Perinatal undernutrition: accumulation of catecholamines in rat brain.

Brains of rats undernourished from midgestation and killed at weaning contained 25 percent less norepinephrine than brains of adequately fed littermates. Perinatal undernutrition also suppressed the accumulation of brain dopamine. Paradoxically, the activity of tyrosine hydroxylase, the enzyme thought to be rate-limiting in catecholamine biosynthesis, was significantly increased in brains from undernourished animals.

Mice with conditional inactivation of fibroblast growth factor receptor-2 signaling in oligodendrocytes have normal myelin but display dramatic hyperactivity when combined with Cnp1 inactivation.

Fibroblast growth factor receptors (Fgfr) comprise a widely expressed family of developmental regulators implicated in oligodendrocyte (OL) maturation of the CNS. Fgfr2 is expressed by OLs in myelinated fiber tracks. In vitro, Fgfr2 is highly upregulated during OL terminal differentiation, and its activation leads to enhanced growth of OL processes and the formation of myelin-like membranes. To investigate the in vivo function of Fgfr2 signaling by myelinating glial cells, we inactivated the floxed Fgfr2 gene in mice that coexpress Cre recombinase (cre) as a knock-in gene into the OL-specific 2',3'-cyclic nucleotide phosphodiesterase (Cnp1) locus. Surprisingly, no obvious defects were detected in brain development of these conditional mutants, including the number of OLs, the onset and extent of myelination, the ultrastructure of myelin, and the expression level of myelin proteins. However, unexpectedly, a subset of these conditional Fgfr2 knock-out mice that are homozygous for cre and therefore are also Cnp1 null, displayed a dramatic hyperactive behavior starting at approximately 2 weeks of age. This hyperactivity was abolished by treatment with dopamine receptor antagonists or catecholamine biosynthesis inhibitors, suggesting that the symptoms involve a dysregulation of the dopaminergic system. Although the molecular mechanisms are presently unknown, this novel mouse model of hyperactivity demonstrates the potential involvement of OLs in neuropsychiatric disorders, as well as the nonpredictable role of genetic interactions in the behavioral phenotype of mice.

Senescent changes in a neurobiological model system: cerebellar Purkinje cell electrophysiology and correlative anatomy.

A within-subjects design was used to assess age changes in cerebellar Purkinje neurons. Four groups of naive male Sprague-Dawley rats, aged 3, 10, 20, and 28 months. underwent single cell recording for electrophysiological assessment of Purkinje cell firing patterns, followed by perfusion for glyoxylic acid induced catecholamine fluorescence. Cerebellar sections were photographed first by fluorescence microscope for catecholamines, and 2-3 weeks later for quantification of lipofuscin autofluorescence. Finally, these same tissues were treated with cresyl violet and photographed a third time to permit quantitative estimates of age changes in the number of Nissl staining Purkinje neurons. Electrophysiological studies revealed significant effect of age on a number of Purkinje cell firing parameters: in particular, increasing numbers of aberrant, very slow-firing cells were encountered in older animals. These cells showed normal climbing fiber mediated burst activity, but spontaneous simple spike firing rates 3-5 times less than normal. Rats exhibiting the highest numbers of such abnormal cells also exhibited the poorest Nissl staining. Conversely, good Nissl staining of Purkinje neurons in an old rat was a reliable predictor of relatively normal Purkinje cell firing. Lipofuscin was found to accumulate measurably in Purkinje neurons by 20 months of age, and to increase significantly thereafter. Deposition of the substance occurred almost exclusively at the apical pole of the soma. Our data suggest, however, that accumulation of lipofuscin in Purkinje neurons, as well as its reported accumulation in the inferior olive, is not a primary cause of electrophysiological dysfunction. There was no apparent age change in glyoxylic acid induced catecholamine fluorescence nor, in separate pharmacological studies, could any senescent alteration in cerebellar catecholamine levels be found.

