Histochemical changes in the blood cells of schizophrenic patients under pimozide treatment.

Chromatin structure and nucleohistone pattern were investigated histochemically in the neutrophils of 11 schizophrenics and 16 healthy controls. Compared to controls, all schizophrenic parents prior to medication showed a distinctly different histochemical pattern consisting of increased concentration and abnormal distribution of nucleohistones. This pattern has been attributed to an increase of arginine-rich histones in schizophrenics. Pimozide administration exerted a normalizing effect on the nucleohistone distribution pattern. These findings further support our view that genomic expression abnormalities may be related to schizophrenic illness.

Histochemical protein markers of monoamine cell bodies in man.

Neurons in brainstem and hypothalamus of postmortem, formaldehyde-fixed tissue of 25 healthy controls were studied with a double-staining procedure for the consecutive demonstration of Nissl material and of cytoplasmic spherical bodies, which are rich in basic proteins. The investigation established that the protein bodies are located in cells that correspond to the aminergic neurons demonstrable by histofluorescence. Protein bodies were also found in the neurons of the inferior olive, which are not as a rule included in the aminergic cell groups. The localization of protein bodies is perikaryal and dendritic and, thus, parallels the distribution of monoamines and monoamine-synthesizing enzymes reported in experimental animals. The invariable presence of protein bodies in all aminergic perikarya of control human brains and their absence from nonaminergic neurons permits us to consider them markers of monoamine neurons in man.

Protein-rich cytoplasmic bodies of substantia nigra and locus ceruleus. A comparative study in parkinsonian and normal brain.

A histochemical study of substantia nigra and locus ceruleus from postmortem brains showed the presence of small spherical cytoplasmic bodies stained selectively by the anionic phosphotungstic acid-hematoxylin (PTAH) stain at a pH of 2.5. The metachromatic reaction to PTAH indicates that these protein bodies contain a protein rich in free basic amino groups. The protein bodies are localized within the neuronal perikaryon as well as in their dendritic processes. These bodies abundantly present in the substantia nigra and locus ceruleus of normal brains were noticeably reduced or absent in parkinsonian brains. Lewy bodies when present show that their core gives the same metachromatic reaction to PTAH as do the protein bodies. These findings suggest that an abnormality of protein synthesis in the substantia nigra and locus ceruleus of parkinsonian brains may be related to the absence of protein bodies and the formation of Lewy bodies and play a role in pathogenesis of the parkinsonian state.

Arginine-rich proteins in spherical inclusions of human locus coeruleus neurons demonstrated by benzil modification.

Previous studies have shown that aminergic neurons in the normal human brain contain acidophilic cytoplasmic inclusions--called protein bodies (PBs)--that are reduced or absent in parkinsonism and disrupted in depression. The purpose of the present study was to elucidate the constitution of PBs in five formalin-fixed normal human brains using histochemical methods specific for histones, protamines, and the amino acid arginine. PBs were revealed with alkaline fast green and bromphenol blue, exhibiting a high content in histones and in protamines. They developed blue metachromasia with phosphotungstic acid-hematoxylin and green fluorescence with phenanthrenequinone, which established the presence of arginyl residues. Using benzil, which selectively modifies the guanido group of arginine, staining was blocked for each of the above two methods. The application of Mallory's trichrome procedure after benzil differentiated the PBs into an unstained core and a still fuchsinophilic rim. Since the fuchsinophilia of the rim was shown to persist after acetylation as well, we suggest that this rim probably contains acidic macromolecules that attach to the basic charges of the amphoteric acid fuchsin. We conclude that the PB are complex structures consisting of a core segregating arginine-rich proteins and a rim which probably contains macromolecules of an acidic nature.

Tyrosine hydroxylase-immunoreactive neurons in paraventricular and supraoptic nuclei of the human brain demonstrated by a method adapted to prolonged formalin fixation.

We studied the distribution of tyrosine hydroxylase (TH) immunoreactive (IR) neurons in the adult human hypothalamus using a modification of the peroxidase-antiperoxidase immunohistochemical method which can be applied on autopsy brain material following prolonged formalin fixation. We observed that most of the TH-IR perikarya localized within the paraventricular (PVN) and supraoptic (SON) nuclei were large and showed homogeneous staining over the entire cytoplasm and processes. These results show that in the human brain a large population of neurons within the neurosecretory nuclei are able to synthesize a catecholamine.

