Long-term alcohol self-administration and alcohol withdrawal differentially modulate microtubule-associated protein 2 (MAP2) gene expression in the rat brain

Chronic alcohol intoxication is known to produce neuronal degeneration in the central and peripheral nervous system of experimental animals and of humans. It is suggested that various components of the cytoskeleton undergo profound changes following chronic alcohol use and misuse. Here we studied the expression of the neuronal cytoskeletal microtubule-associated protein 2 (MAP2) following long-term alcohol consumption and subsequent alcohol withdrawal. Alcohol-preferring AA (Alko Alkohol) rats with a high voluntary alcohol consumption for a period of 16 months were compared with age-matched control rats without prior experience with alcohol. For comparison, in a second experiment, heterogeneous Wistar rats that also had voluntary access to alcohol for 8 months were examined following alcohol consumption and withdrawal. In situ hybridization and subsequent dot blot and Northern blot analysis for further quantification revealed that chronically alcoholized animals exhibit markedly decreased MAP2 mRNA levels in several parts of the extrapyramidal system (mainly in the caudate putamen, the substantia nigra pars compacta and the globus pallidus), the mesolimbic system, in several hypothalamic nuclei and in the nucleus inferior colliculus. Other areas such as the hippocampus, frontoparietal cortex and cerebellum were less affected by chronic alcohol intake, however, in these regions the MAP2 mRNA levels were increased during alcohol withdrawal. These results suggest that long-term alcohol self-administration affects central neurons involved in motor control via the influence on the integrity of the cytoskeleton and may thus induce motor dysfunction.     

Lithium regulation of protein phosphorylation in rat cerebral cortex slices in vitro

Histamine type 2 receptor antagonists (H2RAs) have been found to alter gastric motility. The aims of this study were to determine if H2RAs affect antral contractility in vitro and the mechanism of this effect. Guinea pig antral muscle strips were pinned in an organ bath after removing the mucosa, and circular muscle tension was measured using an isometric force transducer. Gastric myocytes were isolated from guinea pig stomach using collagenase digestion, and cell lengths were measured using an image analysis system. In muscle strips, ranitidine and nizatidine increased the amplitude of spontaneous phasic antral contractions in a concentration-dependent fashion with threshold concentrations of 5 microM. The order of potency for the H2RAs was ranitidine = nizatidine > cimetidine > famotidine. The contractile effects of ranitidine and nizatidine were reduced, but not abolished, by tetrodotoxin and omega-conotoxin GVIA and nearly abolished by atropine. In isolated cells, ranitidine and nizatidine, but not famotidine or cimetidine, induced concentration-dependent cell shortening, with maximal shortening at 10 microM. These contractile effects of ranitidine and nizatidine in isolated cells were inhibited by atropine. Ranitidine and nizatidine increase antral contractility; this effect appears to be mediated by an interaction between ranitidine and nizatidine on cholinergic pathways with both direct effects on smooth muscle cholinergic receptors and indirect effects by increasing cholinergic neurotransmission.     

NT-4/5 and LIF, but not NT-3 and BDNF, promote NPY mRNA expression in cortical neurons in the absence of spontaneous bioelectrical activity

