Immunohistochemical localization of the catecholamine-synthesizing enzymes,substance P and enkephalin in the human fetal sympathetic ganglion

Summary The human fetal sympathetic ganglia were studied using the indirect peroxidase-antiperoxidase PAP method for immunocytochemical demonstration of three catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) as well as the neuropeptides leucine (Leu⁵)-enkephalin and substance P. The neuroblasts of the ganglia showed intense peroxidase immunoreactivity for TH, moderate reaction to DBH, and no reaction to PNMT. The small intensely fluorescent (SIF) cells situated along the blood vessels also showed positive labelling for only two enzymes, TH and DBH. The immunocytochemical localization of these enzymes suggests that both neuroblasts and SIF cells synthesize noradrenalin. Neither the neuroblasts nor SIF cells showed a reaction to substance P, and only the SIF cells contained enkephalin-like immunoreactivity. The role of enkephalin in the noradrenalin-containing SIF cells is unknown, but may be related to neuromodulation of ganglionic transmission.     

Developmental change of an enzyme activity oxidizing γ-aminobutyraldehyde to γ-aminobutyric acid in the chick embryonic brain

An enzyme activity oxidizing -aminobutyraldehyde (ABAL) to GABA reflecting an alternative pathway for GABA synthesis was assayed in the developing chick embryonic brain and was compared with glutamate decarboxylase (GAD) activity. An enzyme activity oxidizing ABAL to GABA showed almost constant level during development in the chick embryonic brain, and was present at low levels compared with GAD activity. The results indicate that GABA synthesis via an alternative pathway is always much less than synthesis via the GAD-dependent pathway in the developing chick embryonic brain.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Phenylethanolamine-N-methyltransferase and dopamine-β-hydroxylase immunoreactivity and the occurrence of noradrenaline and adrenaline in the nervous system of the snail Helix aspersa

Summary The presence of dopamine--hydroxylase (DBH) and phenylethanol-amine-N-methyltransferase (PNMT) immunoreactivity in specific neurones of the snail Helix aspersa has been demonstrated. In addition, high performance liquid chromatography and electrochemical detection have revealed the presence of noradrenaline and adrenaline in the snail central nervous system, although the major catecholamine is dopamine. These results suggest that adrenaline, and perhaps noradrenaline, have transmitter or modulatory functions in the snail nervous system.     

Effect of hydrocortisone on catecholamines and the enzymes synthesizing them in the developing sympathetic ganglion

Summary Newborn rats were daily injected with 0.2 mg hydrocortisone acetate for seven days. They were killed 1, 7 or 21 days after the last injection, together with untreated controls. Hydrocortisone caused a great increase in the number of the small, intensely fluorescent (SIF) cells and the appearance of similar small cells with intense immunohistochemical reactions for tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine (noradrenaline)N-methyltransferase (PNMT) in the superior cervical ganglion. At the same time, the adrenaline content and the PNMT activity of the ganglion greatly increased, while no significant changes were observed in the dopamine or noradrenaline content or TH or DBH activity. All these changes essentially disappeared after a recovery period of seven or 21 days.It is concluded that hydrocortisone causes a temporary increase in the number of SIF cells by causing a synthesis of TH, DBH and PNMT in previously existing small, non-fluorescent cells, which start to synthesize and store adrenaline, thus becoming intensely fluorescent SIF cells. These SIF cells are different from the normal SIF cells of the same ganglion, most of which appear at a later stage of postnatal development when response to hydrocortisone is lost, which contain TH but neither DBH nor PNMT, and which permanently remain in the ganglion.     

Immunocytological localization of dopamine in the guinea pig retina

We examined dopaminergic neurons in the guinea pig retina; antisera against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and an antiserum against gamma-aminobutyric acid (GABA) were used. In the present study, two types of amacrine cells were labeled with an anti-TH antiserum. However, no DBH and PNMT immunoreactivities were seen. The type 1 cell had a larger-sized soma located in the inner nuclear layer with processes ramifying mainly in stratum 1 of the inner plexiform layer (IPL). The type 2 cell had a smaller-sized soma and processes branching in stratum 3 of the IPL. The mean densities were 56.4 +/- 11.5/mm2 for the type 1 cell and 166.6 +/- 30.3/mm2 for the type 2 cell. Double immunocytochemistry using an antiserum against GABA revealed that while none of the type 1 cells showed GABA immunoreactivity, all of the type 2 cells displayed GABA immunoreactivity. Our results suggest that, in the guinea pig retina, the type 1 amacrine cells are pure dopaminergic and the type 2 cells are dopaminergic elements that use GABA as their second transmitter.     

