Unexpected cell type-dependent effects of autophagy on polyglutamine aggregation revealed by natural genetic variation in C. elegans.

BACKGROUND: Monogenic protein aggregation diseases, in addition to cell selectivity, exhibit clinical variation in the age of onset and progression, driven in part by inter-individual genetic variation. While natural genetic variants may pinpoint plastic networks amenable to intervention, the mechanisms by which they impact individual susceptibility to proteotoxicity are still largely unknown. RESULTS: We have previously shown that natural variation modifies polyglutamine (polyQ) aggregation phenotypes in C. elegans muscle cells. Here, we find that a genomic locus from C. elegans wild isolate DR1350 causes two genetically separable aggregation phenotypes, without changing the basal activity of muscle proteostasis pathways known to affect polyQ aggregation. We find that the increased aggregation phenotype was due to regulatory variants in the gene encoding a conserved autophagy protein ATG-5. The atg-5 gene itself conferred dosage-dependent enhancement of aggregation, with the DR1350-derived allele behaving as hypermorph. Surprisingly, increased aggregation in animals carrying the modifier locus was accompanied by enhanced autophagy activation in response to activating treatment. Because autophagy is expected to clear, not increase, protein aggregates, we activated autophagy in three different polyQ models and found a striking tissue-dependent effect: activation of autophagy decreased polyQ aggregation in neurons and intestine, but increased it in the muscle cells. CONCLUSIONS: Our data show that cryptic natural variants in genes encoding proteostasis components, although not causing detectable phenotypes in wild-type individuals, can have profound effects on aggregation-prone proteins. Clinical applications of autophagy activators for aggregation diseases may need to consider the unexpected divergent effects of autophagy in different cell types.

Profile of gastrointestinal involvement in patients with systemic sclerosis.

Systemic sclerosis (SSc) is a chronic autoimmune connective tissue disease. Of the numerous organ manifestations, involvement of the upper and lower gastrointestinal tract (GIT) appears to be the most frequent with regard to the clinical symptoms. However, as the frequency and clinical relevance of GI involvement in patients with SSc are not known in detail, the German network of the systemic sclerosis (DNSS) has developed a detailed questionnaire to evaluate the extent and profile of gastrointestinal involvement in SSc patients. The multi-symptom questionnaire was used at baseline and after 1 year in registered patients of the DNSS. In addition, the results were compared with gastrointestinal disorders in patients with SSc and other rheumatic diseases, as well as with the medical history of the patients. In total, 90 patients were included in the study. The results of the study show that in reality, a much higher (nearly all) percentage of (98,9%) patients than expected suffer from GI-symptoms, regardless of the stage of their disease. Of these, meteorism (87,8%) was the most common followed by coughing/sore voice (77,8%), heartburn (daytime 68,9%, nighttime 53,3%), diarrhea (67,8%), stomach ache (68,9%) and nausea (61,1%). Although SSc patients were treated according to the respective recommendations, only limited improvements with regard to GI-symptoms could be achieved after 1 year of follow-up. In addition, the study revealed that the multi-symptom questionnaire is a useful tool to contribute to identify the gastrointestinal sequelae in systemic sclerosis.

[Transgastric-, transhepatic endosonography-guided biliary drainage (EUCD) in a patient with locally advanced cholangiocarcinoma]. / Endosonografisch gesteuerte transgastrisch-, transhepatische Cholangiodrainage (EUCD) bei einer Patientin mit lokal fortgeschrittenem Gallengangskarzinom.

We report on a 63-year-old female patient with locally advanced cholangiocarcinoma of the extrahepatic biliary tract. She was admitted with progressive obstructive jaundice, initiating cholangitis and distinctive itching. The biliodigestive anastomosis was secondarily barred by tumour infiltration and not accessible via an endoscopic route. Because the patient asked expressively for internal drainage, we successfully performed an endosonography-guided transgastric, transhepatic internal biliary drainage (EUCD). The jaundice and itching were regredient and the patient was discharged in a stable condition.

Interleukin family member ST2 and mortality in acute dyspnoea.