Epigenetic factors are known to influence the differentiation of neocortical neurons. The present study analyses the role of spontaneous bioelectrical activity (SBA) and neurotrophic factors on the expression of neuropeptide Y (NPY) in rat visual cortical neurons using organotypic monocultures prepared from newborn animals and in situ hybridization to detect the NPY messenger ribonucleic acid (mRNA). Spontaneously active cortex cultures display NPY mRNA expression in about 7% of all cortical neurons from 10 days in vitro (DIV) on. Blocking the SBA by chronic application of 10 mM Mg2+ for 3-30 DIV reduces the percentage of NPY neurons to about 2%. Allowing an initial phase of SBA (1-20 DIV) followed by an SBA blockade (for 21-50 DIV) results in 2% labelled neurons, indicating a dramatic reduction of NPY mRNA expression in the absence of SBA. Surprisingly, the reverse experiment (a period of SBA blockade for 1-20 DIV followed by a period of SBA recovery for 21-40 DIV) does not cause an upregulation of NPY mRNA expression. However, allowing cultures to differentiate as spontaneously active cultures, then applying a transient period of SBA blockade which is followed by a second period of SBA, does rescue the NPY mRNA expression in 7% of the cortical neurons. We conclude that SBA is a main trigger for NPY mRNA expression and it is particularly important during an early postnatal period of differentiation. We then analysed whether neurotrophic factors known to modulate cortical neuropeptide expression are able to do so in the absence of SBA. Supplementing chronically blocked cultures with the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and the cytokine, leukaemia inhibitory factor (LIF), reveals that BDNF and NT-3 are unable to increase the percentage of NPY neurons. In contrast, LIF and NT-4/5 increase the percentage of NPY neurons to 4 and 6-7%, respectively. Moreover, neurons treated with NT-4/5 display a very high level of NPY mRNA expression in somata and in the dendritic trees. The data suggest a complex interplay and a hierarchy of epigenetic factors in regulating the neurochemical architecture of the developing neocortex.     

Rapid increase of NGF, BDNF and NT-3 mRNAs in inflamed bladder

Nerve growth factor (NGF) is a mediator of hyperalgesia and has been previously associated with sensory and reflex changes after inflammation of the urinary bladder. A sensitive assay was developed to examine neurotrophin gene expression after bladder inflammation by turpentine, which causes a short-lived inflammatory response. Two hours, but not 6 or 24 h after induction of inflammation, there were significant increases in levels of NGF, brain-derived neurotrophic factor and neurotrophin-3 mRNAs. NGF immunoreactivity was elevated with a similar time course to its mRNA. Our results suggest that during bladder inflammation, endogenous NGF is rapidly up-regulated and released to mediating sensory and reflex changes. Brain-derived neurotrophic factor and neurotrophin-3 may also have a role in the inflammatory response.     

Regional specificity of alterations in NGF, BDNF and NT-3 levels in Alzheimer's disease

Using two-site enzyme immunoassays (EIAs), we measured the levels of neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) simultaneously in three brain regions (motor cortex, dentate gyrus and entorhinal cortex) of patients with Alzheimer's disease (AD) and control individuals. Significant differences between the neurotrophin levels of these two groups were found in the different brain regions depending on the neurotrophin. The NGF level in the dentate gyrus of AD patients was higher, whereas the BDNF level in the entorhinal cortex and the NT-3 level in the motor cortex were lower than the corresponding control levels. These results indicate that protein levels of individual neurotrophins in different brain regions are affected differently by AD, and such differential changes may contribute to the complex pathology of AD.     

Expression and subcellular distribution of glutamate receptor subunits 2/3 in the developing cerebellar cortex

The expression and subcellular location of glutamate receptor subunits 2&3 was investigated in the developing postnatal cerebellum. Immunoblotting revealed that glutamate receptor subunits 2/3 is expressed in an identical pattern of immunoreactive bands of approximately 108 kDa from postnatal day zero to adult animals. Light microscopy showed that within the cerebellar cortex, GluR 2/3 immunoreactivity was essentially confined to Purkinje neurons. Strong immunostaining could be observed at postnatal days 1-3 within Purkinje cell bodies and primary dendrites. With ongoing development, the cell body and an increasingly elaborate dendritic tree was outlined by immunoreaction product. In adult animals, staining of Purkinje cell dendrites was patchy, and staining intensity of the cell body, in particular, was greatly reduced. Ultrastructural analysis revealed that during early postnatal development, immunoreaction product was localized to the cell membrane, but was not confined to postsynaptic densities. From the second postnatal week, glutamate receptor subunits 2/3 immunoreactivity was largely restricted to postsynaptic densities. These observations reveal a developmentally regulated refinement of the subcellular distribution of defining subunits of the AMPA-type glutamate receptor. The presence of membrane bond receptors prior to the formation of synapses also provides a rationale for the known transmitter-mediated modulation of Purkinje cell dendritogenesis.     

