Estrogen-stimulated transcytosis of desialylated ligands and alpha2 macroglobulin in rat liver.

Previous studies have shown that treatment of rats with 17 alpha-ethynylestradiol (EE) causes the appearance in bile of intravenously injected, desialylated ligands, including asialofetuin and low density lipoprotein (LDL). Here we show that activated alpha2-macroglobulin (alpha2-M*), but not insulin, transferrin or acetylated LDL, shows the same phenomenon. Alpha2-M* appearance in bile in EE-treated rats was inhibited by receptor associated protein, but not unlabelled asialofetuin, strongly implicating the alpha2-macroglobulin receptor (alpha2MR/LRP) receptor in this process. Asialofetuin, apolipoprotein B (ApoB) of LDL and alpha2-M* appeared undegraded in the bile of EE-treated but not control rats. When LDL was injected, not only was intact apolipoprotein B detected in bile, but the profile of cholesterol esters appearing in bile was characteristic of the injected human LDL rather than rat lipoproteins. After floatation of the bile on KBr gradients, intact Apo B and cholesterol esters characteristic of human LDL were found at the normal density of LDL suggesting that the majority of the lipoprotein particle remains intact. Stimulation of transcytosis was specific to estrogens, and was highest with 17alpha-ethynylestradiol. After subcutaneous injection of 0.05 mg/kg body weight of ethynylestradiol, sufficient to give a measurable increase in transcytosis, the plasma concentration of ethynylestradiol rose to 2.2 nM. Thus estrogen-stimulated transcytosis of desialylated ligands and alpha2-M* would be expected at physiological estrogen concentrations.

A role for tyrosine phosphorylation in both activation and inhibition of the insulin receptor tyrosine kinase in vivo.

Upon insulin binding, a conformational change in the insulin receptor (IR) leads to IR beta-subunit autophosphorylation, an increase in IR beta-subunit exogenous tyrosine kinase activity, and the rapid endocytosis of the ligand-receptor complex into endosomes. Previous work has shown that upon internalization, rat hepatic endosomal IRs manifest increased autophosphorylating and exogenous tyrosine kinase activity compared to IRs located at the plasma membrane. As this period of enhanced activity is associated with reduced endosomal IR beta-subunit phosphotyrosine content, it has been proposed that partial dephosphorylation of the internalized IR beta-subunit by an endosomally located phosphotyrosine phosphatase(s) [PTPase(s)] mediates this effect. To test whether endosomal PTPase activity was required for internalization-dependent augmentation of IR tyrosine kinase activity, the present study used the peroxovanadium PTPase inhibitor, bisperoxo(1,10-phenanthroline)oxovanadate anion [bpV(phen)], to block IR dephosphorylation within this subcellular compartment. Rats were pretreated with bpV(phen) before receiving insulin (1.5 micrograms/100 g BW). bpV(phen) inhibited the dephosphorylation of 32P-labeled hepatic endosomal IR by approximately 97% at 15 min post-bpV(phen) injection and prevented a decrease in IR beta-subunit phosphotyrosine content after IR internalization. Fifteen-minute bpV(phen) pretreatment produced a significant reduction (75%; P < 0.001) in maximal insulin-stimulated endosomal IR exogenous kinase activity and decreased IR autophosphorylating activity by 4.3-fold in this subcellular fraction. In conclusion, these findings suggest that an hepatic endosomal PTPase(s) regulates internalization-dependent increases in IR exogenous tyrosine kinase activity.

Control of p62 binding to TGN38/41 by phosphorylation.

TGN38/41 cycles between the trans-Golgi network (TGN) and plasma membrane, traversing three sorting compartments: the TGN, plasma membrane and early endosome. The targeting signals responsible for this complex itinerary reside in a short cytoplasmic domain of 33 amino acid residues. We show that phosphorylation of the cytoplasmic domain of TGN38 prevents binding of p62--a cytoplasmic protein essential for exocytic vesicle formation. Thus the cycle of TGN38/41 traffic, and by implication the pathway of exocytosis, could be controlled by phosphorylation of the TGN38 cytoplasmic domain.

Desialylated LDL uptake in human and mouse macrophages can be mediated by a lectin receptor.

