonstrated a safe, rapid (within hours), and long-lasting (several weeks) improvement of positive, negative, anxiety, and depressive symptoms in patients with schizophrenia after a single intravenous injection of sodium nitroprusside at a randomized, placebo-controlled trial

Sodium nitroprusside is an antihypertensive drug that has vasodilatation-effects. It owes its principal activity to being a NO donor, so that it raises NO synthesis. When administered to schizophrenic patients via infusion, NO production escalates, so the tissue levels increase directly, without mediation by NMDA receptors in brain. It is a prominent finding that a single dose administration of nitroprusside in schizophrenia patients provides amelioration in symptoms that lasts up to 2 weeks. It is highly likely that a single dose sodium nitroprusside infusion given to treatment resistant schizophrenia patients yielded a rapid and weeks long improvement just like ketamine, an NMDA receptor antagonist, provided in treatment resistant depression. The effects of nitroprusside on schizophrenia symptoms could be explained by an increase in cerebral perfusion due to vasodilatation. Studies have proven that the blood stream in frontal and temporal cortex, which are concluded to be related to the negative symptoms of schizophrenia, decay in schizophrenia patients when compared to healthy controls” [1]

“Sodium nitroprusside (SNP) alone produced no changes in any of the behaviors evaluated. Ketamine produced hyperlocomotion and stereotypies. However, pretreatment with SNP for up to 1 week before ketamine administration significantly prevented the emergence of hyperactivity induced by ketamine, and pretreatment with SNP for up to 1 day before ketamine administration significantly prevented the emergence of stereotypies induced by ketamine.

The precise mechanism by which the SNP shows antipsychotic effects after its application is finished is not completely understood. A plausible explanation would be the ability of SNP, as a NO donor, to modulate protein kinases, transcription factors and other gene production factors through cGMP enhancement, making its cascading effects last even when SNP itself is no longer acting directly. As an up-regulation on NOS-expressing neurons in the rat hippocampus has been observed after ketamine administration (Keilhoff et al., 2004), another possible explanation to our findings might be related to SNP’s capacity to promote a feedback inhibition of NOS, correcting the up-regulation reported above. Indeed, SNP has been shown to inhibit the enhancement of NOS activity induced in rat neutrophils by treatment with lipopolysaccharide (LPS), an endotoxin. This SNP capacity could be explained through both an inhibition of NOS expression in the neutrophils caused by the nitrosonium ion (NO +) derived from SNP and an increase in NO that could be released directly from SNP (Mariotto et al., 1995).

SNP can also interfere directly with the capacity of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), a protein complex that controls transcription of DNA, to translocate into the nucleus. SNP inactivates NF-κB by nitration of the p65 subunit at Tyr-66 and Tyr-152, suppressing iNOS mRNA expression and preventing the activation of NF-κB target genes by TNFα (Tumor necrosis factor alpha), a cytokine involved in systemic inflammation which stimulates the acute phase reaction (Godínez-Rubí et al., 2013).

There is also evidence that SNP acts directly on NMDA receptors, which could correct the NMDA receptor dysfunction observed in schizophrenia (Oliveira et al., 2008 and Dhami et al., 2013), and that NO exerts a tonic inhibitory effect on dopamine transporters, which could correct a reduced activity of dopamine in the prefrontal cortex and, through feedback cascades, correct dopaminergic hyperactivity in accumbens and striatum, both findings reported in schizophrenia patients (Pycock et al., 1980).

Acute SNP effects in both glutamatergic and dopaminergic pharmacological models of psychosis had already been suggested by other pre-clinical studies. In 2000, Bujas-Bobanovic et al. found that SNP was able to abolish the psychotic behavior and brain c-fos expression induced in rats by PCP, a NMDA antagonist drug (Bujas-Bobanovic et al., 2000). Recently, it was demonstrated that treatment with SNP attenuated the schizophrenia-like changes on prepulse inhibition (PPI) induced in rats by amphetamine, a dopaminergic agonist drug (Issy et al., 2014), and that SNP administration produced a break in the pattern of the sleep–wake cycle similar to that found in animals depleted of dopamine in mice treated with ketamine, another NMDA antagonist drug (Maia-de-Oliveira et al., 2014a).

Interactions between NO and the dopaminergic system also have been reported in some clinical studies. Lee and Kim (2008) found decreased serum NO in schizophrenic patients compared with a control group, and reported that six-week treatment with the antipsychotic risperidone increased NO levels and that this increase was associated with symptom improvement. Among the patients who improved clinically (≥ 30% improvement in PANSS score), NO levels significantly increased after risperidone. However, no significant changes in NO levels were found in the non-responders (Lee and Kim, 2008). A recent meta-analysis described that patients taking antipsychotic medications have higher NO serum levels than those of controls (effect size g = 0.663, 95%CI = 0.365 to 0.961, p < 0.001) (Maia-de-Oliveira et al., 2012), and some interesting research reported enhanced cGMP concentrations in the plasma of patients with schizophrenia after the use of antipsychotic drugs (as mentioned before, NO stimulates cGMP synthesis) (Ebstein et al., 1976, Smith et al., 1976 and Ziimmer et al., 1980).

Furthermore, our group recently observed an improvement of positive and negative symptoms after 4 h of SNP infusion, at a dose of 0.5 μg/kg per minute, in 2 well-documented patients with clozapine-refractory schizophrenia”

To our knowledge, the results described here indicate for the first time that SNP, an NO donor, may present preventive antipsychotic effects. With regard to translation to the clinic, these findings with rats, which have an even faster metabolic rate than humans, suggest that SNP on its own could be responsible for the therapeutic effects up to 4 weeks found in our previous clinical study. If these preventive antipsychotic effects of SNP are replicated in further studies, SNP could be a significant agent for improving patient care outcomes in the near future. We believe that one of the next steps should be the investigation of the effects of SNP in patients at clinical high risk for psychosis. [2]

For more information:

The Effects of Sodium Nitroprusside Treatment on Cognitive Deficits in Schizophrenia: A Pilot Study.

Sodium nitroprusside treatment of clozapine-refractory schizophrenia.

Sodium nitroprusside, a nitric oxide donor for novel treatment of schizophrenia, may also modulate dopaminergic systems.

Nitroprusside single-dose prevents the psychosis-like behavior induced by ketamine in rats for up to one week