Altered expression of microtubule-associated protein 1B in cerebral cortical structures of pentylenetetrazole-treated rats

Using Northern blot, immunoblotting, immunocytochemistry, and in situ hybridization, we show that a single administration of the convulsant pentylenetetrazole leads to robust, long-term changes in microtubule-associated protein 1B and its mRNA, in the adult rat brain. The first increases in MAP1B mRNA were detected at 15 hr following pentylenetetrazole administration in the temporal (Te2) and perirhinal cortex followed by increases in microtubule-associated protein 1B immunoreactivity at 72 hr postseizure. In contrast, the levels of microtubule-associated protein 1B mRNA and protein in layers I-II of the retrosplenial and parietal cortex (Par2) declined visibly by 24 hr and 72 h, respectively, post-seizure. The changes included loss of staining in layers I-II and development of structures resembling "strings-of-beads" along the fibers of projection neurons of layer V. The levels of microtubule-associated protein 1B mRNA in the entorhinal cortex peaked at later times (72 h), especially in layers II-III, and returned to control levels by 10 days. Whereas the levels of microtubule-associated protein 1B immunoreactivity in the retrosplenial and parietal cortex recovered by 5-10 days, it persisted at high levels through day 35 in layer V of the temporal cortex (Te2), layers II-III of the perirhinal cortex and layers I-II of the lateral entorhinal cortex. These results indicate that seizure activity leads to long-term upregulation of genes coding for structural elements that are characteristic of the immature brain such as microtubule-associated protein 1B.     

Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons

The phosphorylation state of cytoskeletal proteins, including certain microtubule-associated proteins, may influence the development and plasticity of axons and dendrites in mammalian neuron in response to appropriate extracellular stimuli. In particular, high molecular weight microtubule-associated protein 2, has been implicated in dendrite growth and synaptic plasticity and is thought to be modulated by phosphorylation and dephosphorylation. We have previously determined that threonines 1620/1623 on the microtubule-associated protein 2 molecule become phosphorylated in vivo and are targets for proline-directed protein kinases in vitro. Using the phosphorylated site-specific antibody 305, we now report the decreased phosphorylation state of high molecular weight microtubule-associated protein 2 during the development of cultured cerebellar granule neurons. Phosphorylation of high molecular weight microtubule-associated protein 2 at this site is significantly inhibited by lithium in short-term cultured neurons, which suggests the implication of glycogen synthase kinase-3. In long-term cultured neurons, it is also partially inhibited by PD098059, an inhibitor of extracellular signal-regulated protein kinase activation, which indicates an additional contribution of this kinase to high molecular weight microtubule-associated protein 2 phosphorylation at this stage. Both in short-term and long-term cultured neurons, okadaic acid augments high molecular weight microtubule-associated protein 2 phosphorylation at this site through the inhibition of protein phosphatases 1 and/or 2A. Finally, glutamate receptor activation leads to a dephosphorylation of high molecular weight microtubule-associated protein 2 at this site which can also be effectively prevented by okadaic acid. These results are consistent with the participation of glycogen synthase kinase-3, extracellular signal-regulated protein kinases and protein phosphatases 1 and 2A, in the regulation of microtubule-associated protein 2 phosphorylation within living neurons, which may be modulated by extracellular signals like the neurotransmitter glutamate.     