Catecholamines and catecholamine-synthesizing enzymes in guinea-pig sensory ganglia

Summary Cranial and spinal sensory ganglia of the guinea-pig were investigated by means of histochemistry and biochemistry for the presence of catecholamines and catecholamine-synthesizing enzymes. Sensory neurons exhibiting immunoreactivity to the rate-limiting enzyme of catecholamine synthesis, tyrosine nydroxylase (TH), were detected by immunohistochemistry in lumbo-sacral dorsal root ganglia, the nodose ganglion and the petrosal/jugular ganglion complex. The carotid body was identified as a target of TH-like-immunoreactive (TH-LI) neurons by the use of combined retrograde tracing and immunohistochemistry. Double-labelling immunofluorescence revealed that most TH-LI neurons also contained somatostatin-LI, but TH-LI did not coexist with either calcitonin gene-related peptide- or substance P-LI. TH-LI neurons did not react with antibodies to other enzymes involved in catecholamine synthesis, i.e., aromatic amino acid decarboxylase (AADC), dopamine--hydroxylase (DH), and phenylethanolamine-N-methyltransferase (PNMT). Petrosal neurons as well as their endings in the carotid body lacked dopamine- and L-DOPA-LI. Sensory neurons did not display glyoxylic acid-induced catecholamine fluorescence. Ganglia containing TH-LI neurons were kept in short-term organ culture after crushing their roots and the exiting nerve in order to enrich intra-axonal transmitter content at the ganglionic side of the crush. However, even under these conditions, catecholamine fluorescence was not detected in axons projecting peripherally or centrally from the ganglia. Sympathetic noradrenergic nerves entered the ganglia and terminated within them. Accordingly, biochemical analyses of guinea-pig sensory ganglia revealed noradrenaline but no dopamine. In conclusion, catecholamines within guinea-pig sensory ganglia are confined to sympathetic nerves, which fulfill presently unknown functions. The TH-LI neurons themselves, however, lack any additional sign of catecholamine synthesis, and the presence of enzymatically active TH within these neurons is questionable.     

Protonmotive force in Brevibacterium flavum: the lack of intracellular pH homeostasis

Brevibacterium flavum 22LD-P cells were shown to maintain a transmembrane pH gradient (pH) from 0.6 to 1.8–2 units and a transmembrane electric potential difference () from 0 to 200 mV depending on the pH and ionic composition of the incubation medium, grwoth substrate and concentration of cells. decreased from 120–140 mV to 0 when medium pH was lowered from neutral to 5.0–5.5 and increased to 180–200 mV when medium pH was raised to 8–9 in cells utilizing acetate or endogenous substrate. Cells growing on sucrose, kept around 100–120 mV at neutral as well as acidic medium pH. Intracellular pH in the acetate utilizing or endogenously respiring cells was maintained with the range of 8.9 to 5.5 at medium pH ranging from 9.1 to 4.0, respectively. Sucrose grown cells were able to maintain a more stable intracellular pH. Endogenously respiring cells in potassium phosphate buffer at high biomass concentrations maintained larger pH and relatively smaller , than the same cells in diluted suspensions. Cells in sodium phosphate buffer possessed larger and almost no pH, but was still dependent on biomass concentration.The lack of intracellular pH homeostasis and the collapse of at acid medium pH are discussed in the context of cell membrane proton permeability.     