Peripheral blood lymphocytes of bipolar affective patients have a histone synthetic profile indicative of an active cell state.

1. Although abnormalities of the immune system have been described in depression, no information exists regarding the biochemical parameters which could characterize the physiological state of lymphocytes from patients with bipolar affective disorder. 2. Lymphocytes of normal control subjects are known to be in the Go resting phase of the cell cycle. Histone synthesis is characteristically different during the Go, G1/G2 and the S phases of the cell cycle. As such, it can be used as a biochemical marker with which to distinguish between cycling and noncycling cells. 3. In order to investigate the possibility of whether or not the lymphocytes of patients with bipolar affective disorder are in an activated state, typical of cycling cells, total histone and histone variant synthesis were analysed in peripheral blood lymphocytes of a group of 12 patients with bipolar affective disorder and 7 normal controls. 4. According to the histone variant synthesis pattern, lymphocytes of patients in normothymia have values similar to those of controls, i.e., of noncycling cells, while patients in either the depressed or the manic phase have values intermediate to those of resting and cycling cells. 5. This study shows that histone synthesis can perhaps be used as a biochemical parameter of possible significance in differentiating amongst the three phases of the illness.

Distribution of ubiquitin immunoreactivity in dopamine neurons of chronic alcoholics.

In animal experiments drugs of abuse, including alcohol, have been shown to stimulate and affect dopamine neurons, which mediate their rewarding effects. In this study, the aim was to establish cellular effects of alcohol on human dopamine neurons, by investigating midbrain from 27 chronic alcoholics (33-84 years old) and 10 matched controls. We used polyclonal antibodies to ubiquitin, a marker which localizes lesions of the cellular stress caused by alcohol, on paraffin sections. Results showed that the grading of ubiquitin immunoreactivity (UBQ-IR) distribution differentiated two groups of alcoholics. The first was characterized by low average age and liver cirrhosis, the second by greater average age and absence of cirrhosis. In the first group UBQ-IR was distributed in dendrites and cell bodies of dopamine neurons, mostly along their membranes. In the second group dopamine neurons were negative, UBQ-IR was confined to dystrophic neurites in the neuropil. These data indicate that human dopamine neurons in chronic alcoholics of the first group, are metabolic targets of the stressful action of alcohol, revealed by UBQ-IR. The results also provide evidence that the second group of users, lacking cellular localization of UBQ-IR, have developed protective compensatory mechanisms to the membrane disruption caused by alcohol, due to a different genetic constitution.

The fine structure of large dense-core organelles in human locus coeruleus neurons.

Protein bodies, the characteristic spherical organelles present in human monoamine neurons, have been shown in previous electron microscope studies to originate as dense bodies in mitochondria. This study was designed to investigate the presence of catecholamine reaction products in the dense bodies of locus coeruleus neurons, in frozen fresh post-mortem brain tissue with the use of potassium permanganate (KMnO4) fixation. This fixation procedure forms a dense KMnO4/catecholamine reaction product, visible in the electron microscope, in the large dense-core vesicles of experimental animals. Our results demonstrate the localization of KMnO4 dense product in the cores of double membrane-bound spherical organelles, as well as in spherical structures in the matrix of typical mitochondria. No typical large dense-core vesicles were observed in these catecholamine neurons of the tissues studied. Our findings are consistent with the notion that altered mitochondria may contribute to the formation of a new type of large dense-core vesicle in the locus coeruleus neurons of man, which is probably an evolutionary adaptation of amine-storing organelles.

Similarities between neuronal Lewy bodies in parkinsonism and hepatic Mallory bodies in alcoholism.

The aim of the present study was to identify components of the Lewy body, which is a characteristic neuronal lesion in idiopathic Parkinsonism, using histochemical methods that selectively stain the Mallory body, a characteristic lesion of the hepatocyte in alcoholism. Our observation that Lewy bodies stain with phosphotungstic acid hematoxylin, the dye originally used for demonstrating alcoholic hyaline (Mallory bodies), promoted this study. The material consisted of formalin-fixed, brain stem tissue from Parkinsonian subjects, and of similarly preserved liver tissues from alcoholic individuals. The methods selected were Roque's chromotrope 2R-aniline blue, and Liisberg's rhodamine B, which stains Mallory bodies due to its affinity for sites of tissue keratinization. Hence, skin was also included in this study as control tissue. Our results showed that Lewy bodies in the brain, Mallory bodies in the liver and stratum corneum in the skin have identical staining properties with the dyes used, indicating the presence of histochemically similar components. Taking into account the reactions of these dyes with model substances, we suggest that the similar components shared by Lewy bodies and Mallory bodies are arginine-rich proteins and lipids associated with keratinization. Similar findings in both, a toxin-induced lesion of the liver, and a spontaneous lesion of the brain may offer clues for understanding the latter's mode of formation.

