The periaqueductal gray (PAG) is important for the organization of organismal response to different types of stress and painful stimuli. Its dorsolateral (dlPAG) column is distinctly characterized by the presence of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), which in many brain regions, is an indication of constitutive nitric oxide (NO) synthase (NOS)-containing neurons. Different stress paradigms activate the dlPAG NOS machinery presumably by a presynaptic influence of NO on dlPAG neurons to modulate the nuclear dynamics to elicit an appropriate response. Since presynaptic components of synapses reside in axonal varicosities, this study assessed the number of varicosities and inter-varicosity spacing of NADPH-d neurons in the dlPAG of free-behaving (control) and acutely restrained male rats. The study tested the hypothesis that stress-induced increase in endogenous NO synthesis involved changes in synaptic density and inter-varicosity spacing and therefore, a non-synaptic component of NO involvement in the dlPAG response to stress. Compared with control, the number of NADPH-d-positive cells, the staining intensity and the number of varicosities per microgram tissue were significantly higher in restrained animals. Also, the inter-varicosity spacing was significantly higher in control than restrained rats, presumably due to the increase in varicosities induced by restraint. Since neural connectivity and synaptogenesis depend on mean varicosity spacing and pattern of varicosity, respectively, the present observations suggest a mechanism whereby restraint stress induces increased activity via synaptic and non-synaptic NO-mediated neurotransmission within the dlPAG.