NT-3, but not BDNF, prevents atrophy and death of axotomized spinal cord projection neurons

Following spinal cord injury, projection neurons are frequently axotomized and many of the cells subsequently die. One goal in spinal injury research is to preserve damaged neurons so that ultimately they are accessible to regeneration-promoting strategies. Here we ask if neurotrophin treatment can prevent atrophy and death of axotomized sensory projection neurons. In adult rats, a hemisection was made in the thoracic spinal cord and axotomized neurons were retrogradely labelled with Fluoro-Gold. Four distinct populations of cells were identified in the lumbar spinal cord, and both numbers and sizes of labelled cells were assessed at different time points postlesion. A progressive and significant degeneration was observed over time with severe atrophy apparent in all cell populations and significant cell loss evident by 4 weeks postlesion. This time point was used to assess neurotrophin effects. Hemisected rats were treated with either neurotrophin 3 (NT-3) or brain-derived neurotrophic factor (BDNF, 12 microg/day for each), or a vehicle solution, delivered continuously to the lesion site via an osmotic minipump. Treatment with NT-3, but not BDNF, completely reversed cell atrophy in three of the four cell populations and also induced a significant increase in the number of surviving cells. In situ hybridization experiments showed trkB and trkC mRNA to be expressed in the majority of ascending spinal projection neurons, suggesting that these cells should be responsive to both BDNF and NT-3. However, only NT-3 treatment was neuroprotective, indicating that BDNF may not have reached the cell bodies of injured neurons. These results demonstrate that NT-3 may be of benefit in preventing the secondary cell loss that occurs following spinal injury.     

NT-3 regulates expression of Brn3a but not Brn3b in developing mouse trigeminal sensory neurons

To further elucidate the nature of the molecular interactions of surfactant apoprotein B (SP-B) with phospholipid (PL) membranes, we studied the binding of SP-B to PL membranes and the lipid-dependency of its subsequent effects on leakage and fusion of membranes. SP-B binding to membranes was studied by labeling the protein with the fluorophore 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) and measuring the fluorescence of the labeled protein in the presence of varying amounts of dipalmitoylphosphatidylcholine-egg phosphatidylglycerol (DPPC-eggPG; 7-3). Leakage of contents from liposomes made of DPPC and varying molar fraction of egg phosphatidylcholine (eggPC) or eggPG was assessed by measuring the fluorescence of entrapped water-soluble probes ANTS and DPX. Fusion of membranes was assessed by measuring the fluorescence of membrane-bound NBD-phosphatidylethanolamine (NBD-PE) and rhodamine-PE (RHO-PE). We found that SP-B bound to PL membranes with high affinity and appeared to irreversibly cluster at the membrane surface, leading to graded release of the vesicle contents and eventually fusion of the membranes with increasing protein-lipid ratios. All lipid mixtures tested were susceptible to the membrane disruptive effects of SP-B, but DPPC-eggPG membranes displayed a biphasic response to increasing molar fractions of eggPG, whereas increasing fractions of eggPC elicited a monotonic response.     

Molecular heterogeneity of progenitors and radial migration in the developing cerebral cortex revealed by transgene expression

We have analyzed the developmental pattern of beta-galactosidase (beta-gal) expression in the cerebral cortex of the beta 2nZ3'1 transgenic mouse line, which was generated using regulatory elements of the beta 2-microglobulin gene and shows ectopic expression in nervous tissue. From embryonic day 10 onward, beta-gal was expressed in the medial and dorsal cortices, including the hippocampal region, whereas lateral cortical areas were devoid of labeling. During the period of cortical neurogenesis (embryonic days 11-17), beta-gal was expressed by selective precursors in the proliferative ventricular zone of the neocortex and hippocampus, as well as by a number of migrating and postmigratory neurons arranged into narrow radial stripes above the labeled progenitors. Thus, the transgene labels a subset of cortical progenitors and their progeny. Postnatally, radial clusters of beta-gal-positive neurons were discernible until postpartum day 10. At this age, the clusters were 250 to 500 microns wide, composed of neurons spanning all the cortical layers and exhibiting several neuronal phenotypes. These data suggest molecular heterogeneity of cortical progenitors and of the cohorts of postmitotic neurons originating from them, which implies intrinsic molecular mosaicism in both cortical progenitors and developing neurons. Furthermore, the data show that neurons committed to the expression of the transgene migrate along very narrow, radial stripes.     

