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. 2019 Apr 17;39(16):3094-3107.
doi: 10.1523/JNEUROSCI.2451-18.2018. Epub 2019 Feb 4.

Glutamate Within the Marmoset Anterior Hippocampus Interacts with Area 25 to Regulate the Behavioral and Cardiovascular Correlates of High-Trait Anxiety

Jorge L Zeredo  1   2 , Shaun K L Quah  2 , Chloe U Wallis  2 , Laith Alexander  2 , Gemma J Cockcroft  2 , Andrea M Santangelo  2 , Jing Xia  3 , Yoshiro Shiba  2 , Jeffrey W Dalley  3   4 , Rudolf N Cardinal  4   5 , Angela C Roberts  2 , Hannah F Clarke  6
Affiliations

Glutamate Within the Marmoset Anterior Hippocampus Interacts with Area 25 to Regulate the Behavioral and Cardiovascular Correlates of High-Trait Anxiety

Jorge L Zeredo et al. J Neurosci. .

Abstract

High-trait anxiety is a risk factor for the development of affective disorders and has been associated with decreased cardiovascular and behavioral responsivity to acute stressors in humans that may increase the risk of developing cardiovascular disease. Although human neuroimaging studies of high-trait anxiety reveals dysregulation in primate cingulate areas 25 and 32 and the anterior hippocampus (aHipp) and rodent studies reveal the importance of aHipp glutamatergic hypofunction, the causal involvement of aHipp glutamate and its interaction with these areas in the primate brain is unknown. Accordingly, we correlated marmoset trait anxiety scores to their postmortem aHipp glutamate levels and showed that low glutamate in the right aHipp is associated with high-trait anxiety in marmosets. Moreover, pharmacologically increasing aHipp glutamate reduced anxiety levels in highly anxious marmosets in two uncertainty-based tests of anxiety: exposure to a human intruder with uncertain intent and unpredictable loud noise. In the human intruder test, increasing aHipp glutamate decreased anxiety by increasing approach to the intruder. In the unpredictable threat test, animals showed blunted behavioral and cardiovascular responsivity after control infusions, which was normalized by increasing aHipp glutamate. However, this aHipp-mediated anxiolytic effect was blocked by simultaneous pharmacological inactivation of area 25, but not area 32, areas which when inactivated independently reduced and had no effect on anxiety, respectively. These findings provide causal evidence in male and female primates that aHipp glutamatergic hypofunction and its regulation by area 25 contribute to the behavioral and cardiovascular symptoms of endogenous high-trait anxiety.SIGNIFICANCE STATEMENT High-trait anxiety predisposes sufferers to the development of anxiety and depression. Although neuroimaging of these disorders and rodent modeling implicate dysregulation in hippocampal glutamate and the subgenual/perigenual cingulate cortices (areas 25/32), the causal involvement of these structures in endogenous high-trait anxiety and their interaction are unknown. Here, we demonstrate that increased trait anxiety in marmoset monkeys correlates with reduced hippocampal glutamate and that increasing hippocampal glutamate release in high-trait-anxious monkeys normalizes the aberrant behavioral and cardiovascular responsivity to potential threats. This normalization was blocked by simultaneous inactivation of area 25, but not area 32. These findings provide casual evidence in primates that hippocampal glutamatergic hypofunction regulates endogenous high-trait anxiety and the hippocampal-area 25 circuit is a potential therapeutic _target.

Keywords: anxiety; area 25; cardiovascular; glutamate; hippocampus; marmoset.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
Measures of high-trait anxiety correlate with glutamate levels within the right aHipp. A, Schematic of the human intruder test apparatus detailing the depth (front, middle, back) and height (floor, low, middle, high, top of nest box) locations that the monkey could occupy. B, Schematic showing the dissection of the hippocampus from AP 4 to AP 6.5 before glutamate analysis. Decreased levels of hippocampal glutamate levels correlated with increased EFA score (C), increased head and body bobbing (D), but not TSAF (E).
Figure 2.
Figure 2.
aHipp LY/CGP treatment reversibly increases the TSAF and reduces the EFA score in the human intruder test. A, Schematic showing the position of the cannula tips and cannula locations for each animal. All aHipp cannulae were located within the ranges of AP, 4.8–5.6, respectively, and are plotted here on a single coronal section. Cytoarchitectonic parcellation was based on Burman and Rosa (2009). Circles represent the estimated maximal spread of the muscimol/baclofen or saline infusions (West et al., 2011). B, TSAF is reversibly increased by aHipp LY/CGP infusion, but this is not shown in the composite anxiety score (C) due to habituation between the two saline infusions.*p < 0.05.
Figure 3.
Figure 3.
Unpredictable threat test and cannulae schematics. A, In the unpredictable threat test (n = 6), animals were placed in a familiar environment for 25 min and played two types of auditory stimuli, a novel auditory cue and a US, for the 4 unpredictable threat days. Each cue was 20 s long, 75 dB, and presented 12 times with an ITI of 40–80 s. Each mildly aversive US was 0.4 s long, 117 dB, and presented 12 times with an ITI of 40–80 s. The threat of the aversive US was unpredictable because there was no relationship between the cue and the US. On the ambiguous cue probe (day 5), monkeys were presented with a novel, ambiguous 20 s, 75 dB cue presented 12 times with an ITI of 100–180 s in the same context. No US was presented. All drug manipulations occurred on ambiguous cue probes. The cycle then repeated with the novel cue incorporated into the unpredictable threat training. B, Glass brain illustrating the rostrocaudal locations of the aHipp, area 25, and area 32 and the intracerebral cannulae _targeting each area. C, Representative histological sections with arrows marking the position of the cannula tips and cannula locations for each animal. All aHipp, area 25, and area 32 cannulae were located within the ranges of AP 4.8–5.6, 12.5–14, and 15.8–16.6, respectively, and are plotted here on a single coronal section for each _target area. Cytoarchitectonic parcellation was based on Burman and Rosa (2009). Circles represent the estimated maximal spread of the musbac or saline infusions (West et al., 2011).
Figure 4.
Figure 4.
aHipp LY/CGP ameliorates the behavioral and cardiovascular correlates of cue-induced anxiety, but these effects can be blocked by simultaneous area 25 inactivation. Graphs show the changes in HR (beats per minute), VS, HRV, CSI, and CVI under drug and saline conditions during the cue relative to the baseline (the last 20 s of the immediately preceding ITI. Positive numbers indicate an increase from baseline and negative numbers indicate a decrease compared with baseline. Data are shown as mean ± SEM. *p < 0.05. A, Compared with saline, aHipp LY/CGP infusion altered responding in a cue-dependent manner, as assessed by cue-induced increases in HR, VS, and CSI and decreases in HRV. B, Simultaneous aHipp LY/CGP + area 25 inactivation abolished the increases in HR, VS, and CSI that were seen with aHipp LY/CGP alone. Area 25 inactivation also increased HR and VS by itself. C, Simultaneous aHipp LY/CGP + area 32 inactivation did not alter the changes in VS and cardiovascular activity seen with aHipp LY/CGP alone. Area 32 inactivation also had no effect on its own.
Figure 5.
Figure 5.
Those animals that showed the smallest cue-induced changes in HR also showed the smallest changes in VS. Within-subjects correlation analysis of the cue-induced changes in HR and VS after infusion of saline into the aHipp revealed that those animals that showed the least HR responsivity also showed the smallest changes in VS. ***p < 0.0001.
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