The γ-Glutamyl Transpeptidase Inhibitor Acivicin Preserves Glutathione Released by Astroglial Cells in Culture

The release of glutathione from astroglial cells was investigated using astroglia-rich primary cultures prepared from the brains of newborn rats. These cells release glutathione after onset of an incubation in a glucose-containing minimal medium. The amount of extracellular glutathione increased with the time of incubation, although the accumulation slowed down gradually. An elevated rate of increase of the glutathione concentration in the incubation medium was found if the astroglial ectoenzyme -glutamyl transpeptidase was inhibited by acivicin. The activity of -glutamyl transpeptidase in astroglia-rich primary cultures, which was found to be 1.9 ± 0.3 nmol/(min × mg protein), was markedly reduced if the cells had been incubated in the presence of acivicin. After 2 h of incubation with acivicin half-maximal and maximal inhibition of -glutamyl transpeptidase activity was found at concentrations of about 5 M and 50 M, respectively. In the presence of acivicin at a concentration above 10 M the glutathione content found released from astroglial cells apparently increased almost proportional to time for up to 10 h. Under these conditions the average rate of release was 2.1 ± 0.3 nmol/(h × mg protein) yielding after a 10 h incubation an extracellular glutathione content three times that of the medium of cells incubated without inhibitor. Half-maximal and maximal effects on the level of extracellular glutathione were found at 4 M and 50 M acivicin, respectively. After a 10 h incubation with acivicin the intracellular content of glutathione was reduced to 75% of the level of untreated astroglial cultures. These results suggest that glutathione released from astroglial cells can serve as substrate for the ectoenzyme -glutamyl transpeptidase of these cells.     

Immunocytochemical localization of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method

Summary The subcellular locilazations of tryrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.     

Appearance of tyrosine hydroxylase,aromatic amino-acid decarboxylase,dopamine β-hydroxylase and phenylethanolamine N-methyltransferase during the ontogenesis of the adrenal medulla

Summary The cellular localization of the enzymes tyrosine hydroxylase (TH), aromatic amino-acid decarboxylase (or dopa decarboxylase, DDC), dopamine -hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla of adult rats and rat fetuses (14th, 17th, 18th, 19th and 21st day) was examined. In the prenatal stages the medullary blastema and an adjacent part of the primitive sympathetic trunk were also investigated. Tissues were fixed in ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Cryostat sections (10 m in thickness) were stained by the indirect immunofluorescence technique. Rabbit antibodies to TH (isolated from human pheochromocytoma), DDC, DBH and PNMT (the latter three isolated from bovine adrenal medulla) were used. Sections incubated with serum of non-immunized rabbits were used as controls.In the adult adrenal medulla, two cell types can be distinguished. One cell type contains only TH, DDC and DBH. The other cell type contains PNMT in addition. It is concluded that these cells correspond to the noradrenaline-(NA-) and adrenaline-(A-)storing cells respectively. In all prenatal stages TH, DDC and DBH are found in the primitive sympathetic trunk, in the medullary blastema, and in the medullary cells which have migrated into the cortical anlage. PNMT is observed for the first time on the 18th day. Moreover, PNMT could only be demonstrated inside the adrenal gland. From these observations it is concluded that the capacity to synthesize NA is developed even before the medullary cells have reached the cortical anlage. On the contrary, the capacity to synthesize A seems to be acquired only after this contact is established. The hypothesis is put forward that this phenomenon might indicate the induction of PNMT by glucocorticoids secreted by the fetal cortex.This study was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) and by the Swedish Medical Research Council (04X-2887-10C). Its results have in part been reported at the 105th Meeting of the Dutch Anatomical Society (Abstract: Acta morphologica neerlando-scandinavica, 14, 251, 1976)     

Compartmentation of α- and β-Carbonic Anhydrases in Cells of Halo- and Alkalophilic Cyanobacteria Rhabdoderma lineare

The organization of carbonic anhydrase (CA) system in halo- and alkalophilic cyanobacteria Rhabdoderma lineare was studied by Western blot analysis and immunocytochemical electron microscopy. The presence of extracellular -CA (60 kD) in the glycocalyx, forming a tight sheath around the cell, and of two intracellular -CA is reported. One -CA (60 kD) is associated with polypeptides of photosystem II (PSII) and is a constitutive enzyme. Another -carbonic anhydrase (25 kD) was induced by low content of bicarbonate in the culture medium; this inducible CA was found in the fraction of total soluble proteins. The expressed synthesis of inducible -CA was accompanied by the increase in the intracellular pool of inorganic carbon, which suggests an important role of this enzyme in the functioning of CO₂-concentrating mechanism.     