We have compared the uptake of desialylated low density lipoprotein (LDL) with other modified forms of LDL in mouse peritoneal macrophages and PMA-activated human U937 monocytes. Neuraminidase-treated LDL (NT-LDL) caused significant cholesterol ester accumulation in both cell types, although the efficiency relative to loading with acetylated LDL (AcLDL) was markedly different, suggesting a very different complement of receptors in the cells. We therefore determined the effect of PMA-activation on lipoprotein receptor expression in U937 cells and found that while scavenger receptor concentration was elevated after PMA-activation, there was no significant change in the expression of the LDL receptor. Receptor specificity of NT-LDL uptake was examined by competition experiments using the degradation assay. This showed that 125I-labelled NT-LDL uptake in U937 cells could largely be accounted for by the persistent expression of the LDL receptor in these cells. In contrast, in mouse peritoneal macrophages where LDL receptor expression is very low, 125I-labelled NT-LDL degradation was also effectively competed by asialofetuin. Surprisingly, 125I-labelled NT-LDL degradation was also effectively competed by AcLDL. Measurement of sialic acid content of AcLDL showed that approximately 14% of the LDL sialic acid, equivalent to 2 to 3 residues per particle, was lost during acetylation of LDL with acetic anhydride. Thus competition between 125I-labelled NT-LDL and AcLDL could be due to lectin receptor binding rather than competition for scavenger receptor binding.

Recovery of function after neonatal or adult hemispherectomy in cats. II. Limb bias and development, paw usage, locomotion and rehabilitative effects of exercise.

To investigate the relationship of age-related processes in the recovery of complex motor functions of the limbs, a variety of neurobehavioral assessments were applied to cats with either neonatal (n = 9) or adult (n = 11) removal of the entire left hemitelencephalon (hemispherectomy). Neonatal-lesioned kittens showed no paw preference in reaching for a manipulandum between 5 and 8 weeks of age; thereafter they developed a preference (5.6%) for the unimpaired left limb which persisted throughout adulthood. Adult-lesioned cats showed a significantly greater left bias (13.9%) than neonatal-lesioned cats and they exhibited more abnormal movements and postures when reaching with the impaired limb. Exercising the impaired limb, was effective in reversing the paw preference bias in all lesioned cats. To master a food retrieval task with the impaired limb, adult-lesioned cats required more trials than the neonatal-lesioned group, extensive food deprivation and at least 1 month of postsurgical recovery. However, after mastering this task all cats could continue to perform it indefinitely in their home cages. In a paw print analysis of locomotion, only adult-lesioned cats showed abnormalities including splayed paws, decreased stride length, and adduction of the right hind limb. The results support the 'Kennard Principle' of enhanced recovery following neonatal vs late brain lesions for the present complex motor patterns and are interpreted in the context of neural plasticity and anatomical reorganization during development.

Recovery of functions after neonatal or adult hemispherectomy in cats. III. Complex functions: open field exploration, social interactions, maze and holeboard performances.

Complex behavioral patterns were studied in cats with removal of the entire left cerebral hemisphere either as neonates (n = 10) or adults (n = 11), and in intact control cats (n = 24). Adult-lesioned cats showed decreased open field activity in locomotion, rearing and sniffing. Lesioned kittens showed similar deficits at 100 days of age, but by 150 days of age they resembled normal littermates in all 3 measures. In the presence of another cat, adult-hemispherectomized cats violated species-typical body-buffer space, approaching and attacking other cats. By comparison, normal cats never attacked and seldom approached in the open field. Neonatal-lesioned adults showed only occasional approach and a tendency to sit or stiff-walk in the presence of other cats; attacks were rare. Adult-lesioned cats responded poorly to reversal training for preferred arm of a T-maze, whereas neonatal-lesioned adults were significantly more trainable. Similarly, adult-lesioned cats exhibited a search deficit in a baited holeboard, while neonatal-lesioned adults searched normally. Overall, these results demonstrate that in this animal model, enhanced recovery following early vs adult lesions can also be found in relatively complex, spontaneous behavioral responses not previously studied in this regard.

Recovery of function after neonatal or adult hemispherectomy in cats: I. Time course, movement, posture and sensorimotor tests.

