ADHD is a neurodevelopmental disorder that affects 2-15% of children, characterized by symptoms like inattention, hyperactivity, impulsivity, and anxiety (Koirala et al 2024). Symptoms often manifest in early childhood, and severity can vary. It is more often diagnosed in males compared to females due to diagnostic bias and women being more likely to internalise and hide symptoms, resulting in symptoms needing to be more prominent in females. For example, males are more likely to exhibit external behaviors such as hyperactivity, while females exhibit internal symptoms such as inattentiveness, which is less disruptive and thus less likely to be noticed and diagnosed (Mowlem 2019). There are also disparities in race, with asian, black, and hispanic children being significantly less likely to be diagnosed and treated for ADHD in comparison to white children. This disparity likely stems from a complex interplay of factors, including cultural stigma surrounding mental health, socioeconomic barriers to accessing healthcare, and implicit biases among clinicians, which influence diagnostic and treatment practices (Shi 2021). Several environmental factors have been linked to increased risk of ADHD: in utero events such as maternal stress, prenatal drug exposure, exposure to lead, low birthrate/prematurity, and high levels of family conflict (Ouakil 2011). ADHD is becoming more recognized and understood, but is still very understudied, and to develop effective interventions, it must be studied more.

The main neurobiological mechanism of ADHD is unknown. Different genes have been implicated, as well as certain environmental factors, but there is no definite known cause. However, research points towards a combination of genetic and environmental factors. Twin studies suggest that ADHD is highly heritable; by comparing rates of ADHD diagnosis in monozygotic and dizygotic twins, they have found a 76% heritability estimate in children and adolescents, with higher rates of ADHD in biological relatives of others with ADHD (Ouakil 2011). However, there is established evidence that dysfunction in ADHD brains centers around the prefrontal cortex and hippocampus, brain areas that are associated with learning, working memory, and reward, and are regulated mainly by norepinephrine and dopamine. These brain regions are particularly sensitive to disruptions during critical periods of neurodevelopment, possibly explaining why early life stressors have such a profound impact on ADHD (Koirala et al 2024). Currently, primary treatment options for ADHD vary from stimulant options like methylphenidate and amphetamine to rarer, nonstimulant options like guanfacine. Methylphenidate blocks dopamine and norepinephrine transporters, thus generating an increase in catecholamine availability in the synaptic terminal. The increase in dopamine and norepinephrine availability affects executive function, reward regulation, the emotional circuit, and decision making, all of which relate to ADHD symptoms, but come with side effects such as sleep disturbance or even cardiovascular complications (Koirala et al 2024). Additionally, emerging studies indicate that methylphenidate also regulates the glutamate system; methylphenidate increases AMPAR surface expression in the hippocampus CA1 area through a PKA-dependent pathway, thus providing another pathway to alleviate cognitive dysfunction and hyperactivity in individuals with ADHD. This is in agreement with studies that have demonstrated that psychostimulants work differently in normal versus diseased conditions (Cheng 2017). However, methylphenidate is only tolerated by 30-50% of adult patients, leading to exploration of alternative treatment options like guanfacine, a norepinephrine α2A-adrenoceptor agonist whose stimulation strengthens functional connectivity of the prefrontal cortex, while blockade of the receptors leads to less working memory and more impulsivity—both of which line up with symptoms of ADHD (Koirala et al 2024). 

This paper will examine the role of glutamate in ADHD pathology. Glutamate is the brain’s most abundant excitatory neurotransmitter, playing crucial roles in synaptic plasticity, learning, and memory—all areas implicated in ADHD pathology. Most cells in the central nervous system express at least one type of glutamate receptor—the main two being AMPA and NMDA—underscoring how crucial glutamate is to a healthy brain (Pal 2021). Studies have shown that disrupted glutamate transmission/function can lead to ADHD pathology, and that methylphenidate, which is an norepinephrine and dopamine reuptake inhibitor, also increases glutamate signalling, but the mechanisms of action are more uncertain (Elia 2020). As ADHD does not have one linked genetic cause, it is studied in mouse models that are bred with ADHD-like phenotypes. The main three used are the spontaneously-hypertensive rat model (SHR), which has the same ADHD symptoms as humans, and the two control models: Wistar-Kyoto (WKY) and Sprague-Daley (SD).