OBJECTIVES: The study objective was to investigate the prognostic utility and patient-specific characteristics of ST2 (suppression of tumorigenicity 2), assessed with a novel sensitive assay. BACKGROUND: Suppression of tumorigenicity 2 signalling has been shown to be associated with death in cardiac and pulmonary diseases. DESIGN/SUBJECTS: In an international multicentre cohort design, we prospectively enrolled 1091 patients presenting with acute dyspnoea to the emergency department (ED). ST2 was measured in a blinded fashion using a novel assay and compared to B-type natriuretic peptide (BNP) and NT-proBNP. The primary end-point was mortality within 30 days and 1 year. The prognostic value of ST2 was evaluated in comparison and in addition to BNP and NT-proBNP. RESULTS: Suppression of tumorigenicity 2 concentrations was higher amongst decedents than among survivors (median 85 vs. 43 U mL⁻¹, P < 0.001) and also higher in patients with impaired left ventricular ejection fraction (LVEF) when compared with preserved LVEF (P < 0.001). In receiver operator characteristics analysis, the area under the curve (AUC) for ST2, BNP and NT-proBNP to predict 30-day and 1-year mortality were 0.76, 0.63 and 0.71, and 0.72, 0.71 and 0.73, respectively. The combinations of ST2 with BNP or NT-proBNP improved prediction of mortality provided by BNP or NT-proBNP alone. After multivariable adjustment, ST2 values above the median (50 U mL⁻¹) significantly predicted 1-year mortality (HR 2.3, P < 0.001). CONCLUSION: In patients presenting to the ED with acute dyspnoea, ST2 is a strong and independent predictor of 30-day and 1-year mortality and might improve risk stratification already provided by BNP or NT-proBNP.

Some neurons of the rat central nervous system contain aromatic-L-amino-acid decarboxylase but not monoamines.

Neurons containing the enzyme aromatic-L-amino-acid decarboxylase (AADC) but lacking either tyrosine hydroxylase or serotonin were found in the spinal cord of neonatal and adult rats by light and electron microscopic immunocytochemistry. The majority of these neurons localized to area X of Rexed contact ependyma. Thus, spinal AADC neurons have the enzymatic capacity to catalyze directly the conversion of the amino acids tyrosine, tryptophan, or phenylalanine to their respective amines tyramine, tryptamine, or phenylethylamine. These amines normally present in the central nervous system may be of potential clinical significance as endogenous psychotomimetics.

Insulin binding to antibodies is a risk factor for inexplicable severe hypoglycaemia in children with type-1 diabetes mellitus.

BACKGROUND: Type-1 diabetic individuals differ with regard to both, the formation of circulating insulin antibodies, and the incidence of severe hypoglycaemia. AIM OF THE STUDY: To assess the association of insulin binding to antibodies with the incidence of severe hypoglycaemia. PATIENTS AND METHODS: In a cross sectional study, 73 children with type-1 diabetes mellitus (median age 14 years, duration of diabetes 6 years) were investigated, 22 of whom had experienced severe hypoglycaemia during the past 18 months, and 51 had never experienced severe hypoglycaemia. Of the patients with severe hypoglycaemia 16 had experienced severe unexplained hypoglycaemias, and 6 had experienced severe hypoglycaemias which were explicable (by missed meals, unplanned physical exercise etc.). Insulin binding was measured in a blinded central laboratory by radioimmunoassay, and expressed as ratio bound/unbound insulin; a binding >15% was considered relevant insulin binding. RESULTS: A total of 38 patients displayed relevant insulin binding (17 of whom had experienced severe hypoglycaemia), and 35 patients did not (5 of whom had experienced severe hypoglycaemia; p=0.0055, Fisher's exact test). Patients with relevant insulin binding were younger (12.2 vs 14.5 years, p=0.006) than patients without relevant insulin binding. From the 16 patients with inexplicable severe hypoglycaemia, 15 displayed relevant insulin binding, compared to 2 of the 6 patients with explicable severe hypoglycaemia (p=0.009). The association of any severe hypoglycaemia, and of inexplicable severe hypoglycaemia, with relevant insulin binding was significant (odds ratio 4.8 (95%CI 1.5-15.2), and 22.1(95%CI 2.7-179.6), p<0.006). Patients with/without relevant insulin binding, or with/without severe hypoglycaemia, did not differ significantly regarding sex, duration of diabetes, number of insulin injections per day, HbA1c and C-peptide levels (ANOVA). CONCLUSION: Insulin binding to antibodies >15% appears to be a strong risk factor for inexplicable severe hypoglycaemias in type-1 diabetic children.