Cats with removal of the left hemitelencephalon (hemispherectomy) as neonates (n = 12) or in adulthood (n = 14), were compared using a battery of 16 neurological and behavioral tests given when they were young adults (kittens) or at least 5 months after the lesion (adults). The neonatal-lesioned subjects grew normally and performed markedly and significantly better than adult-lesioned cats in 13 tests covering the wide range of movement, posture and sensory functions which were assessed. None of the animals recovered tactile placing of the right forelimb or a normal vision in the right visual field. However, the overall recovery was outstanding for all cats such that the neonatal-lesioned were hard to differentiate from intact controls in their spontaneous, daily activities. Because the lesions were similar in the two age-at-lesion groups, and since numerous functions were followed for prolonged, comparable postlesion time, we conclude that, after hemispherectomy in the cat, there definitely is greater functional recovery if the lesion is sustained early in life. We propose that the enhanced recovery of function in neonatal-lesioned cats is largely due to the extensive anatomical reorganization which we have demonstrated in ongoing studies, and which contrasts with a lesser remodeling in adult-lesioned cats.

Chronic exposure to caffeine during early development increases dendritic spine and branch formation in midbrain optic tectum.

Sibling juvenile jewel fish were chronically exposed to caffeine solution (14 mg/l) between 50 and 100 days after fertilization. Some experimental animals were sacrificed at this time, together with control siblings reared without drugs; others were allowed to recover in the absence of caffeine for 18 months. Golgi-stained preparations showed increased formation of dendritic spines on apical stem dendrites of pyriform tectal neurons in juveniles exposed to caffeine. Adults showed minimal effects on the stem dendrite, but caffeine adults formed more primary dendritic branches and more spines on branches than controls did. This study demonstrates the facilitation of neuronal growth and complexity by chronic administration of a chemical agent commonly present during early development in humans.

Rapid effect of biologically relevant stimulation on tectal neurons: changes in dendritic spine morphology after nine minutes are retained for twenty-four hours.

Flight from a threatening stimulus was used as a natural analog of tetanizing electrical stimulation to determine whether brief, intense stimulation can alter dendritic spine morphology. Juvenile jewel fish were pretreated in a dark, sound-proofed room for 48 h, at which time unstimulated controls were sacrificed by hypothermic anesthesia. Remaining fish were forced to flee for 9 min and then either sacrificed immediately (9-min stimulated group) or returned to the dark for 24 h (24 h recovery group). Morphometric quantification of dendritic spines on the basal 120 micrometers of the apical dendrites of tectal pyriform interneurons revealed substantial changes in 3 measures of spine shape: spine head width, overall spine length, and spine stem length. Compared with unstimulated controls, 9 min of stimulation changed the relative frequencies of spine head width, overall spine length and spine stem length. Morphological effects were evident after 24 h, and changes in relative spine length and spine stem length frequencies were more pronounced in the upper strata of neurons 24 h after stimulation. Thus brief, biologically relevant stimulation can cause both immediate and persistent changes in spine morphology; this plasticity is discussed in the context of related anatomical studies.

There is less thalamic degeneration in neonatal-lesioned than in adult-lesioned cats after cerebral hemispherectomy.

In order to study age-related processes of degeneration and recovery, the left hemitelencephalon was surgically removed in 5 adult cats and 5 neonatal kittens which were compared to 5 intact controls. After long survival, brains were sectioned in the coronal plane and thionine-stained. Drawings of gross and microscopic thalamic structures were made at 4 planes and computer-digitized to provide 7 measures: total thalamic area at planes A 7.5 and A 8.5; counts of neuroglia, small neurons (31-100 microns2) and large neurons (101-1000 microns2); area of neuroglia, small neurons and large neurons. All cellular measurements were in the ventrobasal complex at planes A 8.0 and A 10.0. Morphological changes were found bilaterally in all lesioned cats. Ipsilateral to the ablation in adult-lesioned cats, thalamic area, large neuron count and glia cells size were markedly and significantly decreased (P less than 0.01), while in neonatal-lesioned cats these changes were present but significantly reduced in magnitude compared to adult-lesioned animals. In addition, adult-lesioned cats showed a marked increase in glial cell numbers (P less than 0.01) and a decrease in small neuron size (P less than 0.01), while kitten-lesioned animals did not show changes in these measures. In the intact side of the brain, the thalamus of adult-lesioned cats was decreased in size (P less than 0.01), and glial cells were decreased in number and size (P less than 0.05), while in kitten-lesioned brains there were few changes. In both lesioned groups large neurons showed a significant increase in size (P less than 0.01). We conclude that neonatal hemispherectomy results in markedly less thalamic atrophy, retrograde neuronal degeneration and gliosis than the equivalent lesion in adults. The changes are discussed in the context of the increased neuroanatomical reorganization and functional recovery which were reported in neonatal- vs adult-lesioned animals.

