Early specification and autonomous development of cortical fields in the mouse hippocampus

Studies of the specification of distinct areas in the developing cerebral cortex have until now focused mainly on neocortex. We demonstrate that the hippocampus, an archicortical structure, offers an elegant, alternative system in which to explore cortical area specification. Individual hippocampal areas, called CA fields, display striking molecular differences in maturity. We use these distinct patterns of gene expression as markers of CA field identity, and show that the two major hippocampal fields, CA1 and CA3, are specified early in hippocampal development, during the period of neurogenesis. Two field-specific markers display consistent patterns of expression from the embryo to the adult. Presumptive CA1 and CA3 fields (Pca1, Pca3) can therefore be identified between embryonic days 14.5 and 15.5 in the mouse, a week before the fields are morphologically distinct. No other individual cortical areas have been detected by gene expression as early in development. Indeed, other features that distinguish between the CA fields appear after birth, indicating that mature CA field identity is acquired over at least 3 weeks. To determine if Pca1 and Pca3 are already specified to acquire mature CA field identities, the embryonic fields were isolated from further potential specification cues by maintaining them in slice culture. CA field development proceeds in slices of the entire embryonic hippocampus. More strikingly, slices restricted to Pca1 or Pca3 alone also develop appropriate mature features of CA1 or CA3. Pca1 and Pca3 are therefore able to develop complex characteristics of mature CA field identity autonomously, that is, without contact or innervation from other fields or other parts of the brain. Because Pca1 and Pca3 can be identified before major afferents grow into the hippocampus, innervation may also be unnecessary for the initial division of the hippocampus into separate fields. Providing a clue to the source of the true specifying signals, the earliest field markers appear first at the poles of the hippocampus, then progress inwards. General hippocampal development does not follow this pronounced pattern. We suggest that the sources of signals that specify hippocampal field identity lie close to the hippocampal poles, and that the signals operate first on cells at the poles, then move inwards.     

Aspects of cortical organization related to the geometry of neurons with intra-cortical axons

Using the Golgi method, cells with intra-cortical axons in the visual cortex of young mice were classified according to defined geometrical axonal shapes. This study principally describes a computer technique and its application to the study of neuronal morphology. Neurons were converted in a sequence of three-coordinate points which were stored in digital form on magnetic tape. From the stored data and total real length in space of dendrites and axons was obtained and the results compared in two groups of mice raised under different conditions. Preliminary observations show short axonal lengths in mice raised in darkness. Using Eulerian coordinate transformations, reconstructions of individual neurons and of groups of several neurons and fibres were obtained by generating displays of different views after rotation around the horizontal axis. Reconstructed pictures were compared with their corresponding original drawings in order to describe particular aspects of cortical organization.     

Somatic mutation in the neonatal mouse

Several mechanisms that diversify the adult immune repertoire, such as terminal deoxynucleotidyl transferase-dependent N region addition, are not available to the neonatal mouse. One important process that contributes to protective immunity in the adult is somatic mutation, which plays a major role in the generation of high affinity memory B cells. It is not clear whether B cells in the neonatal mouse can activate the somatic mutation machinery. To investigate this, we immunized neonates with poly(L-Tyr,L-Glu)-poly-D,L-Ala-poly-L-Lys complexed with methylated BSA, or (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma-globulin. Eight to fourteen days after priming, V(D)J rearrangements of known V(H) genes (V(H)SM7 family) were screened for mutations using a temperature-melt hybridization assay and oligonucleotide probes specific for complementarity-determining regions I and II; possible mutations were confirmed by sequence analysis. More mutations per sequence were found in heavy chains from neonates immunized with (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma-globulin than in those from neonates immunized with poly(L-Tyr,L-Glu)-poly-D,L-Ala-poly-L-Lys complexed with methylated BSA. Mutations were found in heavy chains lacking N regions, suggesting that B cells of the putative fetal lineage can somatically mutate and diversify an initially limited repertoire. Since neonates immunized as early as 1 or 2 days after birth had mutations, the somatic mutation machinery can be activated soon after birth, suggesting that early vaccination should result in affinity maturation and protective immunity in the neonate.     

Extent of recovery from neonatal damage to the cortical visual system in cats.