Senescent change in tissue weight and immunoreactive beta-endorphin, enkephalin, and vasopressin in eight regions of C57BL/6J mouse brain and pituitary.

Tissue weights and immunoreactive (IR) content and concentration of beta-endorphin, enkephalin, and vasopressin were assayed for senescent change in anterior pituitary, neurointermediate pituitary, hypothalamus, hippocampus, striatum, dorsolateral cortex, and pons-medulla, as well as residual brain samples remaining after the other dissections. Groups of naive male C57BL/6J mice, 8-12 months old and 28-32 months old, served as subjects. Old mice exhibited significant decline in anterior pituitary and hippocampus weight. Significant increases with age were found in pons-medulla weight. IR beta-endorphin content decreased in hypothalamus and neurointermediate pituitary. IR enkephalin decreased in striatum and dorsolateral cortex. IR vasopressin content increased in hypothalamus and residual brain. Assays were replicated in later experiments using additional subjects, for total Ns of up to 54 mice. Although significant overall results were always consistent in direction from replication to replication, the magnitude of age change exhibited considerable variability. Such results suggest that single experiments on age changes in neuropeptides, particularly those giving negative results, should be carefully scrutinized and replicated before their acceptance as evidence for a transmitter's stability or instability throughout the lifespan.

Evidence for changes in the Alzheimer's disease brain cortical membrane structure mediated by cholesterol.

Small angle X-ray diffraction analysis of Alzheimer's disease (AD) lipid membranes extracted from cortical gray matter showed significant, reproducible structure changes relative to age-matched control samples. Specifically, there was an average 4 A reduction in the lipid bilayer width and significant changes in the membrane electron density profiles of AD cortical samples. There were no significant structure differences in the membrane bilayers isolated from an unaffected region (cerebellum) of the AD brain. Lipid and protein analysis of 6 AD and 6 age-matched controls showed that the phospholipid:protein mass ratio was unchanged but that the unesterified cholesterol:phospholipid (C:PL) mole ratio decreased by 30% in the AD temporal gyrus relative to age-matched controls. By contrast, the C:PL mole ratio in the cerebellum did not change significantly. X-ray diffraction analysis of a cholesterol enriched AD sample demonstrated a virtual restoration of the normal membrane bilayer width and electron density profile, suggesting that the cholesterol deficit played a major role in the AD lipid membrane structure perturbation. Alterations in the composition and structure of the membrane bilayer may play an important role in the pathophysiology of AD by altering the activity and catabolism of membrane-bound proteins, including the beta-amyloid precursor protein.

Innervation of embryonic rat cerebral cortex by catecholamine-containing fibers.

Catecholamine-containing neuronal processes penetrate to the outer superficial layers of the developing neocortex via the lateral neocortex anlage by embryonic day (ED) 16, only 48 hours after the final cell division of the catecholamine neurons in the mesencephalon and pons-medulla. Sagittal sections were taken from a series of perfused embryos at precise time-intervals after insemination. The corpus striatum receives a large catecholamine input and serves as a reference for tracing the very fine varicose processes that course through the caudate nucleus to innervate the more rostral structures of the developing neocortex cerebri. The input fibers to the neocortex arrive via three to four small fiber bundles, entering chiefly at the ventro-rostral aspect. The bundles than bifurcate into the deep and superficial layers of the cortex. Between ED16 to ED21 the innervation progresses in ventral to dorsal and rostral to caudal directions. Embryonically, fluorescent fibers are observed in the outermost superficial layer and in the intermediate zone, below the cortical plate; only rarely are they seen crossing the cortical plate. The demonstration of monoaminergic neuronal fibers reaching neocortical structures by ED16 adds further weight to the speculation that they may play a role in induction and differentiation, and suggests that post-natal experimental manipulations using ascending-bundle lesions will have been performed at least five days after the arrival of catecholamine fibers at their cortical destinations.

Methodological considerations in culturing peptidergic neurons.