IgG subclass distribution of autoantibodies in pediatric opsoclonus-myoclonus syndrome.

Opsoclonus-myoclonus syndrome (OMS) in children is a rare disorder including a severe eye movement disturbance, myoclonia, ataxia and often developmental retardation. Both OMS forms, idiopathic or neuroblastoma-associated (paraneoplastic), have been suspected to be autoimmune. Recently, autoantibodies have been found in OMS sera. We here show that autoantibodies in OMS, both intracellular and surface binding, belong mainly to the IgG3 subclass, although the total serum IgG3 level is normal. These results support the autoimmune hypothesis and point to a protein autoantigen as antigenic target.

Progressive islet graft failure occurs significantly earlier in autoantibody-positive than in autoantibody-negative IDDM recipients of intrahepatic islet allografts.

Alloimmunity has been uncovered to be a cause of graft loss representing a major barrier for clinical islet transplantation, and several studies are designed to evaluate new strategies for immunosuppression to prevent alloimmunity. In contrast, the significance for autoimmune destruction of transplanted beta-cells has remained somewhat controversial. Recently, two case reports based on histological findings have suggested recurrent autoimmune insulitis despite immunosuppressive therapy both in clinical pancreas and in islet transplantation. In the present study, in 23 islet-grafted patients with IDDM receiving standard immunosuppressive therapy, we demonstrate that progressive impairment of islet graft function occurs significantly earlier in those individuals positive for autoantibodies as a typical stigma of diabetes-associated autoimmunity that is well established in the prediabetic periods of IDDM. Intraportal infusion of allogeneic islets was performed in 23 C-peptide-negative IDDM patients, according to the clinical transplantation categories defined as islet after kidney (IAK) or simultaneous islet and kidney (SIK). Complete islet graft failure was defined as the 1st day of permanent C-peptide negativity in the serum (<0.2 ng/ml) and C-peptide negativity in the urine (<2 microg/dl). The median observation period following islet transplantation was 12 months (range 1-50) with a cumulative follow-up of 336 months. Islet cell antibodies (ICAs) and GAD65 antibodies were monitored before and regularly after islet transplantation. Kaplan-Meier survival analysis and log-rank statistics revealed a significant (P < 0.05) difference in cumulative islet graft survival depending on the presence of islet cell and/or GAD65 antibodies. These results strongly suggest that recurrent autoimmunity directed to transplanted beta-cells contributes to islet graft failure despite sustained immunosuppression. For successful clinical islet transplantation in the future, new immunosuppressive therapies are needed to prevent both alloimmunity and autoimmunity.

Glutamic acid decarboxylase antibodies are more frequent than islet cell antibodies in islet transplanted IDDM patients and persist or occur despite immunosuppression.