Early development of spiny neurons in fish and mouse: morphometric measures of dendritic spine formation pattern.

Positions of spines on apical dendrites were evaluated using 4 pattern analysis techniques: spine counts, variance/mean ratio, Lloyd's patchiness index, and nearest neighbor distance matrix. Spiny tectal interneurons from jewel fish (100, 130, 160 and 1550 days old), and layer V pyramidal cells in layer IV auditory cortex of CBA/J mice (100 and 450 days old) were studied. Fish's spines became more numerous and more clumped on distal dendritic strata during development, while mice lost dendritic spines with age. Both species developed a significantly regular spacing pattern between neighboring spines during development. These changes are explained in the context of spine function and a biophysical model of dendritic spine patterns.

Crowded jewel fish show changes in dendritic spine density and spine morphology.

This study demonstrates that neuronal development can be adversely affected by short-term crowding. Compared with uncrowded siblings (15 fish/73-litre), juvenile jewel fish (15 fish/3.6-litre) crowded for 60 days exhibited 50-58% fewer dendritic spines and spines with narrower heads on pyriform interneurons in deep tectal strata. These findings indicate that crowded environments can produce aberrant neuronal development similar to that seen in socially deprived jewel fish.

Neurocognitive impairment in dramatic personalities: histrionic, narcissistic, borderline, and antisocial disorders.

Thirty-seven patients with personalities in the dramatic cluster (DSM-III-R histrionic, narcissistic, borderline, and antisocial) and 40 controls matched for age and gender were evaluated on 16 neurocognitive variables. The evaluation screened for deficits in functions of attention, memory, language, abstraction, and behavior planning/sequencing. Analysis of variance revealed significant deficits in neurocognitive performance among patients with dramatic personalities, particularly in subtests requiring multi-step, multi-element associative operations.

The development of social group formation in normal and mentally retarded persons from early childhood through old age: neuropsychiatric, developmental, and cognitive implications.

To test the hypothesis that mentally retarded persons exhibit identifiable styles of social grouping, the size and sex composition of spontaneous social groups were studied in 570 mentally retarded and 3,030 nonretarded persons from preschool-elderly ages. Both populations showed similar developmental trends: group sizes increased through childhood, decreased thereafter until middle age, and increased again in the elderly. However, mentally retarded persons formed smaller groups at all ages: solitary individuals predominated with few groups larger than dyads. A subgroup of lower-functioning retarded persons formed even smaller groups. Nonretarded females were more aggregative than males, but mentally retarded persons showed no such sex differences.

Relationship of depression and cognitive impairment to self-injury in borderline personality disorder, major depression, and schizophrenia.

Self-injury was studied in 64 adults with borderline personality disorder, major depression, or chronic paranoid schizophrenia. Subjects were rated according to acute depression, chronic depression, self-injurious behaviors, and neurocognitive deficits, as measured by cognitive function examination. Borderline patients showed more self-injurious behaviors and more chronic depressive symptoms than the major depression or schizophrenia groups. Self-injury was not significantly correlated with acute or chronic depression in any group, but self-injury was correlated with neurocognitive deficits in borderline and schizophrenic groups. The results are explained in the context of a neurocognitive model of psychotic thought process in borderline disorder and schizophrenia.

Chronic exposure to caffeine during early development modifies spatial behavior in juvenile jewel fish schools.

Thirty sibling African jewel fish (Hemichromis bimaculatus) were either chronically exposed to caffeine between 50 to 100 days of age (at a concentration of 14 mg/l) or reared without drug in a control environment. After caffeine was withdrawn, fish in each group were videotaped for 30 minutes while schooling in a large tank. From the video record, mean distances and coefficients of variation of spacing were computed for each fish and its 1st to 9th nearest neighbors in all observations. Spacing distances were nearly identical for the two groups, but the caffeine fish exhibited much more variability in their spacing behavior (p less than 0.00001). This study demonstrates an effect on behavior of moderate caffeine levels present throughout the early developmental period, and illustrates a quantitative method to test for such effects.

The spacing of rhesus monkey troops changes when a few group members receive delta 9THC or D-amphetamine.

