Critical steps in the early evolution of the isocortex: Insights from developmental biology

This article proposes a comprehensive view of the origin of the mammalian brain. We discuss i) from which region in the brain of a reptilian-like ancestor did the isocortex originate, and ii) the origin of the multilayered structure of the isocortex from a simple-layered structure like that observed in the cortex of present-day reptiles. Regarding question i there have been two alternative hypotheses, one suggesting that most or all the isocortex originated from the dorsal pallium, and the other suggesting that part of the isocortex originated from a ventral pallial component. The latter implies that a massive tangential migration of cells from the ventral pallium to the dorsal pallium takes place in isocortical development, something that has not been shown. Question ii refers to the origin of the six-layered isocortex from a primitive three-layered cortex. It is argued that the superficial isocortical layers can be considered to be an evolutionary acquisition of the mammalian brain, since no equivalent structures can be found in the reptilian brain. Furthermore, a characteristic of the isocortex is that it develops according to an inside-out neurogenetic gradient, in which late-produced cells migrate past layers of early-produced cells. It is proposed that the inside-out neurogenetic gradient was partly achieved by the activation of a signaling pathway associated with the Cdk5 kinase and its activator p35, while an extracellular protein called reelin (secreted in the marginal zone during development) may have prevented migrating cells from penetrating into the developing marginal zone (future layer I).

A brain microdialysis study on 5-HT release in freely moving rat lines selectively bred for differential 5-HT1A receptor function

Breeding for high and low hypothermic responses to systemic administration of a serotonin1A (5-HT1A) receptor agonist (8-hydroxy-2-(di-n-propylamino)tetralin, 8-OH-DPAT) has resulted in high DPAT-sensitive (HDS) and low DPAT-sensitive (LDS) lines of rats, respectively. These lines also differ in several behavioral measures associated with stress. In the present microdialysis study we observed that basal 5-HT concentrations in the prefrontal cortex and dorsal hippocampus did not differ significantly between HDS and LDS rats. Thus, behavioral differences between the HDS and LDS lines might not be attributed to differences in basal 5-HT release. However, both lines had lower basal levels of 5-HT release than their randomly bred control group (random DPAT-sensitive, RDS) in the prefrontal cortex (mean ± SEM, pg/20 µl, was 3.0 ± 0.4 for LDS, 3.8 ± 0.3 for HDS and 6.4 ± 0.6 for RDS; F(2,59) = 5.8, P<0.005). The administration of (±)-fenfluramine (10 mg/kg) induced a greater increase in hippocampal 5-HT levels in HDS rats (500%) as compared with LDS (248%) or RDS (243%) rats (P<0.0001). There were no significant differences in the prefrontal cortex among lines, with a fenfluramine-induced 5-HT increase of about 900% in the three groups. This differential response to fenfluramine may be due to functional alterations of hippocampal 5-HT reuptake sites in the HDS line.

Substance P immunoreactivity in the lumbar spinal cord of the turtle Trachemys dorbigni following peripheral nerve injury

Immunoreactive substance P was investigated in turtle lumbar spinal cord after sciatic nerve transection. In control animals immunoreactive fibers were densest in synaptic field Ia, where the longest axons invaded synaptic field III. Positive neuronal bodies were identified in the lateral column of the dorsal horn and substance P immunoreactive varicosities were observed in the ventral horn, in close relationship with presumed motoneurons. Other varicosities appeared in the lateral and anterior funiculi. After axotomy, substance P immunoreactive fibers were reduced slightly on the side of the lesion, which was located in long fibers that invaded synaptic field III and in the varicosities of the lateral and anterior funiculus. The changes were observed at 7 days after axonal injury and persisted at 15, 30, 60 and 90 days after the lesion. These findings show that turtles should be considered as a model to study the role of substance P in peripheral axonal injury, since the distribution and temporal changes of substance P were similar to those found in mammals.

