Glutamate – a new target for anxiety?

Anxiety is a fact of life. We all feel it at times. Indeed, it serves to protect us from danger. Trait anxiety, however, refers to an individual’s stable and persistent disposition to interpret ambiguous or neutral environmental events as threatening, where those low in trait anxiety would not. Those high in trait anxiety are more likely to develop affective disorders including anxiety and depression¹ and are more likely to present with cardiovascular disease^2,3.

Typical drug treatments for anxiety include SSRI antidepressants like Prozac or Zoloft – which come with a host of unwanted side effects – or addictive GABAergic tranquilisers such as the benzodiazepines (Xanax, Valium etc.). Recent research, however, has uncovered a potential new target for future therapies – the glutamatergic system.

Glutamate is the primary excitatory neurotransmitter in the brain. Disrupted glutamatergic signalling pathways have been associated with anxiety and depression-like behaviours^4,5, and previous studies have linked anxious/depressive phenotypes with decreased glutamate release in the hippocampus⁶. The hippocampus modulates anxious behaviour via a network of regions in the medial prefrontal cortex (mPFC) including Brodmann’s areas 25 and 32 (homologous to the rodent infralimbic and prelimbic subregions respectively^7–9), and changes in connectivity between these prefrontal regions, the amygdala and the hippocampus are implicated in anxiety/mood disorders¹⁰.

The anterior hippocampus projects neurons to both the mPFC and the amygdala through which both regions can be modulated simultaneously¹³; neuroimaging studies have highlighted the importance of these projections in the regulation of negative emotional behaviours such as fear and anxiety^13–15.

In the current study, a subpopulation of marmosets with an endogenous high-trait anxiety phenotype was used to study the relevance of these glutamatergic connections in two common behavioural tests for anxiety – the human intruder paradigm and an unpredictable threat test. The marmoset, which has previously been used in similar experiments^16,17, mirrors many of the neurobiological correlates of pathological anxiety seen in humans including blunted amygdala serotonin function and reduced dorsal anterior cingulate cortex volume.

The researchers therefore investigated the effect of pharmacologically increasing glutamate in the anterior hippocampus in vivo – via implanted intracerebral cannulae – on the performance of the marmosets in the behavioural tests of anxiety. This was done using a drug combination known as the LY/CGP cocktail, which increases presynaptic glutamate release by inhibiting mGlu2/3 and GABA_B receptors¹⁸.

The human intruder test.
Zeredo et al. (2019)

In the human intruder test, an unfamiliar human wearing a mask approaches the marmoset and maintains eye contact for two minutes, which tends to produce an anxious response from the marmoset. High-trait anxiety animals commonly retreat to the back of their cage – spending significantly less time at the front of the cage than ‘normal’ animals – and marmosets in particular elicit a number of species-specific behaviours indicative of anxiety such as head bobbing and particular vocalisations, which combined were used to calculate a composite anxiety score for each test.

Glutamate levels – as well as levels of monoamine neurotransmitters – within the right and left anterior hippocampus of marmosets were compared with the composite anxiety scores and specific anxiety behaviours. The researchers found that reduced glutamate levels in the right anterior hippocampus (though not the left) was associated with a greater amount of time spent at the back of the cage versus the front, and with increased head and body bobbing behaviours during the tests. This suggests that glutamate in the hippocampus may play a key role in anxiety (it’s important to note, however, that both serotonin and noradrenaline in the right anterior hippocampus, and dopamine in the left, also showed a positive correlation with the time spent at the back of the cage; therefore this specific avoidance behaviour is not controlled by glutamate alone).

Importantly, when glutamate was pharmacologically increased in these high-trait anxiety monkeys, they showed less behaviours characteristic of a high-trait anxiety phenotype. There was a marked increase in the time spent at the front of the cage – close to the human intruder – after bilateral infusion of LY/CGP in the anterior hippocampus, indicating that anxiety was effectively reduced following the drug infusion. 

Composite anxiety score following drug infusion versus saline. Zeredo et al. (2019)

In the second behavioural test, the unpredictable threat test, the monkeys were placed in a transparent Perspex box and carried to the test environment. The box was equipped with a speaker through which an alarm could be sounded. For the first four days – the training period – the animals were placed in the test environment and presented with a threatening alarm sound, followed by an auditory cue. The animals thus learned to associate the test environment with the ‘unpredictable threat’ (the alarm). On the fifth day – the test day – the experimenters administered either saline or LY/CGP before presenting the monkeys with a novel, ambiguous auditory cue, within the same threatening context.

The researchers again measured species-specific behavioural indicators of vigilance/anxiety, though this time they also measured changes in blood pressure as well as heart rate variability (HRV) – the interval between heartbeats. HRV reflects the balance between parasympathetic and sympathetic autonomic cardiac regulation and baroreflex activity; decreased HRV is associated with anxiety disorders and depression³. The normal, healthy cardiovascular response to an ambiguous cue in a potentially threatening environment is a heightened stress response leading to a transient reduction in HRV in preparation for fight-or-flight. However, in high-trait anxious individuals, this ‘cardiac responsivity’ is blunted.

As predicted, the animals which were administered LY/CGP showed a greater, more normal cardiac response in accordance with their vigilance/anxiety behaviours, compared with those given saline. However, simultaneous pharmacological inactivation of mPFC area 25 blocked this improvement. Cardiac responsivity was also improved when area 25 was inactivated alone, but this improvement was again blocked when LY/CGP was simultaneously infused into the hippocampus. This means that inactivation of Brodmann’s area 25 and an increase in presynaptic glutamate in the anterior hippocampus have the same effects independently – a return to a more ‘normal’ cardiac responsivity and a reduction in anxiety – but when combined, these effects are blocked, suggesting that the two regions reciprocally interact to regulate cardiac responsivity in response to stress/anxiety.

While rodent studies have particularly highlighted the importance of hippocampal–prelimbic cortex (homologous to area 32 in primates) connectivity in fear/anxiety^10–12, the current study found no significant effect of area 32 inactivation on anxiety regulation. One possible explanation for this is that hippocampal glutamate differentially regulates mPFC regions in healthy and high-trait anxiety individuals. Indeed, reducing hippocampal glutamate in non-anxious rodents and primates has an anxiolytic effect^19,20, yet increasing hippocampal glutamate in high-trait anxiety animals is also anxiolytic^18,21. Future research will inevitably seek to confirm this hypothesis by repeating the experiments with ‘normal’, non-anxious animals for comparison.

Thus, in both experiments, pharmacological increase in glutamate in the anterior hippocampus of high-trait anxiety animals led to an increased engagement with the potential threat – increased approach behaviour in the human intruder test, and more ‘normal’ vigilance behaviours and cardiac responsivity in the unexpected threat test. 

The results therefore suggest that glutamatergic hypofunction in the anterior hippocampus contributes to maladaptive anxiety behaviours in high-trait anxious individuals, and that pathways underlying the connectivity between the anterior hippocampus and Brodmann’s area 25 could be a potential target for future pharmacological interventions for anxiety – potentially replacing the “dirty”, addictive drugs currently prescribed for anxiety disorders with more targeted, effective treatments.

The results certainly warrant further study – I hope that pharmaceutical companies take note.

References:

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