Cats that received either marginal or marginal plus extramarginal lesions as 3-day-old kittens were assessed on a series of tests of visually guided behavior. These Ss were not conspicuously different from normal controls in avoiding obstacles or in activity level. Yet these same operated Ss were severely impaired in performance on the visual cliff and in visual discrimination learning, even when lesions were limited to the geniculocortical portion of the visual system. Maximum losses in pattern and form discrimination learning were observed only in Ss with severe retrograde degeneration in both the lateral geniculate nucleus and the complex of the pulvinar and nucleus lateralis posterior. Photically evoked potentials were recorded in the lateral regions of the neocortex more reliably from operated Ss that had made fewer errors in discrimination learning than from more severely debilitated cases; this relation was present even among cases with nearly equivalent amounts of retrograde degeneration in the visual thalamus. These findings suggest that in the cat (a) recovery of vision is incomplete after neonatal lesions of the visual cortex, and (b) a cortical system lateral to the geniculocortical projections may be involved in pattern vision. (29 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Synapses from labeled type II axons in the mouse cochlear nucleus

This study investigates the ultrastructure and central targets in the cochlear nucleus of axonal swellings of type II primary afferent neurons. Type II axons comprise only 5-10% of the axons of the auditory nerve of mammals, but they alone provide the afferent innervation of the outer hair cells. In this study, type II axons were labeled with horseradish peroxidase, and serial-section electron microscopy was used to examine their swellings in: (1) the granule-cell lamina at its boundary with posteroventral cochlear nucleus, (2) the rostral anteroventral cochlear nucleus, and (3) the auditory nerve root. Only some (18%) of the type II terminal and en-passant swellings formed synapses. The synapses were asymmetric and contained clear round synaptic vesicles, suggesting that they are excitatory. Type II synapses were compared to those from type I fibers providing the afferent innervation of the inner hair cells. Type II synapses tended to have slightly smaller and fewer synaptic vesicles, had a greater proportion of the membrane apposition accompanied by a postsynaptic density, and often had densities that were discontinuous or 'perforated'. In all cochlear nucleus regions examined, the postsynaptic targets of type II synapses had characteristics of dendrites; in most cases these dendrites could not be traced to their cell bodies of origin. Some evidence suggests, however, that targets may include granule cells, spherical cells, and other cells in the nerve root. These results suggest afferent information from outer hair cells reaches diverse regions and targets within the cochlear nucleus.     

Ontogeny of memory in the neonatal mouse.

Conducted 4 experiments with Swiss-Webster mice (N = 488) to examine the development of memory of an escape response between 3-11 days of age. Ss were given 25 training trials in a straight-alley escape task and then retested at various retention intervals. Results show that 5- and 7-day-old Ss had a retention capacity of less than 6 hr. At 9 days of age, however, retention capacity greatly increased to at least 96 hr., suggesting that a period of maturational development critical to long-term memory processes occurs at 9 days of age in the neonatal mouse. (15 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Ulex europaeus 1 lectin targets microspheres to mouse Peyer''s patch M-cells in vivo

OBJECTIVE: To evaluate the relationship between dose of N-0861 ([+/-]N6-endo-norbornan-2-yl-9-methyladenine), N-0861 plasma concentrations, and antagonism of adenosine-induced slowing of atrioventricular nodal conduction and to evaluate A1-receptor occupancy by antagonist present in plasma of subjects after administration of N-0861 to determine A1-selectivity of these effects. METHODS: The study was conducted in patients undergoing a clinically indicated electrophysiology study to evaluate atrioventricular nodal conduction. Nineteen subjects were enrolled in the study and received adenosine (60 to 140 microg/kg) before or during a bolus dose and maintenance infusion of specific doses of N-0861. Adenosine-induced slowing of atrioventricular nodal conduction was determined by measuring A-H intervals on the intracardiac electrocardiograms. Plasma concentrations of N-0861 were determined with an HPLC method. A1-Receptor occupancy by antagonist present in plasma from identical time points was determined with use of a radioreceptor assay. RESULTS: A linear relationship was shown between plasma concentration and dose of N-0861. A-H interval lengthening by 60 microg/kg adenosine was reduced by administration of N-0861. A linear relationship was observed between A1 occupancy and N-0861 concentration and between occupancy and antagonism of adenosine-induced A-H prolongation. CONCLUSION: The results suggest that the effect of N-0861 on antagonism of adenosine-induced prolongation of A-H interval, at the doses used in this study, were the result of effects at the A1 receptor.     