Anatomical connections of the periaqueductal gray: specific neural substrates for different kinds of fear

The periaqueductal gray (PAG) has been traditionally considered to be an exit relay for defensive responses. Functional mapping of its subdivisions has advanced our knowledge of this structure, but synthesis remains difficult mainly because results from lesion and stimulation studies have not correlated perfectly. After using a strategy that combined both techniques and a reevaluation of the available literature on PAG function and connections, we propose here that freezing could be mediated by different PAG subdivisions depending on the presence of immediate danger or exposure to related signaling cues. These subdivisions are separate functional entities with distinct descending and ascending connections that are likely to play a role in different defensive responses. The existence of ascending connections also suggests that the PAG is not simply a final common path for defensive responses. For example, the possibility that indirect ascending connections to the cingulate cortex could play a role in the expression of freezing evoked by activation of the neural substrate of fear in the dorsal PAG has been considered.

Vascular supply of the central nervous system of the land snail Megalobulimus oblongus (Gastropoda, Pulmonata)

The vascularization of the central nervous system of the snail Megalobulimus oblongus was studied by injection of carmine-gelatin solution into the arterial system and using a histochemical technique for the detection of alkaline phosphatase. The central nervous system of M. oblongus is irrigated by the anterior aorta, from which a series of small branches emerge that supply the subesophageal nervous ganglia. In turn, these branches give rise to a series of smaller vessels that irrigate the buccal bulb, the anterior portion of the foot, the cerebral ganglia, the dorsal body gland, and the anterior portion of the reproductive system. No hemolymph vessels were detected within nervous tissue although such vessels were found in the periganglionic connective sheath. This connective sheath contains vascular loops and had a series of overlaps and projections that follow the contour of the nervous ganglia. This arrangement permits a larger area of interaction between the surface of the nervous tissue and the hemolymph and reduces the distance between the deepest portion of a given ganglion and the hemolymph vessels.

c-Fos expression induced by electroacupuncture at the Zusanli point in rats submitted to repeated immobilization

In laboratory animals, acupuncture needs to be performed on either anesthetized or, if unanesthetized, restrained subjects. Both procedures up-regulate c-Fos expression in several areas of the central nervous system, representing therefore a major pitfall for the assessment of c-Fos expression induced by electroacupuncture. Thus, in order to reduce the effect of acute restraint we used a protocol of repeated restraint for the assessment of the brain areas activated by electroacupuncture in adult male Wistar rats weighing 180-230 g. Repeated immobilization protocols (6 days, 1 h/day and 13 days, 2 h/day) were used to reduce the effect of acute immobilization stress on the c-Fos expression induced by electroacupuncture at the Zusanli point (EA36S). Animals submitted to immobilization alone or to electroacupuncture (100 Hz, 2-4 V, faradic wave) in a non-point region were compared to animals submitted to electroacupuncture at EA36S (4 animals/subgroup). c-Fos expression was measured in 41 brain areas by simple counting of cells and the results are reported as number of c-Fos-immunoreactive cells/10,000 µm². The protocols of repeated immobilization significantly reduced the immobilization-induced c-Fos expression in most of the brain areas analyzed (P < 0.05). Animals of the EA36S groups had significantly higher levels of c-Fos expression in the dorsal raphe nucleus, locus coeruleus, posterior hypothalamus and central medial nucleus of the thalamus. Furthermore, the repeated immobilization protocols intensified the differences between the effects of 36S and non-point stimulation in the dorsal raphe nucleus (P < 0.05). These data suggest that high levels of stress can interact with and mask the evaluation of specific effects of acupuncture in unanesthetized animals.