The hippocampus of an SHR rat model for ADHD demonstrates altered AMPA receptor function. AMPARs are ionotropic receptors that rapidly depolarize the neuron upon glutamate binding comprising four subunits. These subunits—GluA1, GluA2, GluA3, and GluA4—determine the receptor’s functional properties, and minor alterations can have profound effects. AMPARs are also involved in learning and memory via affecting long term potentiation or depression, and studies have found that the function of ionic glutamate receptors such as AMPARs are disrupted in the prefrontal cortex and hippocampus of SHR mice (Bai 2022). Scientists recorded AMPAR mediated synaptic transmission at hippocampal excitatory synapses, as well as used immunogold labelling to quantify the density of the AMPAR subunits GluA1 and GluA2/3 in SHR rats (Medin 2019). Immunogold labelling is a staining technique where colloidal gold particles are attached to secondary antibodies, which are attached to primary antibodies, which bind a specific cell component. They can then be identified via electron microscopy, since gold has high electron density and displays high contrast dark spots. Results showed reduced AMPAR mediated synaptic transmission at pyramidal cell synapses in two parts of the hippocampus: the stratum radiatum, and the stratum oriens, demonstrating that ADHD brains have less glutamate signalling, which corresponds with a reduction in learning and memory (Medin 2019). However, the immunogold labelling showed no statistically significant change in subunit density, demonstrating that despite dysfunctional glutamate signalling, the synapses themselves remained unaltered. This most likely shows some sort of compensatory mechanism in the brain, such that while signalling reduces, density does not.

Scientists looked further at AMPAR regulation, and found that the knockout of AMPAR regulatory protein TARP γ-8, an auxiliary AMPAR subunit led to ADHD-like behaviors in mice, including hyperactivity, impulsivity, memory deficits, and more. TARP γ-8 is enriched in the hippocampus and critical for AMPAR expression, trafficking, localization, and plasticity (Bai 2022). After TARP γ-8 knockout, scientists performed a wide variety of assays used to test various facets of ADHD behavior: the open field test (hyperactivity), elevated plus maze (anxiety), cliff avoidance reaction, object recognition (cognitive performance), contextual fear conditioning, time exploring novel objects, and discrimination ratio. They performed these tests both before and after methylphenidate administration, in order to test whether methylphenidate rescued the effects of TARP γ-8 knockout. methylphenidate ultimately rescued major behavioral deficits and improved synaptic AMPAR function by upregulating other AMPAR auxiliary proteins in hippocampal synaptosomes. However, methylphenidate had little effect on impulsivity, only improving cognition and memory performance, and in fact aggravating the anxiety-like behavior associated with TARP γ-8 knockout (Bai 2022). A possible mechanism of action for methylphenidate affecting the AMPA receptors is via the PKA pathway, though more is unknown (Cheng 2017).

After the tests, the scientists extracted the brains of the mice, collected the tissue from the hippocampus and prefrontal cortex, and used synaptosome proteomics to identify the specific proteins being impacted by TARP γ-8 knockout and explore the synaptic mechanisms underlying the ADHD-like phenotypes of TARP γ-8 knockout mice. Once all the proteins were identified, they used partial least squares discriminant analysis (PLS-DA) to analyze the data—they filtered out the differentially expressed proteins, then uploaded the data onto an online platform that analyzed protein interactions and detected clusters of proteins affected (Bai 2022). They found separation between the TARP γ-8 knockout and wildtype cortical and hippocampal synaptosomes, telling them that TARP γ-8 deletion significantly changes the profiles of proteins expressed. In comparison to wildtype mice, the prefrontal cortex had 224 upregulated proteins and 41 down regulated proteins. The hippocampus had 191 upregulated proteins and 44 down regulated proteins. The dramatic shift in protein expression underscores the wide-ranging impact of TARP γ-8 on synaptic function and signalling, suggesting that its deletion disrupts the overall synaptic balance (Bai 2022). Using the online database, they were then able to identify specific proteins whose expression was changed, offering new insights into possible avenues of treatment for ADHD. A few proteins stood out: Grik2, Slc6a3, and Cnih2. In the TARP γ-8 knockout mice, expression of the proteins Grik2 and Slc6a3 were markedly upregulated, while Cnih2 was downregulated. Methylphenidate administration downregulated the former two proteins and upregulated the last, possibly highlighting an underlying mechanism by which the TARP γ-8 knockout mice respond to ADHD medications. Slc6a3 encodes sodium-dependent dopamine transporters, Grik2 is a glutamatergic receptor, and Cnih2 is an AMPAR auxiliary submit that promotes surface trafficking and AMPAR gating. Grik2—which encodes a subunit for kainate receptors, a less studied subset of glutamate receptors—being upregulated may be a compensatory mechanism in response to diminished AMPAR function, although the precise mechanism is unknown. Similarly, Slc6a3’s upregulation suggests a complex interplay between the glutamate and dopamine systems in ADHD pathology that could be studied more. Cnih2 has the most obvious connection to ADHD, being an AMPAR auxiliary subunit just like TARP γ-8, and it being diminished in ADHD mice indicates a sophisticated relationship between the two that is still unknown (Bai 2022).

