Neuronal Nitric Oxide Synthase Colocalizes with N‐methyl‐D‐aspartate (NMDA) and Alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic Acid (AMPA) Receptors in the Dorsolateral Periaqueductal Gray of Rats

Abstract

The midbrain periaqueductal gray (PAG) plays an important role in cardiovascular regulation, respiratory control, pain modulation, and autonomic responses to stressful stimuli. Among the four columns of the PAG, the dorsolateral PAG (dlPAG) contains the highest density of neurons that express neuronal nitric oxide synthase (nNOS), the enzyme for synthesis of nitric oxide (NO) in neurons. Because inhibition of NO synthesis in the dlPAG produces pressor responses, attenuates fear responses and attenuates stress responses, it has been suggested that NO may modulate cardiovascular regulation and responses to fear and stress. NMDA type and AMPA type glutamate receptors are also expressed in the dlPAG and have been implicated in cardiovascular control and defensive behavior. Physiological studies have suggested that NO and glutamate receptors may interact in the dlPAG in cardiovascular regulation and stress responses. An NO donor injected into the dlPAG has been shown to decrease cardiovascular responses induced by NMDA, while an nNOS inhibitor increased them. NO‐induced flight behavior was prevented by antagonists to NMDA or AMPA receptors in the dlPAG. However, anatomical data to support interactions between NO and glutamate receptors in the dlPAG have been missing. We performed fluorescent immunohistochemistry for nNOS, NMDAR1 (subunit of NMDA receptor) and GluR2 (subunit of AMPA receptor) and examined labeled sections with confocal microscopy to test the hypothesis that nNOS and glutamate receptors colocalize in the rat dlPAG. We found that nNOS and NMDAR1 immunoreactivity (IR) colocalize in dlPAG neurons in rat. We noted 94.9 ± 1.1% nNOS‐IR containing neurons in the dlPAG also contained NMDR1‐IR, while 26.9 ± 4.2% NMDAR1‐IR containing cells also contained nNOS‐IR. Colocalization of nNOS‐IR and GluR2‐IR was also observed in the dlPAG with 91.9 ± 2.3% of nNOS‐IR containing neurons also containing GluR2‐IR and 28.2 ± 5.5% of GluR2‐IR containing cells also containing nNOS‐IR. NMDAR1‐IR and GluR2‐IR also colocalized in the dlPAG. Of the NMDAR1‐IR containing dlPAG cells 78.6 ± 9.1% were positive for GluR2‐IR while 65.7 ± 1.7% of GluR2‐IR containing dlPAG cells were positive for NMDAR1‐IR. Our data support our hypothesis that nNOS colocalizes with NMDA and AMPA receptors in the dlPAG and provide an anatomical basis for interaction between NO and glutamate receptors in regulating cardiovascular function and responses to fear and stress by the dlPAG.Support or Funding InformationNIH R01HL088090 and VA Merit Review