Lithium carbonate and the problem of kidney damage. Intracellular effects in rat kidney slices.

Histological lesions in man, presumably indicating kidney damage, may occur during therapy with lithium carbonate. We studied the cellular effects of lithium carbonate applied in vitro to slices of kidney from adult female Sprague-Dawley rats. Alternate slices were immersed in saline solution containing 1.1 mEq/L of lithium carbonate; the other slices were immersed in ordinary saline solution as controls. Compared with control specimens, a two-hour incubation in lithium carbonate solution showed (1) with toluidine blue 0, in the cells of the collecting ducts a fibrous metachromatic network that was interpreted as indicating microtubule bundles; and (2) with Brookes' stain in the proximal convolutions, increased acidophilia of the hyaline droplets and of the nucleus that implied interaction of lithium carbonate with the protein transport and degradation mechanism located in these cells. The results can be interpreted as being due to polymerizations or to conformational changes of protein components caused by lithium. These changes are consistent with altered functions of the kidney observed in manic-depressive patients receiving treatment with lithium carbonate, which lead to polyuria (collecting ducts) and may lead to nephrotoxicity (proximal convolutions).

Histochemical marker of human catecholamine neurons in ganglion cells and processes of a temporal lobe ganglioglioma.

A temporal lobe ganglioglioma was surgically removed from an 8-year-old boy who had developed temporal lobe paroxysmal convulsions. The present study describes staining reactions of the ganglioglioma tissue with histochemical methods designed to demonstrate the protein bodies, which are markers of catecholamine neurons in humans. Brookes' procedure for basic proteins and rhodamine B for lipids showed identical staining of the following: (1) spherical inclusion bodies in the neoplastic neurons, (2) large eosinophilic globules in the dilated neuronal processes, and (3) the protein bodies in the neurons of human locus ceruleus and sympathetic ganglion. We conclude that the catecholamine neurons of the ganglioglioma possess the same marker of aminergic identity as normal catecholamine neurons in humans. Thus, this marker could be used as an additional diagnostic tool for the identification of this type of tumor.

Histochemical changes of peripheral blood leukocytes during lithium treatment.

The effect of lithium on the histochemistry of blood granulocytes was studied in 16 manic-depressive patients during lithium prophylaxis. Blood smears obtained before, at 2 months and at 12 months after initiation of treatment showed that lithium caused condensation of the chromatin in granulocytes, conformation changes in eosinophilic granules and the disappearance of basic proteins found in the cytoplasm of eosinophils in the drug-free patients. The latter findings are interpreted as indicating the involvement of lithium in histamine metabolism.

Short and long-term effects of neuroleptics in relation to their cellular mechanism of action.

1. Some mechanisms of action of neuroleptics at cellular level are reviewed, mainly the effects on synaptic transmission and the effects on chromatin. 2. As regard to the effects at synaptic level, a brief review is presented on the available evidence in support of the currently prevailing dopamine hypothesis. 3. Studies carried out on the mechanisms and sites of action of neuroleptics on chromatin show that: a. Behavioral changes caused by psychotropic drugs in experimental animals are associated with chromatin alterations and induced macromolecular syntheses. b. Parkinsonian and possibly drug-induced extrapyramidal symptoms are associated with aberrations in protein synthesis. c. Destabilization under "stress" of the heterochromatin in schizophrenics seems to be due to histone modifications and is partly prevented by neuroleptic treatment.

Fine structure of neuronal spherical arginine-rich bodies of substantia nigra and locus coeruleus in the human brain.

