Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions

Basic fibroblast growth factor (bFGF) supports the survival of neurons from many regions of the E18 fetal rat brain. Survival was significantly increased for neurons derived from the hippocampus, entorhinal cortex (EC), frontal cortex, parietal cortex (PC), occipital cortex, striatum, septum, and thalamus, but not from the subiculum (Sb). The proportion of neurons rescued by bFGF varied among brain regions, suggesting the existence of subpopulations of responsive neurons. Like hippocampal neurons, neurons from the EC and PC required about 1 pM bFGF (10-20 pg/ml) for half-maximal response; striatal neurons, in contrast, required about 3 pM bFGF. Neurite outgrowth after 24 hr exposure was significantly increased for neurons from the hippocampus, EC, and PC, while striatal neurons had only a marginal response. Although bFGF stimulated some astrocytic proliferation in the cultures, glial contamination was maintained at 2% or less. Acidic FGF (aFGF) supported smaller numbers of neurons from each region, although it significantly increased survival of neurons from hippocampus, EC, PC, striatum, and Sb. The concentration required for half-maximal survival was around 100-300 pM (2-5 ng/ml). It appears that bFGF and aFGF are potent trophic factors for many populations of CNS neurons and could potentially play a significant role in nervous system development.     

Differential vulnerability of immature murine neurons to oxygen-glucose deprivation

In vivo studies support selective neuronal vulnerability to hypoxia-ischemia (HI) in the developing brain. Since differences in intrinsic properties of neurons might be responsible, pure cultures containing immature neurons (6-8 days in vitro) isolated from mouse cortex and hippocampus, regions chosen for their marked vulnerability to oxidative stress, were studied under in vitro ischemic conditions-oxygen-glucose deprivation (OGD). Twenty-four hours of reoxygenation after 2.5 h of OGD induced significantly greater cell death in hippocampal than in cortical neurons (67.8% vs. 33.4%, P = 0.0068). The expression of neuronal nitric oxide synthase (nNOS) protein, production of nitric oxide (NO), and reactive oxygen species (ROS), as well as glutathione peroxidase (GPx) activity and intracellular levels of reduced glutathione (GSH), were measured as indicators of oxidative stress. Hippocampal neurons had markedly higher nNOS expression than cortical neurons by 24 h of reoxygenation, which coincided with an increase in NO production, and significantly greater ROS accumulation. GPx activity declined significantly in hippocampal but not in cortical neurons at 4 and 24 h after OGD. The decrease in GSH level in hippocampal neurons correlated with the decline of GPx activity. Our data suggest that developing hippocampal neurons are more sensitive to OGD than cortical neurons. This finding supports our in vivo studies showing that mouse hippocampus is more vulnerable than cortex after neonatal HI. An imbalance between excess prooxidant production (increased nNOS expression, and NO and ROS production) and insufficient antioxidant defenses created by reduced GPx activity and GSH levels may, in part, explain the higher susceptibility to OGD of immature hippocampal neurons.     

Age-dependent vulnerability of brain choline acetyltransferase activity to transient cerebral ischemia in rats

Male Fischer-344 rats aged 6, 12, or 24 months were subjected to four-vessel occlusion cerebral ischemia to assess age-dependent ischemic vulnerability of cholinergic and GABAergic neurons based on choline acetyltransferase (EC 2.3.1.6) and glutamic acid decarboxylase (EC 4.1.1.15) activities. Activities of both enzymes were similar (p greater than 0.05) in 6- (n = 5) and 12- (n = 5) month-old rats. Mean +/- SEM choline acetyltransferase activities in the cortex, hippocampus, striatum, and cerebellum of 6-month-old controls were 75 +/- 5, 123 +/- 9, 415 +/- 9, and 50 +/- 4 nmol acetylcholine/hr/mg protein, respectively, and were 20-30% lower (p less than 0.05) in all brain regions except the cerebellum in 24-month-old controls. Choline acetyltransferase activity was unaffected by ischemia in 6- and 12-month-old rats but was reduced by 30-60% in 24-month-old rats. Mean +/- SEM glutamic acid decarboxylase activities in the cortex, hippocampus, striatum, and cerebellum of 6-month-old controls were 98 +/- 8, 86 +/- 7, 144 +/- 13, and 125 +/- 9 nmol gamma-aminobutyric acid/hr/mg protein, respectively, and 25-35% lower in all regions of 24-month-old controls. After 30 minutes of ischemia and 5 days of recovery, glutamic acid decarboxylase activities were reduced (p less than 0.05) in all brain regions and age groups. However, its activity was decreased (p less than 0.05 compared with age-matched controls) by 55% in the cortex and 79% in the hippocampus of 24-month-old rats compared with 30% and 45% in younger rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of Na(+)-K(+)-Cl(-) cotransporter in rat brain during development and its localization in mature astrocytes