Pancreatic islet grafts transplanted into patients with autoimmune diabetes are potentially threatened by two immune responses, allograft rejection and the recurrence of autoimmune insulitis. In the present study we investigated the humoral autoimmune response directed to islet autoantigens by studying islet cell antibodies and glutamic acid decarboxylase (GAD 65) antibodies in twenty-one insulin-dependent diabetes-mellitus (IDDM) patients undergoing intraportal islet allotransplantation. Islet transplantation was performed according to the following recipient categories: Islet after kidney transplantation (n=10), simultaneous islet and kidney transplantation (n=6) and islet transplant alone (n=5). GAD 65 antibodies were detected in a radioligand GAD 65 antibody assay using recombinant, in vitro translated, human 35S-methionin labelled GAD 65 as tracer. Islet cell antibodies were determined by indirect immunofluorescence technique on human pancreas. In six out of twenty-one patients we observed GAD 65 antibodies before islet transplantation and the GAD 65 antibodies persisted despite immunosuppression. In contrast only two subjects were concordantly islet cell antibody positive and the titre decreased post transplantation. In addition we observed occurrence of GAD 65 antibodies in five subjects that were shown to be antibody negative before islet transplantation with three of them subsequently becoming positive for islet cell antibodies. The remaining ten patients were GAD 65 antibody and islet cell antibody negative before islet transplantation and remained negative thereafter. Interestingly none of the patients was exclusively positive for islet cell antibodies without being positive for GAD 65 antibodies. In summary we have demonstrated in twenty-one islet grafted individuals that humoral autoimmunity to islet antigens can persist or occur despite immunosuppression. Islet cell antibodies appear to be less frequent (5 out of 21, 23%) compared to GAD 65 antibodies (11 out of 21, 52%) suggesting that they are more affected by immunosuppressive therapy. We conclude that GAD 65 antibodies are a useful tool to further evaluate a possible link between persistent autoimmunity and early or late graft failure after islet transplantation.

Comparative analysis of organ-specific autoantibodies and celiac disease--associated antibodies in type 1 diabetic patients, their first-degree relatives, and healthy control subjects.

OBJECTIVE: In type 1 diabetes the coexistence with other endocrine diseases and organ-specific autoantibodies has been frequently reported leading to the concept of autoimmune polyendocrine syndrome (APS). In addition, an association of type 1 diabetes with celiac disease has been described. These disorders share a similar genetic background, and first-degree relatives of type 1 diabetic patients may also be affected significantly. Screening for specific antibodies allows early diagnosis of these disorders. RESEARCH DESIGN AND METHODS: In the present cross-sectional study, we analyzed sera from 197 recent-onset type 1 diabetic patients at the time of diagnosis, 882 first-degree relatives, and sera of 150 healthy control subjects for prevalence and co-occurence of the following antibodies (method): insulin autoantibodies (radioimmunoassay); GAD and IA-2 antibodies (radioligand assay); islet cell antibody, anti-adrenal cortex antibodies, and anti-gastric parietal cell antibodies (indirect immunofluorescence); anti-thyroglobulin and anti-thyroid peroxidase antibodies; and gliadin IgG/A and tissue-transglutaminase IgA (enzyme-linked immunosorbent assay). RESULTS: The overall frequency of gastric patietal cell antibodies and adrenal antibodies did not differ significantly among groups. In contrast, type 1 diabetes-associated antibodies and thyroid antibodies were significantly more frequent both in recent-onset type 1 diabetic patients and in the group of first-degree relatives (P < 0.05). The prevalence of gliadin IgG/IgA and transglutaminase IgA was significantly higher in the group of recent-onset type 1 diabetic patients (P < 0.05), but the difference between first-degree relatives and control subjects did not reach statistical significance. Focusing on the coexistence of antibodies, the group of recentonset type 1 diabetic patients presented with 27.4% of the subjects testing antibody-positive-specific for two or more of the envisaged disorders (i.e., type 1 diabetes, autoimmune thyroiditis, and celiac disease) compared with 3.1% in the group of first-degree relatives and 0 of 150 in the control population (P < 0.05). CONCLUSIONS: We conclude that, in an active case-finding strategy, recent-onset type 1 diabetic patients should be routinely screened at least for concomitant autoimmune thyroid disease and additionally for celiac disease. Screening in their first-degree relatives should include at a minimum the search for thyroid autoimmunity in addition to screening for pre-type 1 diabetes.

The membrane-associated protein pKe#192/MAP17 in human keratinocytes.