Immunocytochemistry of GABA and glutamic acid decarboxylase in the thoracic ganglion of the crab Eriphia spinifrons

We have used specific antisera against protein-conjugated -aminobutyric acid (GABA) and rat-brain glutamic acid decarboxylase (GAD) in immunocytochemical preparations to study the distribution of putatively GABAergic neurons in the fused thoracic ganglion of the crab Eriphia spinifrons. In the thoracic neuromeres, about 2000 neurons with somata arranged in clusters or located singly in the cell cortex exhibited both GABA-like and GAD-like immunoreactivity. In addition, more than a hundred cells showed only GABA-like immunoreactivity. Fibrous immunoreactive staining to GAD and GABA was distributed throughout the neuropil of the thoracic ganglion, and several fiber tracts contained immunoreactive processes. Sets of serially homologous neurons exhibited GABA-like and GAD-like immunoreactivity in the thoracic neuromeres. Especially prominent were one medial and four ventro-lateral clusters of somata, together with thirteen individually recognized cells in each neuromere. Six of these cells in the ventro-medial cell cortex may be the somata of inhibitory motoneurons. The leg nerves contained three immunoreactive fibers, corresponding to the previously described common inhibitory motoneuron and the two specific inhibitors. The results present further evidence for GABA being the neurotransmitter of all inhibitory leg motorneurons, and suggest its presence and role as a neurotransmitter in a considerable number of interneurons in the thoracic ganglion of the crab.     

Localization of l-glutamate decarboxylase immunoreactivity in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex of the rat

Summary The localization of l-glutamate decarboxylase (GAD), the GABA-synthesizing enzyme, was studied in the rat major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex by indirect immunofluorescence technique with a specific antiserum raised in rabbits. GAD immunoreactivity was demonstrated in small cells of these ganglia. The GAD-immunoreactive small cells were 10–20 m in diameter and formed clusters or occured as solitary cells. The principal neurons were non-reactive but they were surrounded by immunoreactive processes. Studies on colocalization of GAD with tyrosine hydroxylase (TH), the rate-limiting enzyme of the catecholamine synthesis, in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex indicated that all GAD-immunoreactive small cells were also labelled with TH. In the major pelvic ganglion all TH-immunoreactive SIF cells were also immunoreactive for GAD. However, in the coeliac-superior mesenteric ganglion complex there occured TH-immunoreactive small cells which showed no immunoreactivity to GAD. It is suggested that the small GAD-immunoreactive cells represent small intensely fluorescent (SIF) cells.     

Role of protein phosphorylation in regulation of brainL-glutamate decarboxylase activity

In the brain, the -aminobutyric acid (GABA) level is primarily controlled by the activity of its synthesizing enzyme,L-glutamate decarboxylase (GAD). At present, mechanisms responsible for regulation of GAD activity remain largely unknown. Here we report that GAD activity is inhibited by conditions favoring protein phosphorylation, and this inhibition can be reversed by phosphatase treatment. Furthermore, this inhibition appears to result from the suppression of a Ca²⁺-dependent phosphatase. Phosphorylation of GAD is demonstrated by direct incorporation of³²P into the GAD protein. These results suggest that GAD activity in the brain is inhibited by phosphorylation and activated by dephosphorylation. A model for regulation of GABA synthesis related to neuronal excitation is discussed.     

Immunocytochemical study of tyrosine hydroxylase and dopamine β-hydroxylase immunoreactivities in the rat pancreas

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine -hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory acitivity of the acinar cells, duct cells and endocrine cells.     

Inability to produce a model of dialysis encephalopathy in the rat by aluminum administration

We attempted to produce a rat model of brain aluminum toxicity in order to explore whether or not aluminum accumulation produces the neurochemical changes observed in brains of patients who die with dialysis encephalopathy. Daily subcutaneous injection of Al(OH)₃ caused marked elevation of serum aluminum concentrations, but did not increase brain aluminum contents, either in rats with normal renal function, or in rats with unilateral or 5/6 nephrectomies. LiCl pretreatment, which has been reported to cause irreversible renal failure, did not impair renal function nor aid in achieving elevated brain aluminum contents. No reductions in brain contents of -aminobutyric acid (GABA) or in glutamic acid decarboxylase (GAD, E.C.4.1.1.15) and choline acetyltransferase (ChAT, E.C.2.3.1.6) activities were observed in aluminum-treated rats. We conclude that the rat is not a suitable laboratory animal to explore the role of aluminum toxicity in causing the GABA and ChAT deficits present in brains of hemodialyzed human patients.     