Evidence that urocortin is absent from neurons of the Edinger-Westphal nucleus in pigeons

The Edinger-Westphal nucleus (EWN) is a central preganglionic parasympathetic cell group that gives rise to cholinergic input to the ciliary ganglion, thereby regulating several neurovegetative ocular functions. Recently, the supposed presence of the neuropeptide urocortin (UCN) has been reported in EWN neurons in rodent brain. The purpose of the present study was to examine the distribution of UCN in avian brain and to investigate by immunohistochemical analysis the possible use of this substance as an EWN marker in a non-mammalian class of vertebrates. Brain tissue of pigeons was incubated with a specific antibody against UCN and the results showed labeling of many small neurons, forming a double wing in the dorsal mesodiencephalic transition area. Their size and shape, however, differed from those of EWN neurons, and they were preferentially located rostral to the EWN. Double-label experiments employing an antibody against the enzyme choline acetyltransferase (ChAT) showed that UCN is not localized to the cholinergic cells of the EWN and confirmed the rostral distributionof UCN never overlapping the ChAT+ EWN cells. Taken together, these results suggest that, at least in pigeons, the UCN+ population does not belong to the traditionally defined EWN.

Skin morphology of the mutant hairless USP mouse

The morphology of the skin of the mutant hairless USP mouse was studied by histological, histochemical and immunohistochemical methods and compared to the skin of BALB/c mice. Representative sections of the dorsal skin from mice of both strains aged 18 days, and 1, 3, 6, and 8 months were studied. Sections stained with hematoxylin and eosin showed cystic formations called utricles and dermal cysts in the dermis that increased in size and number during growth. Skin thickness increased significantly at 8 months. Sections stained with picrosirius and examined with polarized light, displayed different colors, suggesting different thicknesses of dermal collagen fibers (probably types I and III). Weigert, Verhoeff and resorcin-fuchsin stains revealed fibers of the elastic system. The PAS and Alcian blue methods revealed neutral and acid glycosaminoglycans in the skin ground substance of both mouse strains. Immunohistochemical staining for fibronectin and laminin did not show differences between the mutant and BALB/c mice. Mast cells stained by the Gomori method and macrophages positive for HAM 56 antibodies were observed in both mouse strains. Except for the presence of enlarged cysts in the hairless strain, no qualitative differences were found during development of the skin of BALB/c and the mutant hairless mice.

Differential role of entorhinal and hippocampal nerve growth factor in short- and long-term memory modulation

We studied the effects of infusion of nerve growth factor (NGF) into the hippocampus and entorhinal cortex of male Wistar rats (250-300 g, N = 11-13 per group) on inhibitory avoidance retention. In order to evaluate the modulation of entorhinal and hippocampal NGF in short- and long-term memory, animals were implanted with cannulae in the CA1 area of the dorsal hippocampus or entorhinal cortex and trained in one-trial step-down inhibitory avoidance (foot shock, 0.4 mA). Retention tests were carried out 1.5 h or 24 h after training to measure short- and long-term memory, respectively. Immediately after training, rats received 5 µl NGF (0.05, 0.5 or 5.0 ng) or saline per side into the CA1 area and entorhinal cortex. The correct position of the cannulae was confirmed by histological analysis. The highest dose of NGF (5.0 ng) into the hippocampus blocked short-term memory (P < 0.05), whereas the doses of 0.5 (P < 0.05) and 5.0 ng (P < 0.01) NGF enhanced long-term memory. NGF administration into the entorhinal cortex improved long-term memory at the dose of 5.0 ng (P < 0.05) and did not alter short-term memory. Taken as a whole, our results suggest a differential modulation by entorhinal and hippocampal NGF of short- and long-term memory.

Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10(6) cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodamine-labeled alpha-bungarotoxin. The development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 ± 1.0 µm; N = 213), the diameter of ectopic fibers (59.1 µm; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.