Neuronal spherical bodies, rich in arginine, of catecholamine neurons in man display staining reactions of mitotic chromosomes and myelin basic protein. They show a unique fine structure and density in the EM after phosphotungstic acid hematoxylin block-staining. With an electron-lucent core, a dense rim and a limiting double membrane they stand out and are differentiated from all other neuronal inclusions, especially melanin. Protein bodies were found inside mitochondria, where they apparently originate as small globules in the matrix. They later enlarge into spheres by obliterating the cristae, but retaining the outer membranes of the parent mitochondrion. The arginine-rich basic protein of the spherical bodies, it is argued, may be involved in the modulation of excitability of the catecholamine neurons in man.

Dispersed and compact chromatin demonstrated with a new EM method: phosphotungstic acid hematoxylin block-staining.

Glutaraldehyde/KMnO4 double fixation and phosphotungstic acid hematoxylin (PTAH) block-staining, before dehydration were found to reveal, with great detail and sharpness, the nuclear distribution of compact heterochromatin masses as electron-lucent patches. By contrast the areas of decondensed and dispersed chromatin acquired a high electron density due to the binding of the large PTAH molecule to basic groups in the loosened chromatin network. The method was tested on human blood leukocytes, on the thymus gland from immature rats, containing mitotic figures, and on mature avian erythrocytes. The results indicated that each cell type acquires a specific pattern of electron densities in the nucleus which depends upon the relative amounts of compact and dispersed chromatin present in that nucleus. Since the tissues are stained in-block immediately after fixation, artifacts of stain localization, due to alcohol dehydration, are avoided. Thus, PTAH block-staining "translates" the state of aggregation of the chromatin into characteristic and specific density patterns of the nuclei. This method may prove useful in differentiating active from inactive portions of the genome, at the ultrastructural level.

Localization of phospholipids in human catecholamine neurons with Baker's acid haematein.

Protein bodies in catecholamine neurons of the normal human brain contain arginine-rich proteins similar to those present in the core of Lewy bodies in Parkinsonian brains. Lewy bodies are known also to contain phospholipids in their core, demonstrable with Baker's acid haematein. We used Baker's procedure on catecholamine neurons of normal brains to test whether the protein bodies also contain phospholipids. Postmortem tissues of three control individuals were used in this study. Locus coeruleus and substantia nigra were dissected out from the fresh brains and two sets of blocks were made from each area. One set was fixed in formol-calcium for the preservation of lipids, the second was fixed in Bouin's solution and treated with hot pyridine for the extraction of lipids. Finally, both sets were subjected to the same chroming and staining procedure according to Baker. In catecholamine neurons the protein bodies were stained with acid haematein, indicating the presence of phospholipids. Since this staining resisted pyridine extraction we conclude that these phospholipids are firmly bound to the basic proteins. Thus, protein bodies in healthy catecholamine neurons give the same positive reaction for phospholipids as that reported for the core of Lewy bodies in damaged neurons in Parkinsonism.

Modifications of female sex chromatin (Barr body) in rat neurons after reserpine administration.

3 groups of rats were sacrificed 30 min, 4 h and 24 h after reserpine (10 mg/kg i.p.) injection. Toluidine blue stained sections showed that in the motor neurons of the spinal cord the drug, at 4 h, had induced a migration of the Barr body from the nucleolus to the nuclear membrane and an increase in its size and RNA concentration. From our findings we suggest that reserpine may have an activating role on X-linked genes.

Lewy bodies in parkinsonism share components with intraneuronal protein bodies of normal brains.

Histochemical characteristics of the Lewy bodies, in catecholamine neurons of 10 Parkinsonian patients, were compared to those of the spherical protein bodies, the basic protein-rich markers of catecholamine neurons in man. Special methods for proteins and lipids showed that the core of the Lewy bodies, in the neurons of the locus coeruleus and the substantia nigra, contains basic proteins and lipids normally found in the protein bodies. Acid fuchsin and the lipid-soluble fluorescent dye rhodamine B stained the entire core of the Lewy body in the parkinsonian brains and the entire sphere of the protein body in the control brains. Bromsulfophthalein, another acidic dye, which selectively binds to the enzyme gluthathione-S-transferase, had affinity only for a ring-like lamina at the outer layer of the core of the Lewy body and for the outer rim of the protein body. These results demonstrate that Lewy bodies and protein bodies contain similar macromolecular components, that is lipids and two different types of proteins, which also show similar stratification in the two structures. On the other hand, the presence in several neurons of the Parkinsonian patients, of aggregates representing transitional forms between protein bodies and Lewy bodies, indicates that abnormalities of protein bodies precede, and are somehow linked to Lewy body production.