Alteration of tubulin-Gi protein interaction in rat cerebral cortex with aging

The ability of the tubulin dimer to interact with and to modulate the Gi function inhibiting adenylyl cyclase was examined in cerebral cortex membranes from 2-month-old and 24-month-old rats. The hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp)-dependent inhibition of adenylyl cyclase was significantly decreased in cerebral cortex membranes from 24-month-old rats. Tubulin, prepared from rat brains by polymerization with GppNHp, caused inhibition of adenylyl cyclase (approximately 28%) in 2-month-old rats. Tubulin-GppNHp-dependent inhibition of adenylyl cyclase in 24-month-old rats was significantly attenuated (approximately 15%). In 2-month-old rats, when tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to Gi alpha. Transfer of AAGTP from tubulin to Gi alpha was reduced in 24-month-old rats. Furthermore, photoaffinity labeling of [32P]AAGTP to Gi alpha in cortex membranes was significantly decreased in 24-month-old rats. No differences were observed in the amounts of Gs alpha, Gi alpha, or G beta subunits and tubulin, estimated by immunoblotting, in cortex membranes from 2-month-old and 24-month-old rats. These results suggest that the ability of tubulin to interact with Gi and thereby modulate the inhibitory regulation of adenylyl cyclase is reduced in the cerebral cortex of 24-month-old rats.     

Emx1 and Emx2 show different patterns of expression during proliferation and differentiation of the developing cerebral cortex in the mouse

Insights into the complex structure of the forebrain and its regulation have recently come from the analysis of the expression of genes that are likely to be involved in regionalization of this structure. We cloned four new homeo box genes, Emx1, Emx2, Otx1 and Otx2, and showed that in day 10 mouse embryos their expression domains are continuous regions of the developing brain contained within each other in the sequence Emx1 < Emx2 < Otx1 < Otx2. Recently Otx1 has been found to be specifically expressed during neurogenesis of layer 5 and 6 in the developing cerebral cortex. In order to better understand the role of Emx1 and Emx2 in the maturation of the cortex we analysed by in situ hybridization their expression patterns in the developing mouse cerebral cortex, from embryonic day 12.5 to adulthood. We found that Emx2 is expressed exclusively in proliferating cells of the ventricular zone whereas Emx1 is expressed in both proliferating and differentiated neurons, throughout the cortical layers and during all the developmental stages examined. Therefore, Emx2 gene products might control some biological parameters of the proliferation of cortical neuroblasts or of the subsequent cell migration of postmitotic neurons, leaving the cortical germinal zone. Conversely, Emx1 expression, which is confined exclusively to the dorsal telencephalon, characterizes most cortical neurons during proliferation, differentiation, migration and postnatal development and maturation.     

Heat-shock protein 72 expression in excitotoxic versus penetrating injuries of the rodent cerebral cortex

The induction of heat shock protein 72 (hsp72) has been described in various experimental models of brain injury. The present study examined hsp72 expression patterns within the rodent cerebral cortex in experimental paradigms designed to mimic two mechanisms of damage produced by penetration of the cerebral cortex: (1) tissue tearing from the missile track and (2) diffuse excitotoxicity during temporary cavitation and shock wave formation. Adult male Spaque-Dawley rats received controlled penetration (stab) or injection of the NMDA receptor excitotoxin, quinolinic acid (QA), into the frontal cortex and were killed 1-24 h later. Tissue from the lesioned, sham-operated, or contralateral uninjected cortex was processed for Western and immunocytochemical analyses of hsp72 protein expression. By 12 h, both controlled penetration and excitotoxic brain injuries produced significant increases in hsp72 immunoreactivity, which decreased toward control levels at 24 h. However, the severity and regional distribution of hsp72 expression varied between the two models. Specifically, the controlled penetration injury produced many hsp72-expressing cells near the needle track, while immunoreactive cells within the QA-injected cortex were found in the periphery of the lesion site. Morphological assessment of brain sections subjected to dual-labeling procedures demonstrated that cells expressing hsp72 were primarily neuronal in both models of injury. These results suggest that although controlled penetration and diffuse excitotoxicity may induce similar temporal and cellular patterns of hsp72 expression, the spatial location of hsp72-immunoreactive cells may differ between the two models.     