UNLABELLED: In order to isolate genes that are upregulated in human keratinocytes upon loss of cell/matrix contact, a subtractive cDNA library was constructed from dispase-treated versus untreated keratinocytes. Among the cloned cDNAs one was pKe#192 having an open reading frame of 411 bp. By database analysis pKe#192 was found to be identical with the gene "MAP17" previously isolated from human kidney. Kyte-Doolittle hydrophobicity analyzes showed a hydrophobic amino terminus of 13 amino acids, a transmembrane region and a 61 amino acid hydrophilic carboxy-terminus and two potential phosphorylation sites. In order to study regulation of pKe#192/MAP17 expression, RNA was extracted from resting human keratinocytes and from keratinocytes stimulated by dispase-induced detachment from the growth substratum. Reverse transcription polymerase chain reaction did not reveal specific mRNA in resting keratinocytes, whereas mRNA was detectable after detachment. For further characterization poly- and monoclonal antibodies were generated against a recombinant fusion protein. Immunohistologic studies using the mono- and polyclonal antibodies showed staining of the upper layers of the stratum granulosum in normal human epidermis. The staining was colocalized with involucrin. Immunhistologic staining of frozen sections derived from lesional skin of bullous pemphigoid und pemphigus vulgaris indicated that pKe#192/MAP17 was upregulated in the epidermis adjacent to the blister. Taken together, the data demonstrate that pKe#192/MAP17 is expressed in keratinocytes and may be involved in epidermal physiology and pathology. KEYWORDS: bullous diseases/differentiation.

Cytoarchitectonics of substantia nigra grafts: a light and electron microscopic study of immunocytochemically identified dopaminergic neurons and fibrous astrocytes.

Maturation of dopaminergic (DA) neurons and astroglia was studied in transplants of the substantia nigra grown for up to 7 months in the brain of rats. The investigation had three specific aims. The first was to observe effects of different transplant positions on the longevity of DA neurons. Second, the grafts were examined for changes of synaptic interactions and associations between DA neurons and astroglia. Third, an answer was sought to the question whether transplanted DA neurons migrate into the adjacent host brain. The grafts were taken from the ventral mesencephalon of rat embryos of different ages (day 14 to 18 of gestation) and placed into the cerebral cortex, tectum, cerebellum, or ventricles of newborn host animals. Following different times of survival the immunocytochemical localization of tyrosine hydroxylase (TH) and of glia filament protein (GFA) in the transplants were observed. In all of the transplantation sites, except for one, neurons of different morphologies that contained TH were found in the grafts. The cerebellar white matter of the host brain failed to support the long-term survival of DA neurons. The overall structure of mature substantia nigra grafts had some resemblance to intact substantia nigra (SN). On the ultrastructural level, it was found that morphological expression of some immature features of DA neurons, such as glial sheaths, somatic spines, and lack of oligodendroglia, persisted in mature grafts. Specific associations of DA neurons and astroglia in the grafts suggested that the cytoarchitectonic appearance of a given brain region may be related to the existence of particular neuron glia relationships. In contrast to intact SN, transplants revealed deficiencies in unlabeled pleomorphic boutons and contained some TH-immunoreactive terminals. Migration of DA neurons and their processes into the adjacent host brain was rarely observed.

Transplantation of embryonic occipital cortex to the tectal region of newborn rats: a light microscopic study of organization and connectivity of the transplants.

Occipital cortex was taken from fetal rats and transplanted to the tectal region of newborn rats, where it developed a specific structural identity reflecting in part its cortical origin. The implants showed locally distributing intratransplant connections, and the majority formed connections with defined regions of te host cerebral cortex and the brainstem. A sparse afferent projection from the host had its origin in visual, somatosensory, and cingulate areas of te cortex, pretectum, superior colliculus, central gray, hypothalamus, pontine reticular formation, raphe nuclei, and the locus coeruleus. No input was identified from either the retina or the dorsal thalamus. Efferent fibers were observed in normal fiber preparations as compact bundles running through the host brainstem along two main routes, one group of bundles in a dorsal position and a second group more ventral. Efferent fibers traveling rostrally along the first pathway distributed in the lower part of the stratum griseum superficiale and in the intermediate laminae of the superior colliculus, and in some cases they reached the pretectum and the lateral posterior thalamic nucleus. Deep efferent fibers ran rostrally and caudally in the central gray, and in some cases laterally directed fibers were seen to distribute in the midbrain tegmentum and reticular formation, in one case reaching the pontine gray. The finding that most afferent and many efferent connections of cortical transplants are uncharacteristic of normal cortex stands in marked contrast to retina and tectum, which, when transplanted to the same region, make relatively normal connections.