Induction and suppression of reagins in the neonatal mouse

Neonatal SWR mice are capable of synthesizing reagins when immunized with a mixture of concanavalin and ovalbumin or a mixture of Bordetella pertussis, alum and ovalbumin. Reaginic antibody-forming cells can be found in the spleen, lymph nodes, bone marrow and Peyer's patches. Tolerance with respect to IgE can be induced by the injection of deaggregated ovalbumin into neonatal mice.     

Initial trajectories of sensory axons toward laminar targets in the developing mouse spinal cord

Quantitative ultrasonic tissue characterization using backscattered high-frequency intravascular ultrasound could provide a basis for the objective identification of lesions in vivo. Representation of local measurements of quantitative ultrasonic parameters in a conventional image format should facilitate their interpretation and thus increase their clinical utility. Toward this goal, the apparent integrated backscatter, the slope of attenuation (25-56 MHz) and the value of the attenuation on the linear fit at 37.5 MHz were measured using the backscattered radio frequency signals from in vitro human aortae. Local estimations of these ultrasonic parameters from both normal and atherosclerotic aortic segments were displayed in a B-scan format. The morphological features of these parametric images corresponded well to features of histological images of the same regions. The attenuation from 25-56 MHz of seven segments of the medial layer (both with and without overlying atheroma) were measured using the multinarrow-band backscatter method. The average attenuation in the media at 24 degrees C +/- 3 degrees C was 45 +/- 16 dB/cm at 25 MHz and 102 +/- 13 dB/cm at 50 MHz. This work represents progress toward the development of quantitative imaging methods for intravascular applications.     

Dendritic alterations in cortical pyramidal cells in the sparse fur mouse

Although the transfer of cholesteryl ester (CE) from high-density lipoprotein (HDL) to the apolipoprotein B-containing lipoproteins (very-low-density lipoproteins + low-density lipoproteins) has been shown to be abnormally increased in a number of conditions associated with increased cardiovascular risk, it has not been studied in patients with essential hypertension (EH). To determine whether subjects with EH have increased CE transport, CE transfer (CET) was estimated isotopically and lipoprotein lipid and phospholipid composition determined in a group of 14 untreated normolipidemic (triglycerides 116+/-46, cholesterol 185+/-30, HDL 38+/-10 mg/dl) otherwise healthy ethnically diverse EH subjects. CET was significantly increased in EH subjects compared to a similar group of normotensive controls (EH: k = 0.27+/- 0.09 vs. control k = 0.11+/-0.02: P < 0.01). Lipoprotein concentration and composition were comparable in the two groups and closely resembled that of an age- and sex-matched reference group. The abnormal increase in CET persisted (k = 0.25+/-0.12) after 3 months of treatment with the angiotensin converting enzyme (ACE) inhibitor ramipril without a change in either plasma or lipoprotein lipids. Thus, CET is increased in normolipidemic subjects with EH and is not affected by the ACE inhibitor ramipril.     

Analysis of Huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons

Huntingtin, the protein product of the Huntington's disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin-associated protein 1 (HAP-1), a brain-enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP-1 is unknown, but recent evidence supports a role in microtubule-dependent organelle transport. We examined the subcellular localization of HAP-1 with an antibody made against the NH2-terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP-1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP-1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP-1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP-1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP-1 localized to membrane-bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP-1 was also strongly associated with an unusual large "dense" organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP-1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP-1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons.     

Early walking in the neonatal rat: a kinematic study

The development of the early stage of locomotion (between Postnatal Days 3 and 10) was studied in newborn rats. At this age, rats are known to perform limited locomotor activities, consisting of an inefficient nonpostural gait termed crawling. By providing appropriate olfactory stimulation, it was possible to override the pups' reluctance to walk and to discover their actual locomotor abilities. The step period decreased from 1,200 ms to 900 ms from Postnatal Days 4 to 9, showing both a regular decrease in the swing and a discontinuous decrease in the stance phase. The fore- and hindlimb periods stabilized early on an alternate pattern of coupling. The ipsilateral coupling shifted progressively from 220 degrees to 260 degrees in relation with the change in the gait pattern. In parallel with the change in timing, the newborn rats showed gradual changes in the foot position and in the interlimb spatial coordination. These results show that quadruped locomotion develops before postural control is acquired, in a continuous process as the nervous system develops.     