Both explant and dispersed cell culture preparations of brain tissue provide a means to directly assess the functions of neuropeptide-containing brain cells isolated from the complex influences present in vivo. The validity of the approach depends on reproducibility of observations. In dispersed cell cultures, we find that cell responses, as determined by secretion of the peptide somatostatin, have remained relatively constant both quantitatively and qualitatively over numerous preparations. In addition, for pharmacologic studies on somatostatin secretion, data from several laboratories are in good agreement. The validity of the dispersed cell approach also depends on whether the pharmacologic and physiologic behavior of cells parallels that expected of excitable tissue. The variability of the explant cultures from culture dish to culture dish makes quantitative experiments, such as demonstrated with the dispersed cultures, difficult. On the other hand, explant cultures better maintain the integrity of the tissue components, so that interactions between neurons and glial cells could occur as in vivo. The long-term health and viability of neuropeptidergic cells in explant and dispersed culture make both preparations potentially useful models to examine central nervous system physiology. Future work with such preparations must eventually address the problems of culturing adult brain tissue, the precise nutrient and hormonal requirements of brain cells, so that undefined medium components, such as serum, can be eliminated from the culture environment, and the general question of whether observations made in vitro facilitate our understanding of intact brain physiology.

Chemical and physiological aspects of the actions of lithium and antidepressant drugs.

The possible mechanisms underlying the anti-manic actions of lithium have been examined in a variety of interdisciplinary experiments. The possibility that lithium can regulate the sensitivity changes in dopaminergic transmission produced by chronic treatment with haloperidol has been tested. Although a modest modification of behavioral responses to the dopamine agonist apomorphine was found, there was no evidence that this action of lithium reflected alterations of the binding parameters of dopamine-related ligands. In other studies, consistent, dose-dependent increases in brain enkephalin content were found after rats consumed a specially manufactured lithium diet for 2-3 weeks. Not only were brain enkephalin levels increased after this treatment, but some signs of basal analgesic responsiveness also suggested that the elevated levels of enkephalins were functionally significant. To test the possibility that the effects of lithium may not be seen in normal rats, the effects of lithium were compared on spontaneously hypertensive and unaffected, normotensive rats of a related strain. Treatment with lithium altered blood pressure in the hypertensive strain but did not affect blood pressure in the controls. These studies suggest that multiple brain systems may be regulated by treatment with lithium but that the critical pathophysiological process may not be demonstrable in the normal rat.

Effect of isolation conditions on brain regional enkephalin and beta-endorphin levels and vocalizations in 10-day-old rat pups.

Young rat pups were isolated from their dams under different conditions. The endogenous opioid peptides were measured in brain regions after isolation. Because there is no uptake mechanism for peptides released at the synapse and because released peptide is rapidly degraded enzymatically, decreases in peptide levels over this time course can be interpreted as release from terminals. No change was observed in either peptide in the hypothalamus, septum, or amygdala after isolation compared with controls. Significant decreases were seen in the midbrain after isolation. A comparison of peptide levels and ultrasonic vocalizations in the pups isolated in familiar, novel, or control conditions was also performed. Enkephalin levels in the midbrain were decreased in familiar and novel conditions, but in the brainstem opioid peptides were decreased only in the familiar condition. The greater involvement of the opioid peptides in the pups isolated in familiar conditions may contribute to the ability of naltrexone to block vocalization.

Repeated isolation in the neonatal rat produces alterations in behavior and ventral striatal dopamine release in the juvenile after amphetamine challenge.

Rat pups were isolated from the mother and nest for 1 hr per day from Postnatal Day (PN) 2 to 9 At PN 27, rats were tested for behavioral responsiveness to 2.0 or 7.5 mg/kg amphetamine. Only isolated rats receiving the 7.5 mg/kg dose displayed increased activity scores, compared with nonisolated and nonhandled controls. Their increased activity is attributed to a slower latency to enter into stereotypy. In a second experiment, similarly treated groups were challenged by the 7.5 mg/kg dose during a session in which a microdialysis probe implanted in the ventral striatum was being perfused. The challenge drug elicited a much greater increase in dialysate dopamine in isolated vs. nonisolated groups. Results are discussed with regard to dissociation between sensitized and subsensitized responses.