Transplantation of embryonic occipital cortex to the brain of newborn rats: a Golgi study of mature and developing transplants.

Segments of the occipital cortex were taken from rat embryos (E16-E19) and transplanted to the cerebral cortex or the tectal region of a newborn rat host. With the aid of Golgi impregnation techniques, neuron morphology was studied in cortical transplants which had survived for 1 week or more in the host brain. In mature transplants (greater than 4 weeks) three main groups of neurons, termed groups I-III, were identified. Group I neurons resembled pyramidal neurons of the intact cerebral cortex. No preferential orientation of either soma or dendrites of group I neurons was observed in the transplants, and some group I neurons had curved apical dendrites. Group II neurons had predominantly stellate form and their dendrites were densely covered with spines. Paucity or absence of dendritic spines characterized group III neurons which exhibited various dendritic topologies. Different neuron types were also recognized in immature transplants growing for 1 and 2 weeks in the host brain. The sequence of dendritic maturation of transplanted cortical neurons is similar to that seen in intact cortex, although the stage reached related more to the actual age of the transplant than to that of the host. Thus, group I neurons in the 1-week-old transplants taken from E16 embryos had not attained the same complexity of branching as pyramidal neurons in the surrounding host cortex, but rather resembled slightly younger cells more like those found in the cerebral cortex of the newborn rat. These results show, therefore, that at least the basic cell classes identified in intact visual cortex can also be recognized in the cortical transplants. This will provide a foundation for studies defining which cells project to the host brain and which are involved in particular intrinsic connections.

The effect of forebrain lesions in the neonatal rat: survival of midbrain dopaminergic neurons and the crossed nigrostriatal projection.

Lesions were placed in the striatum and the olfactory tubercle of 1-day-old rat pups. Control and experimental animals were raised to adulthood. Efferent projections of mesencephalic neurons were examined by injecting the retrograde tracers horseradish peroxidase or Fast Blue into the undamaged striata of some experimental animals. The survival of the mesencephalic dopaminergic neurons was monitored by using immunocytochemical localization of tyrosine hydroxylase. Small lesions in the caudate-putamen had no appreciable effect on the survival of tyrosine hydroxylase-containing neurons in the mesencephalon, but the density of dopaminergic terminals adjacent to the lesion increased in the remaining caudate-putamen. Striatal lesions that involved an estimated area of more than one-third resulted in loss of dopamine neurons of the substantia nigra compacta. Rostral lesions in the striatum affected mostly rostrally positioned neurons in the substantia nigra. Dorsal lesions of the caudate-putamen resulted in disappearance of dorsal A9 neurons. Reduction of the A10 and A8 dopamine neuron groups occurred if the neonatal lesions involved the olfactory tubercle and nucleus accumbens. Some tyrosine hydroxylase-containing neurons persisted even after the largest lesions. These dopaminergic neurons formed a crossed nigrostriatal pathway which was confirmed by retrogradely transported tracers. The density of this crossed projection in the adult appeared unaffected by the neonatal lesion. We concluded that dopaminergic neurons form topographically ordered projections with their targets in the newborn rat. Rearrangement of these fibers appeared limited, but compensatory increase of axon terminal density was evident in partially lesioned target areas.

Islet cell antibodies and glutamic acid decarboxylase antibodies in patients with insulin-dependent diabetes mellitus undergoing kidney and islet-after-kidney transplantation.

