To study cholinergic input from the brainstem reticular formation (RF) in schizophrenia, we used computerized neuromorphometry to determine the number of nicotinamide adenosine dinucleotide phosphate-diaphorase (NADPHd)-labeled neurons in the pedunculopontine nucleus (PPN), a major RF cholinergic necleus. Schizophrenic patients had 60% more NADPHd+ PPN neurons than controls (p < 0.002), consistent with an increase in RF cholinergic tone. RF cholinergic systems in schizophrenia were also studied using molecular measures of the concentration of choline acetyltransferase (ChAT), an accepted index of cholinergic neurotransmission, in the mesopontine region extending beyond the limits of the PPN into more posterior areas. Young and elderly schizophrenics had reductions in the level of ChAT in the RF of 40% and 70%, respectively. Moreover, in elderly schizophrenic subjects assessed antemortem with a diagnostic battery, RF ChAT levels significantly correlated with indices of cognitive performance in several domains (r/s = 0.82 - 0.98) and tended to correlate inversely with the degree of positive psychotic symptoms (r/s = -0.64). Deregulation of RF cholinergic systems in schizophrenia may, therefore, relate to the cognitive impairment and, perhaps, the psychotic features of schizophrenia. We propose to continue our investigations in schizophrenia of RF neural systems featuring NADPHd+ and/or ChAT containing neurons using computerized neuromorphometry and Western immunoblot analyses. Enlarging the study population will permit us to validate previous neuropathological results and examine clinicopathological correlations such as that between RF ChAT and cognitive performance. We also propose to add Western immunoblot measures of nitric oxide synthase (NOS) concentration and extend our morphometric analyses of NADPHd+ neurons to the cerebellum and hippocampus because of evidence that NADPHd is a NOS that synthesizes nitric oxide (NO) which is a free radical with potential metabolic and neurotransmitter actions that could be relevant in schizophrenia. Hence, we have replicated our initial finding of an increase in the number of NADPHd+ PPN neurons in schizophrenia and complemented it with a possibly related finding of decreased RF ChAT levels in the wider mesopontine region. We now seek to examine how these changes relate to the clinical signs of schizophrenia, and to determine the possible role of NOS containing neurons in the RF and in other brain regions in schizophrenia.