Adult rats stressed as neonates show exaggerated behavioral responses to both pharmacological and environmental challenges.

Adult rats that were isolated from the mother and nest for 1 hr per day from Postnatal Day 2 to 9 were studied. Controls consisted of handled littermates as well as separate litters that were never handled. As adults, animals were given either a pharmacological challenge (1.0 or 2.0 mg/kg amphetamine) or an environmental challenge (restraint). Previously isolated animals demonstrated increased activity compared to controls at both drug doses. Similarly, isolated animals manifested exaggerated inhibition of activity after restraint. Previously isolated animals usually did not show differences compared to controls under baseline conditions (saline injection or no restraint). The neuroplastic changes that result from the neonatal experience are long lasting and appear when the system is challenged.

Repeated isolation stress in the neonatal rat: relation to brain dopamine systems in the 10-day-old rat.

Isolation of the rat pup from the nest and dam for one hour per day from PN 2-9 is a useful paradigm for producing stress in the neonate. These previously isolated rats respond to an amphetamine challenge with alterations in activity at the juvenile stage or as adults. Furthermore, when dopamine release is measured in the nucleus accumbens, juveniles release 3 times more dopamine after amphetamine than do controls. This study describes changes in behavior and brain dopamine systems at PN 10. Experiment 1 determined an appropriate amphetamine dose that could be used for behavioral activation at PN 10. Experiment 2 produced significant evidence of enhanced behavioral activation after the isolation paradigm and indicated that brain regions innervated by the mesolimbic dopamine system, septum, and hypothalamus display increased dopamine turnover and that the nigrostriatal pathway is less active. Likewise, in Experiment 3, in vivo microdialysis of the nucleus accumbens indicated that previously isolated pups respond to an amphetamine challenge with a several-fold increase in dopamine release over a 4-hour session.

Ethanol attenuation of morphine dependence: comparison to dizocilpine.

Recent studies indicate that morphine dependence, assessed as the severity of naloxone-precipitated opiate withdrawal in rats, is attenuated by dizocipline, a non-competitive, excitatory amino acid antagonist. Because ethanol is a putative excitatory amino acid antagonist, the present study compared the effects of co-administration of ethanol to that of dizocilpine on morphine dependence. Rats were administered morphine (10 mg/kg) twice daily for 9 days. One group received ethanol (1 g/kg) co-administration, another received dizocilpine (0.05 mg/kg) co-administration, and a third served as vehicle controls. On day 10, all rats received naloxone (4 mg/kg) injections and ratings of several classic signs of opiate withdrawal were made. Both ethanol- and dizocilpine-treated rats showed significantly less severe precipitated opiate withdrawal overall, with the ethanol group showing reduced ratings of some specific signs. These results demonstrate that ethanol, like dizocilpine, attenuates the development of morphine dependence. The results are consistent with the action of ethanol at glutamate receptors.

X-ray diffraction analysis of brain lipid membrane structure in Alzheimer's disease and beta-amyloid peptide interactions.

Small angle x-ray diffraction analysis of Alzheimer's disease (AD) lipid membranes reconstituted from cortical gray matter showed significant, reproducible structure changes relative to age-matched control samples. Specifically, there was an average 4 A reduction in the lipid bilayer width and marked changes in membrane electron density profiles of AD cortical samples. There were no significant structure differences in the membrane bilayers isolated from an unaffected region (cerebellum) of the AD brain. Lipid and protein analysis of six AD and six age-matched controls showed that the phospholipid:protein mass ratio was unchanged, but that the unesterified cholesterol:phospholipid (C:PL) mole ratio decreased by 30% in the AD temporal gyrus relative to age-matched controls. The C:PL mole ratio was not significantly different for samples prepared from cerebellum of AD versus control patients. X-ray diffraction analysis of a cholesterol-enriched AD sample demonstrated a virtual restoration of the normal membrane bilayer width and electron density profile, suggesting that the cholesterol deficit played a major role in the AD lipid membrane structure perturbation. Addition of beta-amyloid peptide to bovine brain phospholipid membranes significantly changed the electron density associated with the hydrocarbon core. Alterations in the composition and structure of the membrane bilayer may play an important role in the pathophysiology of AD by altering the activity and catabolism of membrane-bound proteins, including the beta-amyloid precursor protein.

