Adequate fixative in immunohistochemistry requires not only a rapid and total immobilization of the antigen, but also a sufficient preservation of its immunoreactivity and maintenance of its accessibility to the immunochemical reagents for localization. Thus, the optimal fixation condition for a specific antigen necessitates a compromise between these opposing variables and can be determined by the preparation of a series of tissues with a progressively increasing degree of fixation. Unless the results of localization using such a series is available, one must be satisfied with adequate but less than optimal results. In the present study, this principle is demonstrated using the localization of tyrosine hydroxylase in the dopaminergic system with formaldehyde as the fixative. The rate and degree of fixation with formaldehyde was shown to be highly pH dependent. By perfusing the tissue with formaldehyde at pH 6.5 (where the rate of fixation is extremely slow) it is possible to rapidly distribute the fixative homogeneously into the tissue. By suddenly changing to a formaldehyde perfusate of higher pH, the cross-linking reaction is rapidly increased. This two-step fixation procedure provides a means of obtaining a rapid and uniform immobilization of the antigen, so that its translocation can be avoided. The final degree of fixation is controlled by the duration and pH of the second fixative solution. The results obtained by increasing the pH of the second solution demonstrated that complete fixation of tyrosine hydroxylase in the dopaminergic system with formaldehyde maybe obtained using a very basic formaldehyde solution (pH 11) while still retaining immunoreactivity of the enzyme. The localization that was achieved at lower pH appeared adequate until it was compared to the results obtained by perfusion at pH 11 in the second step.

Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase.

An antiserum to monoamine oxidase B (MAO-B) was used to define the distribution of this metabolic enzyme in the adult rat brain immunocytochemically. MAO-B is specifically located in two major central nervous system cell classes, astrocytes and serotonin-containing neurons. Double-immunofluorescence experiments using antisera to glial fibrillary acidic protein and MAO-B showed that both protoplasmic and fibrillary astrocytes throughout the brain contain MAO-B, whereas oligodendrocytes do not contain the enzyme. Areas lacking a blood-brain barrier, such as the specialized circumventricular organs, also contain MAO-B-positive cells. A double-immunofluorescence experiment using antisera to serotonin and MAO-B enabled the positive identification of neurons containing both molecules. The catecholamine-containing neurons of the brain did not contain detectable amounts of MAO-B. The specific distribution of MAO-B in the adult central nervous system indicates that the role of MAO-B in monoamine metabolism may be more specifically defined than previously believed.

https://www.pnas.org/content/pnas/79/20/6385.full.pdf

Immunocytochemical demonstration of monoamine oxidase B in brain astrocytes and serotonergic neurons

Catechol O-methyltransferase mRNA expression in human and rat brain: evidence for a role in cortical neuronal function.

Neurochemistry of the vertebrate olfactory bulb

The COMT (catechol-O-methyltransferase) gene has been linked to a spectrum of human phenotypes, including cognition, anxiety, pain sensitivity and psychosis. Doubts about its clinical impact exist, however, because of the complexity of human COMT polymorphism and clinical variability. We generated transgenic mice overexpressing a human COMT-Val polymorphism (Val-tg), and compared them with mice containing a null COMT mutation. Increased COMT enzyme activity in Val-tg mice resulted in disrupted attentional set-shifting abilities, and impaired working and recognition memory, but blunted stress responses and pain sensitivity. Conversely, COMT disruption improved working memory, but increased stress responses and pain sensitivity. Amphetamine ameliorated recognition memory deficits in COMT-Val-tg mice but disrupted it in wild types, illustrating COMT modulation of the inverted-U relationship between cognition and dopamine. COMT-Val-tg mice showed increased prefrontal cortex (PFC) calcium/calmodulin-dependent protein kinase II (CaMKII) levels, whereas COMT deficiency decreased PFC CaMKII but increased PFC CaMKKβ and CaMKIV levels, suggesting the involvement of PFC CaMK pathways in COMT-regulated cognitive function and adaptive stress responses. Our data indicate a critical role for the COMT gene in an apparent evolutionary trade-off between cognitive and affective functions.

https://www.jneurosci.org/content/jneuro/28/35/8709.full.pdf

Genetic Dissection of the Role of Catechol-O-Methyltransferase in Cognition and Stress Reactivity in Mice

Immunohistochemical demonstration of catechol-o-methyltransferase in mammalian brain.

Immunohistochemical localization of cathechol-O-methyltransferase in circumventricular organs of the rat: Potential variations in the blood-brain barrier to native catechols

An antiserum to rat liver catechol-O-methyltransferase (COMT) was utilized in the immunological characterization of COMT from rat kidney, brain, and choroid plexuses, in addition to rat liver. The presence of anti-COMT activity was confirmed by the direct inhibition of the activity of the enzyme from rat liver by small quantities of the antiserum and by the inhibition of the activity of the enzyme from rat brain. The specificity of the antiserum was demonstrated both by immunoelectrophoresis of rat liver COMT, and by a partial purification of rat liver COMT in which changes in COMT specific activity were correlated with the appearance of a precipitin line in double-immunodiffusion experiments. The antigenic similarity of the enzyme derived from rat liver, kidney, brain, and choroid plexuses was demonstrated by the formation of a precipitin line of identity when preparations from these four tissues were diffused against the antiserum.

Anti-catechol-O-methyltransferase: demonstration of specificity and immunological cross-reactivity with the enzyme from rat liver, kidney, brain, and choroid plexuses.

Catechol-O-methyltransferase (COMT) (EC 2.1.1.6) was localized using fluorescence immunohistochemistry in rat, chinchilla, and bovine brains. Specific fluorescence was visible only in ventricular ependymal cells, cells of the choroid plexuses, astrocytes, and oligodendrocytes. The patterns of localization observed in the non-neuronal elements suggest that this enzyme may function as a barrier to free diffusion of catechol compounds within the central nervous system.

Non-neuronal localization of catechol-o-methyltransferase in brain

Catechol-O-methyltransferase (COMT) has been localized to the non-neuronal elements of the central nervous systems. Its localization to pia-arachnoid and choroid plexuses imply an enzymatic barrier to prevent catechols of peripheral origin from entering the brain or CSF. The presence of COMT in ventricular ependyma suggests a restriction of free diffusion between CSF and brain parenchyma with the exception of specific areas where ependyma lacks the enzyme. Perineuronal oligodendrocytes adjacent to some cortical neurons contain COMT. The processes from these cells may protect specific catechol synapses from the more general effects of catecholamines acting by a neurohumeral mechanism.

Gary P. Kaplan

Papers

Immunohistochemical demonstration of catechol-o-methyltransferase in mammalian brain.

Immunohistochemical localization of cathechol-O-methyltransferase in circumventricular organs of the rat: Potential variations in the blood-brain barrier to native catechols

Anti-catechol-O-methyltransferase: demonstration of specificity and immunological cross-reactivity with the enzyme from rat liver, kidney, brain, and choroid plexuses.

Non-neuronal localization of catechol-o-methyltransferase in brain

Immunohistochemical localization of catechol-O-methyltransferase in brain: potential role as an enzymatic barrier