The humoral immune response to islet autoantigens, here defined by the presence of islet cell antibodies (ICA) and glutamic acid decarboxylase (GAD 65) antibodies, was studied in patients with long-term insulin-dependent diabetes mellitus (IDDM) receiving immunosuppressive therapy following kidney and islet-after-kidney transplantation. In a cross-sectional study of 30 kidney-grafted, long-term IDDM patients and 30 matched, nontransplanted IDDM controls, we observed a significant (P<0.05) decrease in ICA positivity by standard immunosuppressive therapy, but not in frequency or index levels of GAD 65 antibodies. Because of this intriguing finding, we investigated, in a pilot study on seven islet-after-kidney transplant recipients, the time course of frequency and levels of ICAs and GAD 65 antibodies relative to islet graft function. Stable islet graft function was seen in the patients with low GAD 65 antibody index levels, whereas rapid islet graft failure occurred in a patient with high GAD 65 antibody index levels prior to transplantation. In addition, GAD 65 autoimmunity reoccurred in one pretransplant antibody-negative patient 2 months after graft failure was noted. In conclusion, these observations suggest that beta-cell autoimmunity directed to GAD 65 can persist despite immunosuppressive therapy and may adversely affect islet graft function, possibly indicating disease recurrence as a major threat to successful clinical islet transplantation.

Aromatic L-amino acid decarboxylase in the rat brain: immunocytochemical localization during prenatal development.

Immunocytochemically labeled cells containing the enzyme aromatic L-amino acid decarboxylase were localized in the brain of rat embryos at gestational age E15-E19. Cell groups that contained aromatic L-amino acid decarboxylase but lacked either the enzyme tyrosine hydroxylase or the indolamine serotonin were referred to as "D" groups. Anatomical landmarks, cytoarchitectonic structure and histochemical staining for acetylcholinesterase were used to delineate the position of "D" groups. In the E15 embryo three "D" groups existed. The first to appear, named D1, was located in the spinal cord and had been demonstrated before. A large "D" cell cluster was found in the walls of the central forebrain deep to the hypothalamic sulcus. This group distributed dorsally in the ventral dorsal thalamic region and ventrally in the dorsal hypothalamus. The rostral-most "D" group, D14, occurred in the ventral telencephalon just medial to fibers of the nigrostriatal projection. D14 was the smallest of the early groups. In E16 and E17 embryos dorsal di- and mesencephalic "D" groups were first detected. During the course of ontogeny a considerable increase of immunoreactive cells occurred and segregation of the large central forebrain cluster into several rostrally and laterally distributed "D" groups took place. Some "D" groups that occur in the adult brain were not present in the E19 embryo. This study provides a first report of the localization of several unique cell groups in the brain of rat embryos and their appearance at different stages of gestation. It also gives further support to the notion that variations of aromatic L-amino acid decarboxylase staining intensities may be characteristic of different monoamine neurons.

Influence of grafted glia cells and host mossy fibers on anomalously migrated host granule cells surviving in cortical transplants.

Embryonic cerebral cortex was transplanted over the cerebellum of one- to two-day-old rats. In mature rats, clusters of granule cells that failed to migrate into the internal granule cell layer were now found within the graft tissue. Immunocytochemical staining of astrocytic glial cells in the cortical transplants revealed that glial processes were distributed in an unusual polarized orientation in those regions that contained host granule cells. Other areas of the graft exhibited glia cells with processes that projected radially from their cell body, thus resembling fibrous astrocytes. Fibrous astrocytes in transplants, however, were more heavily stained than similar glial cells in the intact cerebral cortex of the host, indicating a quantitative difference in the glia fibrillary acidic protein. Acetylcholinesterase-positive fibers were observed between the clusters of granule cells in the cortical grafts. Such fibers traversed the molecular layer and, since they could be traced from the white matter of the host cerebellum, they were presumed to be mossy fibers. It is concluded that migration of external granule cells in the cerebellum can altered by placing embryonic cerebral cortex next to the developing cerebellum. Granule cells that have migrated into the grafted cerebral cortical tissue nevertheless receive afferent fibres.

Electrophysiology of the mammalian cerebellar cortex in organ culture.