Free ranging troops of 18--25 rhesus macaques were housed in large enclosures where a few higher-ranking monkeys were given psychotropic agents (delta 9THC: 4 mg/kg or d-amphetamine: 2 mg/kg). Measures of group spacing were (1) mean distance between all animals. (2) daily variance in mean distance, (3) nearest neighbor distance, (4) daily variance in nearest neighbor distance, (5) index of clumping (S2/mean ratio), (6) % touching, and (7) % movement/2 sec. When 3--4 animals were drugged, the entire group moved closer together and distances were more variable. Acute changes after the first week of delta 9THC and effects after delta 9THC withdrawal were seen in two experiments. Chronic behavioral changes were found for d-amphetamine and in one delta 9THC experiment.

Reassessing morphine effects in cats: II. Protracted effects on sleep-wakefulness and the EEG.

Adult cats were implanted with standard electrodes to record EEG, EOG, and EMG. After 15 days, morphine sulphate or saline placebo was given IP at 0.5, 1.0, 2.0, 3.0 mg/kg, at least 15 days apart. Cats were continuously recorded for 72 hr postinjection. Wakefulness, drowsiness, NREM and REM sleep percentages were scored from polygraphic features and statistically analysed. There was a dose-dependent suppression of NREM and REM sleep for at least 6 hours postmorphine, with a progressive sleep recovery thereafter. During the insomnia period there was an EEG/behavioral dissociation where bursts of high-voltage waves were seen over a background of desynchrony; meanwhile the animal was first aroused although quiet and later showed stereotypic behavior. There was a prolonged NREM sleep rebound which started later at the higher doses. A significant, relatively brief REM sleep rebound was seen only at the lowest dose. The latency for NREM and REM sleep onset was also dose-dependent. Possible brain sites of morphine actions and similarities with effects in other species are discussed.

Reassessing morphine effects in cats: I. Specific behavioral responses in intact and unilaterally brain-lesioned animals.

Behavioral responses to single low doses of morphine (0.5-3.0 mg/kg IP) were measured in intact cats and in cats with removal of one cerebral hemisphere or one caudate nucleus. Responses were dose-dependent and formed 3 stages: (1) autonomic stage (0-15 min postdrug): with vocalization, salivation, licking, swallowing, retching and vomiting; (2) quiet stage (15-60 min postdrug): sitting, fixed gaze, mydriasis, and pricked pinnae; (3) head movement stage (from 30-60 min postdrug and decreasing by the 5th hr): fully aroused but mostly sitting; showing discrete, complex head movements of a visual-tracking type with pouncing/avoidance paw movements, and with irregular, dose-dependent bouts of rocking, pivoting, and backing. Sleep, grooming, micturition and defecation were suppressed. In hemispherectomized cats the frequency of head movements was increased only towards the side of the ablation, and there was a strong bias for body turning to that side together with a significant bias to move the ispilateral paw. None of these biases were significant in cats with a unilateral caudate ablation. We conclude that the cat is an excellent model for behavioral morphine studies when dose levels below those inducing "feline mania" are used. CNS sites underlying these responses are discussed.

Reassessing morphine effects in cats: III. Responses of intact, caudate nuclei-lesioned and hemispherectomized animals following chronic administration and precipitated withdrawal.

Behavioral response to low doses of morphine (2.0 to 3.0 mg/kg, IP) administered for up to 15 days, and responses to subsequent naloxone challenges, were measured in intact, unilaterally and bilaterally caudate-lesioned (acaudate) cats, and in hemispherectomized cats using a video time-sampling method. For all groups minor tolerance to posture and movement activation patterns was seen, with a reciprocal increase in motor relaxation, which was somewhat more marked for acaudate cats. In contrast to this weak tolerance, all cats showed strong, typical withdrawal manifestations at the beginning of abstinence and a "mini withdrawal" could still be precipitated 15-30 days later when morphine was no longer detectable in the blood. The cats with the unilateral lesions showed whole body turning toward the lesioned side after morphine and away from the lesioned side following naloxone. Only hemispherectomized and acaudate animals showed significant physical deterioration (e.g., weight loss, decreased activity). The comparisons between weak tolerance development versus strong physical dependence and the possible mechanisms involved in shifting the turning biases are discussed. The potential of the cat as a model for studying opiate effects is stressed.