Related Experiment Videos
Alteration of neuronal nitric oxide synthase activity and expression in the cerebellum and the forebrain of
M Tregnago1, M Virgili, B Monti
1Department of Biology, University of Bologna, Via Selmi 3, 40126 Bologna, Italy.
Abstract:
Microencephalic rats were obtained through gestational (for the forebrain) or neonatal (for the cerebellum) administration of the DNA-alkylating agent methylazoxymethanol acetate (MAM), which selectively kills dividing cells during neurogenesis. In the microencephalic cerebellum the specific activity of calcium-dependent nitric oxide synthase (NOS) was decreased by 35-40% at 12, 28 and 70 days of age. Other neurochemical markers not related to granule cells (the neuronal population selectively compromised by neonatal MAM treatment), choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) were not decreased, but actually increased when determined as specific activity. In agreement with the decreased catalytic activity measured in the tube, the expression of neuronal NOS protein was attenuated as judged from immunohistochemistry and Western blotting. In the microencephalic forebrain, the specific calcium-dependent NOS activity measured in homogenates of the whole hemisphere was significantly increased as compared to normal animals. Accordingly, immunohistochemistry for neuronal NOS, as well as NADPH-diaphorase histochemistry revealed an apparent increase in the density of strongly reactive neurons in the underdeveloped cortex and striatum of microencephalic rats. The results reported here demonstrate that permanent alterations of neuronal NOS activity and expression occur when the development of the brain and its neuronal circuits are severely compromised. Furthermore, the permanent downregulation of neuronal NOS in the cerebellum of microencephalic rats may be exploited for the study of the role of NO in mechanisms of synaptic plasticity such as long term depression (LTD).
Insights
Methylazoxmethanol acetate (MAM) induces microcephaly, causing permanent changes in nitric oxide synthase (NOS) activity. Cerebellar NOS decreased, while forebrain NOS increased, offering insights into brain development and synaptic plasticity.
Area of Science:
- Neuroscience
- Developmental Neurobiology
- Neurochemistry
Background:
- Methylazoxmethanol acetate (MAM) is a DNA-alkylating agent used to induce microcephaly in animal models.
- MAM selectively targets dividing cells during neurogenesis, impacting brain development.
- Nitric oxide synthase (NOS) plays a crucial role in neuronal function and development.
Purpose of the Study:
- To investigate the long-term effects of MAM-induced microcephaly on calcium-dependent nitric oxide synthase (NOS) activity and expression in the rat brain.
- To explore the relationship between compromised brain development and alterations in NOS.
- To assess the potential of these alterations for studying synaptic plasticity mechanisms like long-term depression (LTD).
Main Methods:
- Induction of microcephaly in rats via gestational or neonatal administration of MAM.
- Measurement of calcium-dependent NOS specific activity in cerebellar and forebrain homogenates at various ages.
- Assessment of other neurochemical markers, including choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD).
- Evaluation of neuronal NOS protein expression using immunohistochemistry and Western blotting.
- Histochemical analysis using NADPH-diaphorase staining.
Main Results:
- Neonatal MAM treatment led to a sustained 35-40% decrease in cerebellar NOS specific activity and protein expression.
- Forebrain NOS specific activity and the density of NOS-reactive neurons were significantly increased in microcephalic rats.
- ChAT and GAD specific activities were not decreased in the cerebellum, indicating selective effects on NOS.
- Immunohistochemistry and Western blotting confirmed reduced neuronal NOS expression in the microencephalic cerebellum.
- Increased NOS reactivity was observed in the cortex and striatum of the microencephalic forebrain.
Conclusions:
- Severe compromise of brain development during neurogenesis results in permanent alterations of neuronal NOS activity and expression.
- The observed downregulation of cerebellar NOS in microcephalic rats provides a model for studying the role of NO in synaptic plasticity, particularly LTD.
- These findings highlight the critical role of NOS in normal brain development and function.