Neuronal damage and plasticity identified by microtubule-associated protein 2, growth-associated protein 43, and cyclin D1 immunoreactivity after focal cerebral ischemia in rats

BACKGROUND AND PURPOSE: An objective of therapeutic intervention after cerebral ischemia is to promote improved functional outcome. Improved outcome may be associated with a reduction of the volume of cerebral infarction and the promotion of cerebral plasticity. In the developing brain, neuronal growth is concomitant with expression of particular proteins, including microtubule-associated protein 2 (MAP-2), growth-associated protein 43 (GAP-43), and cyclin D1. In the present study we measured the expression of select proteins associated with neurite damage and plasticity (MAP-2 and GAP-43) as well as cell cycle (cyclin D1) after induction of focal cerebral ischemia in the rat. METHODS: Brains from rats (n=28) subjected to 2 hours of middle cerebral artery occlusion and 6 hours, 12 hours, and 2, 7, 14, 21, and 28 days (n=4 per time point) of reperfusion and control sham-operated (n=3) and normal (n=2) rats were processed by immunohistochemistry with antibodies raised against MAP-2, GAP-43, and cyclin D1. Double staining of these proteins for cellular colocalization was also performed. RESULTS: Loss of immunoreactivity of both MAP-2 and GAP-43 was observed in most damaged neurons in the ischemic core. In contrast, MAP-2, GAP-43, and cyclin D1 were selectively increased in morphologically intact or altered neurons localized to the ischemic core at an early stage (eg, 6 hours) of reperfusion and in the boundary zone to the ischemic core (penumbra) during longer reperfusion times. CONCLUSIONS: The selective expressions of the neuronal structural proteins (MAP-2 in dendrites and GAP-43 in axons) and the cyclin D1 cell cycle protein in neurons observed in the boundary zone to the ischemic core are suggestive of compensatory and repair mechanisms in ischemia-damaged neurons after transient focal cerebral ischemia.     

BDNF and NT-3 applied in the whisker pad reverse cortical changes after peripheral deafferentation in neonatal rats

It has been known for a long time that subcortical input drives the specification of cortical areas. Molecular signals mediating this instructive effect from the periphery are poorly understood. In foetal or neonatal rats, ablation of whisker follicles, transection of the infraorbital nerve, inhibition of axonal transport, but not impulse activity blockade, prevent formation of barrels in the primary somatosensory cortex (S1). These findings suggest that a chemical signal, possibly arising from the skin or the follicle, may be responsible for somatotopic pattern formation in S1. Neurotrophins promote survival and differentiation of primary sensory neurons, and are expressed in the whisker pad during development. Neonatal rats received gelfoam impregnated with NGF, BDNF or NT-3 under the whisker pad following surgical denervation of whisker rows D and E on P0. Barrel formation in S1 was assessed on P7 by acetylcholinesterase histochemistry and 5-HT-immunohistochemistry. BDNF and NT-3, but not NGF, promoted development of the cortical barrels corresponding to denervated whiskers. Furthermore, BDNF and NT-3 prevented the lesion-induced expansion of row C barrels, while NGF appeared to promote row C expansion. Our results suggest that BDNF and NT-3 arising from the whisker pad are involved in the formation and/or maintenance of the barrel pattern in S1. These findings are potentially relevant for the prevention of sensory disturbances possibly due to reorganization of central sensory circuits after peripheral nerve lesions in humans.