Effect of hemorrhagic hypotension on cortical oxygen pressure and striatal extracellular dopamine in cat brain

This study investigated the relationships between blood pressure, cortical oxygen pressure, and extracellular striatal dopamine in the brain of adult cats during hemorrhagic hypotension and retransfusion. Oxygen pressure in the blood of the cortex was measured by the oxygen dependent quenching of phosphorescence and extracellular dopamine, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) by in vivo microdialysis. Following a 2 h stabilization period after implantation of the microdialysis probe in the striatum, the mean arterial blood pressure (MAP) was decreased in a stepwise manner from 132 +/- 2 Torr (control) to 90 Torr, 70 Torr and 50 Torr, holding the pressure at each level for 15 min. The whole blood was then retransfused and measurements were continued for 90 min. As the MAP was lowered there was a decrease in arterial pH, from a control value of 7.37 +/- 0.05 to 7.26 +/- 0.06. The PaCO2 decreased during bleeding from 32.3 +/- 4.8 Torr to 19.6 +/- 3.6 Torr and returned to 30.9 +/- 3.9 Torr after retransfusion. The PaO2 was 125.9 +/- 15 Torr during control conditions and did not significantly change during bleeding. Cortical oxygen pressure decreased with decrease in MAP, from 50 +/- 2 Torr (control) to 42 +/- 1 Torr, 31 +/- 2 Torr and 22 +/- 2 Torr, respectively. A statistically significant increase in striatal extracellular dopamine, to 2,580 +/- 714% of control was observed when MAP decreased to below 70 Torr and cortical oxygen pressure decreased to below 31 Torr. When the MAP reached 50 Torr, the concentration of extracellular dopamine increased to 18,359 +/- 2,764% of the control value. A statistically significant decrease in DOPAC and HVA were observed during the last step of bleeding. The data show that decreases in systemic blood pressure result in decrease in oxygen pressure in the microvasculature of the cortex, suggesting vascular dilation is not sufficient to result in a full compensation for the decreased MAP. The decrease in cortical oxygen pressure to below 32 Torr is accompanied by a marked increase in extracellular dopamine in the striatum, indicating that even such mild hypoxia can induce significant disturbance in brain metabolism.     

Interval timing and the encoding of signal duration by ensembles of cortical and striatal neurons.

This study investigated the firing patterns of striatal and cortical neurons in rats in a temporal generalization task. Striatal and cortical ensembles were recorded in rats trained to lever press at 2 possible criterion durations (10 sec or 40 sec from tone onset). Twenty-two percent of striatal and 15% of cortical cells had temporally specific modulations in their firing rate, firing at a significantly different rate around 10 sec compared with 40 sec. On 80% of trials, a post hoc analysis of the trial-by-trial consistency of the firing rates of an ensemble of neurons predicted whether a spike train came from a time window around 10 sec versus around 40 sec. Results suggest that striatal and cortical neurons encode specific durations in their firing rate and thereby serve as components of a neural circuit used to represent duration. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Manipulation of gonadal hormones in neonatal rats alters the morphological response of cortical neurons to brain injury in adulthood.

The authors examined the effects of sex and neonatal hormones on the response of pyramidal cells (Layer III, parietal cortex) to injury of the medial frontal cortex in the adult rat. At birth, males were gonadectomized (GDX) or sham-operated. Females were given testosterone (T) or oil injections. In adulthood, rats that had been left intact at birth were GDX, and they then received bilateral medial frontal cortex lesions or sham surgery. Rats not exposed to T at birth exhibited losses of dendritic arbor (males GDX at birth) or dendritic spine density (oil-treated females). Compensation after cortical injury is dependent on the rat's sex and history of exposure to gonadal steroids. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effect of neonatal thymectomy on follicle numbers in the post-natal mouse ovary

1. Surgical removal of the thymus gland within 48 hours of birth has no effect upon the numbers of small, medium and large ovarian follicles in Bagg albino strain mice during the first 12 weeks of life. 2. 20% of thymectomised mice more than 12 weeks of age developed ovarian atrophy which included the disappearance of corpora lutea and an overall reduction in all fractions of the follicle population. 3. These results do not support the proposal of Nishizuka and Sakakura that the thymus gland specifically maintains the non-growing, small (primordial) follicle pool in the mouse ovary during post-natal and reproductive life.     

Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality

Targeted disruption of the insulin receptor gene (Insr) in the mouse was achieved using the homologous recombination approach. Insr+/- mice were normal as shown by glucose tolerance tests. Normal Insr-/- pups were born at expected rates, indicating that Insr can be dispensable for intrauterine development, growth and metabolism. However, they rapidly developed diabetic ketoacidosis accompanied by a marked post-natal growth retardation (up to 30-40% of littermate size), skeletal muscle hypotrophy and fatty infiltration of the liver and they died within 7 days after birth. Total absence of the insulin receptor (IR), demonstrated in the homozygous mutant mice, also resulted in other metabolic disorders: plasma triglyceride level could increase 6-fold and hepatic glycogen content could be five times less as compared with normal littermates. The very pronounced hyperglycemia in Insr-/- mice could result in an increased plasma insulin level of up to approximately 300 microU/ml, as compared with approximately 25 microU/ml for normal littermates. However, this plasma level was still unexpectedly low when compared with human infants with leprechaunism, who lack IR but who could have extremely high insulinemia (up to > 4000 microU/ml). The pathogenesis resulting from a null mutation in Insr is discussed.     

Wnt-7a maintains appropriate uterine patterning during the development of the mouse female reproductive tract

The murine female reproductive tract differentiates along the anteroposterior axis during postnatal development. This process is marked by the emergence of distinct cell types in the oviduct, uterus, cervix and vagina and is dependent upon specific mesenchymal-epithelial interactions as demonstrated by earlier heterografting experiments. Members of the Wnt family of signaling molecules have been recently identified in this system and an early functional role in reproductive tract development has been demonstrated. Mice were generated using ES-mediated homologous recombination for the Wnt-7a gene (Parr, B. A. and McMahon, A. P. (1995) Nature 374, 350-353). Since Wnt-7a is expressed in the female reproductive tract, we examined the developmental consequences of lack of Wnt-7a in the female reproductive tract. We observe that the oviduct lacks a clear demarcation from the anterior uterus, and acquires several cellular and molecular characteristics of the uterine horn. The uterus acquires cellular and molecular characteristics that represent an intermediate state between normal uterus and vagina. Normal vaginas have stratified epithelium and normal uteri have simple columnar epithelium, however, mutant uteri have stratified epithelium. Additionally, Wnt-7a mutant uteri do not form glands. The changes observed in the oviduct and uterus are accompanied by a postnatal loss of hoxa-10 and hoxa-11 expression, revealing that Wnt-7a is not required for early hoxa gene expression, but is required for maintenance of expression. These clustered hox genes have been shown to play a role in anteroposterior patterning in the female reproductive tract. In addition to this global posterior shift in the female reproductive tract, we note that the uterine smooth muscle is disorganized, indicating development along the radial axis is affected. Changes in the boundaries and levels of other Wnt genes are detectable at birth, prior to changes in morphologies. These results suggest that a mechanism whereby Wnt-7a signaling from the epithelium maintains the molecular and morphological boundaries of distinct cellular populations along the anteroposterior and radial axes of the female reproductive tract.     

A comparison of postlesion growth of retinotectal and corticotectal axons after superior colliculus transections in neonatal rats

We examined, in neonatal rats, the postinjury response of two different axonal systems that project to a common target area in the visual system. Transections across the rostral part of the left superior colliculus (SC) were made in 2- or 6-day-old rats (P2, P6). Lesioned animals were randomly selected into short- or long-term groups. The short-term group was used to determine the efficacy of the lesion technique; 2-6 days after transections, right (contralateral) eyes were injected with horseradish peroxidase (HRP). Complete deafferentation of the SC was achieved in 73% of P2 (n = 22) and 53% of P6 (n = 10) short-term animals. In the long-term group (examined 2-7 months after transection), retinotectal and corticotectal projections were assessed in each animal by using [3H]proline and wheat germ agglutin-HRP, respectively. Examination of a series of sagittal sections revealed that the cut had extended across the entire SC in 63% of P2 (n = 19) and 55% of P6 (n = 12) long-term rats. Despite this, retinal and cortical axons were seen in appropriate layers in postlesion SC in all P2 lesioned animals. Cortical projections caudal to the cut were seen in all P6 rats; however, in these animals, the retinal projection was sparse and not always present. Differences in lesion geometry led to consistent differences in the pattern and extent of ingrowth of retinal and cortical axons into postlesion SC neuropil. The two axonal populations also followed different paths as they grew between prelesion and postlesion SC. It is likely that a number of factors influenced the patterns of postlesion growth, including the relative maturity of the axons and the neuropil into which they were growing. There was also, however, clear evidence of competitive interactions between retinal and cortical axons in postlesion SC that consistently led to greater than normal segregation of the two populations and hence restricted their terminal distributions.