A direct comparison was made between the electrical properties of rat Purkinje cells in cerebellar organotype cultures and those in acute slices from age-matched animals. Cultures were prepared from 9-11-day-old animals. Intracellular recordings were made 5-12 days later, at which time the folia architecture of the cerebellum was still well preserved. The resting membrane potentials and input resistances of Purkinje cells in cultured and acute slice preparations from young animals were comparable to those of mature Purkinje cells in slices. Neurons from animals younger than 14 days differed from mature Purkinje cells in that they fired at low frequencies in response to outward current pulses. The latter property was found in all cultured neurons studied, independent of their time in culture. These action potentials were generated by Na+ and Ca2+ conductances as shown by the application of selective channel blockers. Cultured or acute slice preparations from animals younger than 11 days shared other immature electroresponsive features. In both groups, Na+-dependent plateau depolarizations were observed in less than 10% of Purkinje cells unless K-conductances were blocked, and considerable membrane depolarization was often required to elicit Ca2+-dependent action potentials. These findings are compatible with the relative prominence of voltage-dependent outward currents in immature Purkinje cells, a property which may be enhanced in culture. The injection of hyperpolarizing current pulses revealed a marked time-dependent anomalous rectification in all Purkinje cells. At the breaks of such pulses, several events were observed. In all cells, a rebound conductance was identified which could generate post-anodal spike bursts. In cultured neurons, however, hyperpolarizing pulses were also followed by a slow return to resting potential. This membrane potential profile was similar to that produced by the activation of an A conductance. Experiments on acute slices from animals of different ages (P9-P17) showed that this A-like conductance was expressed only during a brief period in Purkinje cell development. A higher level of spontaneous synaptic activity was observed in cultured than in acute slice preparations. Both unitary excitatory postsynaptic potentials and inhibitory postsynaptic potentials could be elicited in the former by parallel fiber stimulation, and could be fully reversed by outward or inward transmembrane current injections, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytology and organization of rat cerebellar organ cultures.

Roller tube cultures of parasagittal cerebellar slices were taken from young rats aged 9-11 days, and maintained in vitro for 1-2 weeks. Morphological aspects of cell types and synaptic relationships in such organ cultures were examined at light and electron microscopic levels. Some neurons were marked by intracellular injections of horseradish peroxidase for subsequent identification of their connection patterns. Cytoarchitecture of the cerebellar cortex was largely preserved in the organ cultures. Dendritic trees of Purkinje cells exhibited isoplanar organizations that often resembled their orientation at the time of explanation. Other cerebellar neurons, namely granule cells, Golgi cells, basket cells, stellate cells, all differentiated within the organ cultures. In addition, some neurons of the deep cerebellar nuclei remained viable during the period of culture. Mossy fibers most probably of cerebellar nuclear origin were found terminating on the dendrites of granule cells and Golgi cells. Quite unexpected were certain types of direct synapses of afferent fibers on short necked spines arising from Purkinje cell smooth dendrites and somata. Such terminals resembled climbing fibers. They were most likely modified mossy fiber afferents, since the organ cultures did not include neurons of the inferior olive which are well spearated from the cerebellar mass at postnatal stages. These "ascending" mossy fibers presumably occupied postsynaptic surfaces that were either vacated by deafferentation or induced by the afferent fibers themselves. Intracellularly labeled Purkinje cells had widely distributed axonal collateral branches. Labeled axons were distributed within the Purkinje cell layer. Several recurrent Purkinje cell axon collaterals stained with reaction products of horseradish peroxidase tracer were followed at the ultrastructural level. In one case, labeled terminals were examined in an area of approximately 2 mm2. Terminals of Purkinje cell collaterals formed symmetric synapses with somata of basket cells and dendrites of Golgi cells, but not Purkinje cell somata. Some large boutons of serially traced Purkinje cell axon collaterals formed asymmetric contacts with profiles interpreted as Golgi cell dendrites. In contrast to the apparent axonal sprouting in cerebellar organ cultures, maturation of dendritic processes remained static. Astroglia cells of diverse shapes were observed following immunocytochemical staining with antisera to glia filament proteins. The distribution patterns of immunoreactive astrocytes changed dramatically in cerebellar slice cultures maintained for 3-6 weeks in vitro.