An acid-sensing ion channel that detects ischemic pain

Ischemic pain occurs when there is insufficient blood flow for the metabolic needs of an organ. The pain of a heart attack is the prototypical example. Multiple compounds released from ischemic muscle likely contribute to this pain by acting on sensory neurons that innervate muscle. One such compound is lactic acid. Here, we show that ASIC3 (acid-sensing ion channel #3) has the appropriate expression pattern and physical properties to be the detector of this lactic acid. In rats, it is expressed only in sensory neurons and then only on a minority (~40%) of these. Nevertheless, it is expressed at extremely high levels on virtually all dorsal root ganglion sensory neurons that innervate the heart. It is extraordinarily sensitive to protons (Hill slope 4, half-activating pH 6.7), allowing it to readily respond to the small changes in extracellular pH (from 7.4 to 7.0) that occur during muscle ischemia. Moreover, both extracellular lactate and extracellular ATP increase the sensitivity of ASIC3 to protons. This final property makes ASIC3 a "coincidence detector" of three molecules that appear during ischemia, thereby allowing it to better detect acidosis caused by ischemia than other forms of systemic acidosis such as hypercapnia.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

An extract of Polygonum multiflorum protects against free radical damage induced by ultraviolet B irradiation of the skin

Over the last decades, the incidence of ultraviolet B (UVB)-related skin problems has been increasing. Damages induced by UVB radiation are related to mutations that occur as a result of direct DNA damage and/or the production of reactive oxygen species. We investigated the anti-oxidant effects of a Polygonum multiflorum thumb extract against skin damage induced by UVB irradiation. Female SKH-1 hairless mice were divided into three groups: control (N = 7), distilled water- (N = 10), and P. multiflorum extract-treated (PM, N = 10) groups. The PM (10 g) was extracted with 100 mL distilled water, cryo-dried and 9.8 g was obtained. The animals received a topical application of 500 µL distilled water or PM extract (1, 2, 4, 8, and 16%, w/v, dissolved in distilled water) for 30 min after UVB irradiation (wavelength 280-320 nm, 300 mJ/cm²; 3 min) of the dorsal kin for 14 days, and skin immunohistochemistry and Cu,Zn-superoxide dismutase (SOD1) activity were determined. SOD1 immunoreactivity, its protein levels and activities in the skin were significantly reduced by 70% in the distilled water-treated group after UVB irradiation compared to control. However, in the PM extract-treated groups, SOD1 immunoreactivity and its protein and activity levels increased in a dose-dependent manner (1-16%, w/v, PM extract) compared to the distilled water-treated group. SOD1 protein levels and activities in the groups treated with 8 and 16%, w/v, PM extract recovered to 80-90% of the control group levels after UVB. These results suggest that PM extract strongly inhibits the destruction of SOD1 by UV radiation and probably contains anti-skin photoaging agents.

Pattern of Wnt ligand expression during chick eye development

The dorsoventral axis of the eye is determined prior to optic cup invagination. A variety of signaling pathways have been implicated in the maintenance of the optic dorsoventral axis, including, but not limited to, bone morphogenetic protein 4, Sonic Hedgehog and retinoic acid. Here, we investigated the possible contribution of Wnt ligands to the establishment or maintenance of the optic axis by analyzing their expression pattern during early chick optic development. We performed in situ hybridization of Wnt-1, Wnt-3a, Wnt-4, and Wnt-5a during the optic vesicle, early optic cup and established optic cup stages and focused our analysis on the optic region. Our data showed that Wnt-5a, but none of the others, is expressed in the dorsal region of the eye starting from the Hamburger and Hamilton stage 14 (HH14). These results are supported by cryosections of the labeled optic region, which further reveal that Wnt-5a is expressed only in the dorsal retinal pigmented epithelium. Thus, we propose that Wnt-5a is a marker for dorsal retinal pigmented epithelium in chick embryos from HH14 to HH19.

Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death

Intrahippocampal administration of kainic acid (KA) induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 ± 8 min in saline controls (N = 4) which increased to 369 ± 71 and 322 ± 63 min in animals receiving herbimycin A (1.74 nmol, N = 4) and K252a (10 pmol, N = 4), respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90% of cell loss in KA-treated animals) in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.