Morphological and functional correlates of VIP neurons in cerebral cortex.

Vasoactive Intestinal Polypeptide (VIP) promotes the hydrolysis of 3H-glycogen newly synthesized from 3H-glucose by mouse cortical slices. This effect occurs rapidly, approximately 50% of the maximal effect being reached within one minute. The maximal effect is achieved after 5 minutes and maintained for at least 25 minutes. Furthermore the glycogenolytic effect of VIP is reversible, and pharmacologically specific. Thus several neuropeptides present in cerebral cortex such as cholecystokinin-8, somatostatin-28, somatostatin-14, met-enkephalin, leu-enkephalin, do not affect 3H-glycogen levels. VIP fragments 6-28, 16-28 and 21-28 are similarly inactive. Furthermore, among the peptides which share structural homologies with VIP, such as glucagon, secretin, PHI-27 and Gastric Inhibitory Peptide, only secretin and PHI-27 promote 3H-glycogen hydrolysis, with EC50 of 500 and 300 nM respectively, compared to an EC50 of 25 nM for VIP. Immunohistochemical observations indicate that each VIP-containing bipolar cell is identified with a unique radical cortical volume, which is generally between 15-60 micrograms in diameter and overlaps with the contiguous domains of neighbouring VIP-containing bipolar cells. Thus this set of biochemical and morphological observations support the notion that VIP neurons have the capacity to regulate the availability of energy substrates in cerebral cortex locally, within circumscribed, contiguous, radial domains.

The distribution of catecholamines and beta-endorphin in the brains of three behaviorally distinct breeds of dogs and their F1 hybrids.

This study examines neurochemical and behavioral differences among three types of domestic dogs and F1 hybrids derived from them. Purebred dogs included Border Collies, representing herding dogs, Shar Plaininetz, representing livestock protecting dogs, and Siberian Huskies, representing Northern dogs. Composite behavioral scores were derived from frequency measures of various components of predatory behavior observed when the dogs were tested with mice. Catecholamine levels, including norepinephrine (NE), dopamine (DA), and epineprine (EPI), were determined in various brain regions by high-performance liquid chromatography (HPLC) with electrochemical detection. beta-endorphin levels were determined in the same regions by RIA. Collies showed the highest levels of non-consummatory behaviors and Huskies the highest levels of consummatory behaviors. Shars were found to have lower levels of NE and DA than Collies and Huskies in several brain regions, including those comprising the nigrostriatal DA system. Positive correlations between neurochemical and behavioral characteristics could be made between Shars and Collies. Comparisons of F1 hybrids with their respective parental breeds revealed no clear pattern of inheritance for these characteristics but suggested that multiple factors, both independent and epistatic, are involved. Based on previous studies on nigrostriatal DA and behavior, the levels of DA in this system may be causally related to the levels of predatory behavior expressed by Collies and Shars.

Dorsal lesions of the prefrontal cortex: effects on alcohol consumption and subcortical monoaminergic systems.

