Stimulation of the periaqueductal gray matter of the midbrain activates enkephalin-releasing neurons that project to the raphe nuclei in the brainstem. 5-HT (serotonin) released from the raphe nuclei descends to the dorsal horn of the spinal cord where it forms excitatory connections with the inhibitory interneurons located in Laminae II (aka the substantia gelatinosa). When activated, these interneurons release either enkephalin or dynorphin (endogenous opioid peptides), which bind to mu and kappa opioid receptors, respectively, on the axons of incoming C and A-delta fibers carrying pain signals from nociceptors activated in the periphery.

The activation of the mu-opioid receptor inhibits the release of substance P from these incoming first-order neurons and, in turn, inhibits the activation of the second-order neuron that is responsible for transmitting the pain signal up the spinothalamic tract to the ventral posterolateral nucleus (VPL) of the thalamus. The nociceptive signal is thus inhibited before reaching the cortical areas that interpret the signal as pain, such as the anterior cingulate. This is sometimes referred to as the gate control theory of pain and is supported by the fact that electrical stimulation of the PAG results in immediate and profound analgesia.^[3] The periaqueductal gray is also activated by viewing distressing images associated with pain.^[4]

Notably, the anterior cingulate cortex is thought to be responsible for emotional responses to pain, including perceived social or emotional pain. Reducing nociceptive signaling to this area not only reduces overall pain signaling, but appears to also reduce sensitivity to pain. Furthermore, activation of mu-opioid receptors has been shown to provide an "analgesic" effect for emotional pain.^[5][6]

Dorsal PAG neurons are activated during various defensive behaviors.^[7] Stimulation of the dorsal and lateral aspects of the PAG can provoke defensive responses characterised by freezing immobility, running, jumping, tachycardia, and increases in blood pressure and muscle tonus. In contrast, stimulation of the caudal ventrolateral PAG can result in an immobile, relaxed posture known as quiescence, whereas its inhibition leads to increased locomotor activity.^{[citation needed]}

Lesions of the caudal ventrolateral PAG can greatly reduce conditioned freezing, whereas lesions of the dorsal aspect can reduce innate defensive behavior, virtually "taming" the animal^{[citation needed]}.

Neurons of the PAG are excited by endorphins and by opiate analgesics. It also plays a role in female copulatory behavior (see lordosis behavior) via a pathway from the ventromedial nucleus of the hypothalamus.^{[citation needed]}

The PAG may be specifically involved in human maternal behavior. The PAG contains a high density of vasopressin and oxytocin receptors, and it has direct connections with the orbitofrontal cortex, which might mediate the role of the PAG in maternal love. The lateral orbitofrontal cortex is activated by pleasant visual, tactile, and olfactory stimuli. Its response depends on pleasantness rather than on intensity of stimulation. Here, its activity is likely to reflect one aspect of the pleasant emotions associated with motherly love.^[8]

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  Schematic representation of the chief ganglionic categories (I to V).
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  Transverse section of mid-brain at level of inferior colliculi.
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  Transverse section of mid-brain at level of superior colliculi.
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  MRI section of human mid-brain showing periaqueductal gray

• Rostral ventromedial medulla
• Emotion

• Stained brain slice images which include the "Periaqueductal gray" at the BrainMaps project