Moving on from AMPAR function in the hippocampus, scientists looked at the prefrontal cortex and found more evidence that disruptions in glutamatergic transmission contributes to ADHD. They found that in SHR rats, AMPAR-mediated synaptic transmission in the pyramidal neurons of the prefrontal cortex was diminished, as was the surface expression of AMPAR subunits. This aligns with earlier findings that the prefrontal cortex is a critical hub for executive functions such as impulse control and decision-making, both of which are impaired in ADHD (Ouakil 2011). To test the effect the behavioral phenotype, they used SHR rats for the ADHD model and SD rats for the control, with four populations—control + saline, control + methylphenidate, SHR + saline, and SHR + methylphenidate—being tested with behavioral tests like the discrimination ratio test, midline crossing before and after methylphenidate, and center entry tests. Methylphenidate administration ultimately ameliorated the behavioral deficits and restored synaptic function, as the SHR mice had diminished AMPAR function (Cheng 2017). After extracting the brains from the rats, they used biotinylation and western blotting to examine the surface expression of AMPAR and NMDAR subunits in SHR cortical slices. Biotinylation is the process of attaching a biotin to the amino acid or carbohydrate of a protein, and western blotting can then be used to identify that specific protein. Lining up with the results of the other studies in this paper, they found a significant reduction in the expression of the GluR1 and GluR2 subunits (Cheng 2017). 

To further ensure that the alterations to the prefrontal cortex pyramidal neurons was being driven by the AMPA receptors, they used DREADD technology to selectively activate prefrontal cortex pyramidal neurons—DREADD stands for “designer receptors exclusively activated by designer drugs,” and they are activated by the ligand clozapine-N-oxide (CNO), which is exogenously administered. DREADD technology is a major advancement in neuroscience due to its unprecedented precision in activating specific neuronal populations (Manvich 2018). This study stereotaxically injected a CaMKII-driven Gq-coupled DREADD adeno-associated virus, hM3D, into the medial prefrontal cortex of the SHR rats. Afterwards, application of CNO, which activated the DREADD, significantly increased the frequency of action potentials in the hM3D expressing prefrontal cortex pyramidal neurons, and the increased AMPAR-EPSC activity restored AMPAR function in the SHR ADHD model. When behavioral tests were performed again on rats with the DREADD chemogenetic technology, the discrimination ratios were significantly improved, and AMPAR function was elevated. While hypoactivity of the prefrontal cortex has long been found in individuals with ADHD, these results only further confirmed that diminished AMPAR transmission and function in the prefrontal cortex underlies behavioral deficits in ADHD and that enhancing prefrontal cortex activity is a possible treatment strategy (Cheng 2017).

In conclusion, a couple changes in the ADHD brain stand out: delayed maturation of prefrontal cortex neurons, and less glutamate transmission in the prefrontal cortex and hippocampus, both of which contribute heavily to ADHD pathology.  These findings suggest that treatments for ADHD need to not just address neurotransmitter imbalance, but address disrupted neuronal development and connectivity as well, requiring a multifaceted approach. A shift is required in the way ADHD is treated, from focusing mainly on catecholamines to incorporating interventions that target other neurotransmitters like glutamate as well. The first study demonstrated that AMPA receptor function is diminished in the ADHD brain. The second focused on a specific regulatory protein, TARP γ-8, whose knockout contributed to ADHD, and which identified three specific proteins that are altered in ADHD pathology: Grik2, Slc6a3, and Cnih2. The final paper focused on diminished glutamate transmission in the pyramidal neurons of the prefrontal cortex, and how rescuing this deficit can rescue ADHD behavior. All three of these papers together indicate that treatments for diminished glutamate transmission are a novel possibility for treating ADHD, and one with a lot of potential. Multiple strategies are already evident for intervention, such as enhancing AMPAR function or modulating regulatory glutamate subunits like TARP γ-8. Glutamate, as the brain’s primary excitatory neurotransmitter, is underexplored in terms of ADHD in comparison to catecholamines like dopamine, but is essential for the prefrontal cortex and hippocampus to function—both brain areas crucial to ADHD. Future studies could examine the three proteins—Grik2, Slc6a3, and Cnih2—in detail as possible treatment avenues, or at translating experimental tools like DREADD technology to be clinically viable in humans. This would require addressing ethical, safety, and technical challenges but holds promise for creating highly targeted interventions with minimal side effects. Ultimately, there is a lot to explore at the intersection of glutamatergic dysfunction with other neurotransmitter systems and parts of the brain. ADHD is a complicated disorder, and understanding it could pave the way for new and better treatment strategies, changing how we treat this disorder.