Glutamic acid decarboxylase-positive neuronal cell bodies and terminals in the human cerebellar cortex

The distribution of -aminobutyric acid (GABA) in the human cerebellar cortex was studied using immunohistochemistry for glutamic acid decarboxylase (GAD), the enzyme that catalyses GABA synthesis. Observations by light microscopy revealed, in all layers of the cerebellar cortex, strong, punctate positivity for GAD, related to putative GABAergic nerve terminals, as well as a diffuse cytoplasmic immunoreactivity within neuronal cell bodies. GAD-positive nerve terminals were found in close relationship with the walls of the cerebellar cortex microvessels. Observations by electron microscopy revealed positive nerve terminals in contact with the astrocyte perivascular sheath of capillaries. GAD immunoreactivity was also detected within astroglial perivascular endfeet and endothelial cells. The findings provide further insights into the GABAergic synapses of the circuitry of the human cerebellar cortex. The detection of vascular GAD immunoreactivities suggests that GABAergic mechanisms may regulate cerebellar microvessel function.     

Convulsions and inhibition of glutamate decarboxylase by pyridoxal phosphate-γ-glutamyl hydrazone in the developing rat

We have previously shown that in the adult rat the inhibition of brain glutamate decarboxylase (GAD) activity by pyridoxal phosphate--glutamyl hydrazone (PLPGH) administration does not result in convulsions, whereas in the adult mouse intense convulsions invariably occur. In the present study we report that, surprisingly, immature rats from 2 to 20 days of age treated with PLPGH (80 mg/kg) showed generalized tonic-clonic convulsions, whereas no convulsions at all were present in 30 days-old or older rats. GAD activity, measured by enzymic determination of GABA formed in forebrain homogenates, was inhibited by about 60% at the time of convulsions in 15 days-old and younger rats, whereas the inhibition was between 40 and 50% in older animals. The addition of the coenzyme pyridoxal 5-phosphate to the incubation medium completely reversed this inhibition. In all treated animals GABA levels were lower compared to controls. The results indicate that the susceptibility of GAD in vivo to a diminished cofactor concentration decreases with age. It seems possible that changes in the expression of enzyme forms are reflected in developmental variations in the susceptibility to seizures induced by vitamin B₆ depletion, but alterations of other B₆-dependent biochemical pathways cannot be discarded.     

The Activities of Six Exo- and Endopeptidases in the Substantia Nigra,Neostriatum, and Cortex of the Rat Brain

We determined the enzymatic activity and crude subcellular distribution of four exopeptidases: Dipeptidylaminopeptidase IV (DAP-IV), Alanyl aminopeptidase (AAP), Prolyl aminopeptidase (PAP) and -Glutamyl transpeptidase (GTP), and two endopeptidases: Postproline endopeptidase (PEP) and Trypsin-like peptidase (T-L P) in pars compacta (SNPC) and pars reticulata (SNPR) of substantia nigra, caudate-putamen (CAU) and cerebral cortex (CC) of the rat brain. We found: 1) DAP-IV activity is comparatively higher in SNPC and it is equally distributed in the postmitochondrial precipitate (PR) and supernatant (SN) fractions of SNPC, CAU and CC but higher in the SN from SNPR. 2) CC shows the highest activity of AAP and its activity is mainly located in the SN from all areas. 3) The activity of PAP is comparatively higher in SNPC and it is exclusively located in the SN from all areas. 4) GTP activity is similar in all areas but its predominance is in the SN for SNPC and SNPR, and in the PR for CAU and CC. 5) CAU has higher PEP activity (higher in the PR) than CC (higher in the SN); no activity is detected in the substantia nigra. 6) The activity of a Trypsin-like peptidase is the highest in SNPC and SNPR; this activity have some predominance in the SN and higher predominance in the same fraction from CAU and CC.