Male Wistar rats were subjected to either bilateral aspiration lesions of the dorsal regions of the prefrontal cortex (PFC) or sham lesions and placed on a 6-week, modified sucrose-fading procedure. At the time of sacrifice, the size of the lesion, both in anterior-posterior and medial-lateral dimensions, was measured. Following sacrifice, levels of dopamine (DA), serotonin (5-HT), norepinephrine (NE), and their metabolites were measured in the midbrain (raphe) and nucleus accumbens (NA). Lesioned animals had reductions in 5-HT in the NA, and DA and NE in the raphe. The lesioned group drank more of a solution of 5% alcohol than controls early in the sucrose fading, and less during the later stages. In the lesioned group, the size of the left- and right-hemisphere lesions predicted 5-HIAA levels in the NA, and 5-HT and 5-HIAA levels in the raphe. A laterality effect was noted, such that the size of left-hemisphere lesions were positively associated with raphe 5-HT and 5-HIAA levels, and negatively associated with 5-HT levels in the NA, while right-hemisphere lesions showed the opposite relationships. In addition, the width of the left-hemisphere lesion predicted some measures of alcohol intake. These results suggest that, in the rat, the dorsal PFC is involved in the regulation of monoamines in subcortical regions known to be important in the regulation of reinforced behaviors, and that this regulation differs between hemispheres and shows a laterality effect. In addition, the dorsal PFC appears to have a subtle involvement in the regulation of alcohol intake.

Catecholamines in mutant mouse cerebellum: fluorescence microscopic and chemical studies.

Catecholamine-containing fibers have been examined in the cerebella of normal and hypocerebellar mutant mice using Falck-Hillarp and glyoxylic acid histofluorescence techniques. The amounts of norepinephrine and dopamine were also determined chemically in the same mutants. Green fluorescent fibers in cerebella of normal mice are similar in size and distribution to those described in the rat. Weanling and adult weaver, reeler and staggerer mice all manifest greatly increased specific catecholamine fluorescence per unit area in cerebellar cortex, but the patterns of fluorescent fibers are distinctive. In weaver, the fibers are of normal diameter, surround Purkinje cell bodies and appear to climb along major dendrites. In reeler, similar fibers form a plexus around cortical and non-cortical Purkinje cells; relatively normal fluorescent fiber patterns are present in well-organized cortical regions, while stouter disoriented fibers course through the shallow molecular layer in disorganized regions. Staggerer cerebellar cortex exhibits the greatest fluorescence with most terminals appearing as matted tangles adjacent cell bodies. Clearly defined fibers, however, can be distinguished in the molecular layer running perpendicular to the pia or for long distances in the coronal plane parallel to the pia. The major catecholamine determined chemically is norepinephrine. Reeler cerebella contain normal absolute levels and a doubled concentration of norepinephrine. In contrast, and despite the fluorescence findings, the total norepinephrine content of weaver and staggerer cerebella is significantly reduced and concentrations are not significantly different from normal.

Antiserum to nerve growth factor does not prevent the increase of brain stem noradrenaline after neonatal 6-hydroxydopa.

Antiserum to nerve growth factor (anti-NGF) given intraventricularly to newborn rats systemically injected with 6-hydroxydopa (6-OH-DOPA), did not prevent the long-term increase of brain stem noradrenaline produced by 6-OH-DOPA when given alone. Since the anti-NGF was biologically active and penetrated into the brain parenchyma, the role played by NGF in the outgrowth of central noradrenergic neurons, responsible for the elevation of brain stem noradrenaline, does not seem to be important.

Perinatal development of the endorphin- and enkephalin-containing systems in the rat brain.

Radioimmunoassay and microdissection procedures were used to study the perinatal development of the endorphin- and enkephalin-containing systems in the rat brain. In contrast to values reported on adult rat, endorphin levels are much higher than enkephalin levels on embryonic day 16. The highest endorphin values are found in the diencephalon, midline telencephalon and medulla-midbrain regions. Perinatally, enkephalin content increases at a faster rate than endorphin in all brain regions, producing a marked drop of the endorphin/enkephalin ratios. Between postnatal days 6 and 25, both endorphin and enkephalin levels increase, approaching their adult distribution pattern. No correlation was found between regional distributions or rates of increase of endorphin and enkephalin in any of these developmental stages, suggesting that the two peptide systems develop independently from each other.