References

Bai, W. J., Luo, X. G., Jin, B. H., Zhu, K. S., Guo, W. Y., Zhu, X. Q., Qin, X., Yang, Z. X., Zhao, J. J., Chen, S. R., Wang, R., Hao, J., Wang, F., Shi, Y. S., Kong, D. Z., & Zhang, W. (2022). Deficiency of transmembrane AMPA receptor regulatory protein γ-8 leads to attention-deficit hyperactivity disorder-like behavior in mice. Zoological research, 43(5), 851–870. https://doi.org/10.24272/j.issn.2095-8137.2022.122

Cheng, J., Liu, A., Shi, M. Y., & Yan, Z. (2017). Disrupted Glutamatergic Transmission in Prefrontal Cortex Contributes to Behavioral Abnormality in an Animal Model of ADHD. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, 42(10), 2096–2104. https://doi.org/10.1038/npp.2017.30

Elia, J., Izaki, Y., Ambrosini, A., & Hakonarson, H. (2020). Glutamatergic Neurotransmission in ADHD: Neurodevelopment and Pharmacological Implications Review Article. Journal of Pediatrics and Neonatology, 2, 1006. https://www.medtextpublications.com/open-access/glutamatergic-neurotransmission-in-adhd-neurodevelopment-and-pharmacological-implications-505.pdf

Koirala, S., Grimsrud, G., Mooney, M.A. et al. (2024). Neurobiology of attention-deficit hyperactivity disorder: historical challenges and emerging frontiers. Nat. Rev. Neurosci. 25, 759–775. https://doi.org/10.1038/s41583-024-00869-z

Manvich, D.F., Webster, K.A., Foster, S.L. et al. (2018). The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice. Sci Rep 8, 3840. https://doi.org/10.1038/s41598-018-22116-z

Medin, T., Jensen, V., Skare, Ø., Storm-Mathisen, J., Hvalby, Ø., & Hildegard Bergensen, L. (2019). Altered α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function and expression in hippocampus in a rat model of attention-deficit/hyperactivity disorder (ADHD). Behavioural Brain Research, 360, 209–215. https://doi.org/10.1016/j.bbr.2018.12.028

Mowlem, F., Agnew-Blais, J., Taylor, E., & Asherson, P. (2019). Do different factors influence whether girls versus boys meet ADHD diagnostic criteria? Sex differences among children with high ADHD symptoms. Psychiatry research, 272, 765–773. https://doi.org/10.1016/j.psychres.2018.12.128

Pal M. M. (2021). Glutamate: The Master Neurotransmitter and Its Implications in Chronic Stress and Mood Disorders. Frontiers in human neuroscience, 15, 722323. https://doi.org/10.3389/fnhum.2021.722323

Purper-Ouakil, D., Ramoz, N., Lepagnol-Bestel, AM. et al. (2011). Neurobiology of Attention Deficit/Hyperactivity Disorder. Pediatr Res 69, 69–76. https://doi.org/10.1203/PDR.0b013e318212b40f

Shi, Y., Hunter Guevara, L. R., Dykhoff, H. J., Sangaralingham, L. R., Phelan, S., Zaccariello, M. J., & Warner, D. O. (2021). Racial Disparities in Diagnosis of Attention-Deficit/Hyperactivity Disorder in a US National Birth Cohort. JAMA network open, 4(3), e210321. https://doi.org/10.1001/jamanetworkopen.2021.0321