Na(+)-K(+)-Cl(-) cotransporter has been proposed to play an important role in the regulation of intracellular Cl(-) concentration in neurons during development. In this study, the expression pattern of the cotransporter in different regions of rat brain was examined at birth (P0), postnatal days 7 (P7), P14, P21, and adult by Western blotting analysis. In cortex, thalamus, cerebellum and striatum, the cotransporter expression level was low at P0 and significantly increased at P14 (P<0.05). The expression peaked at P21 and was maintained at the same level in adulthood. However, in hippocampus, a peak level of the cotransporter expression was detected in adult brain. The immunocytochemistry study of adult rat brain revealed that an intense staining of the Na(+)-K(+)-Cl(-) cotransporter protein was observed in dendritic processes of CA1-CA3 hippocampal pyramidal neurons. In contrast, abundant immuno-reactive signals of the cotransporter were found in somata of thalamic nucleus. Immunofluorescence double staining demonstrates that the Na(+)-K(+)-Cl(-) cotransporter was expressed in astrocytes within cortex, corpus callosum, hippocampus and cerebellum. In addition, co-localization of the cotransporter and glial fibrillary acidic protein (GFAP), or with aquaporin 4, was found in perivascular astrocytes of cortical cortex and white matter. The results indicate that a time-dependent expression of the Na(+)-K(+)-Cl(-) cotransporter protein occurs not only in cortex but also in hippocampus, striatum, thalamus and cerebellum. In addition, the cotransporter is expressed in astrocytes and perivascular astrocytes of adult rat brain.     

Human fetal cortical and striatal neural stem cells generate region-specific neurons in vitro and differentiate extensively to neurons after intrastriatal transplantation in neonatal rats

Human fetal brain is a potential source of neural stem cells (NSCs) for cell replacement therapy in neurodegenerative diseases. We explored whether NSCs isolated from cortex and striatum of human fetuses, aged 6-9 weeks post-conception, maintain their regional identity and differentiate into specific neuron types in culture and after intrastriatal transplantation in neonatal rats. We observed no differences between cortex- and striatum-derived NSCs expanded as neurospheres in proliferative capacity, growth rate, secondary sphere formation, and expression of neural markers. After 4 weeks of differentiation in vitro, cortical and striatal NSCs gave rise to similar numbers of GABAergic and VMAT2- and parvalbumin-containing neurons. However, whereas cortical NSCs produced higher number of glutamatergic and tyrosine hydroxylase- and calretinin-positive neurons, several-fold more neurons expressing the striatal projection neuron marker, DARPP-32, were observed in cultures of striatal NSCs. Human cortical and striatal NSCs survived and migrated equally well after transplantation. The two NSC types also generated similar numbers of mature NeuN-positive neurons, which were several-fold higher at 4 months as compared to at 1 month after grafting. At 4 months, the grafts contained cells with morphologic characteristics of neurons, astrocytes, and oligodendrocytes. Many of neurons were expressing parvalbumin. Our data show that NSCs derived from human fetal cortex and striatum exhibit region-specific differentiation in vitro, and survive, migrate, and form mature neurons to the same extent after intrastriatal transplantation in newborn rats.     

Neuronal differentiation following transplantation of expanded mouse neurosphere cultures derived from different embryonic forebrain regions

In vitro, expanded neurospheres exhibit multipotent properties and can differentiate into neurons, astrocytes and oligodendrocytes. In vivo, cells from neurospheres derived from mouse fetal forebrain have previously been reported to predominantly differentiate into glial cells, and not into neurons. Here we isolated stem/progenitor cells from E13.5 lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE) and cortical primordium, of a green fluorescent protein (GFP)-actin transgenic mouse. Free-floating neurospheres were expanded in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and implanted after five to six passages into the striatum, hippocampus and cortex of neonatal rats. Cell suspensions of primary LGE tissue were prepared and grafted in parallel. Grafted cells derived from the primary tissue displayed widespread incorporation into all regions, as visualized with the mouse-specific antibody M2, or mouse satellite DNA in situ hybridization, and differentiated into both neurons, astrocytes and oligodendrocytes. Grafts of neurosphere cells derived from the LGE, MGE and cortical primordium differentiated primarily into astrocytes, but contained low but significant numbers of GFP-immunoreactive neurons. Neurons derived from LGE neurospheres were of three types: cells with the morphology of medium-sized densely spiny projection neurons in the striatum; cells with interneuron-like morphologies in striatum, cortex and hippocampus; and cells integrating into SVZ and migrating along the RMS to the olfactory bulb. MGE- or cortical primordium-derived neurospheres differentiated into interneuron-like cells in both striatum and hippocampus. The results demonstrate the ability of in vitro expanded neural stem/progenitor cells to generate both neurons and glia after transplantation into neonatal recipients, and differentiate in a region-specific manner into mature neurons with morphological features characteristic for each target site.     

Colocalization of Somatostatin Receptors with DARPP-32 in Cortex and Striatum of Rat Brain

Somatostatin (SST)-positive medium-sized aspiny interneurons are selectively spared in excitotoxicity. The biological effects of SST are mediated via five different receptors, namely somatostatin receptor (SSTR)1-5; however, SSTR subtype spared in excitotoxicity and involved in neuroprotection is not known. Dopamine- and cAMP-regulated phosphoprotein (DARPP-32) is predominantly expressed in medium-sized projection neurons that are most vulnerable in excitotoxicity. In the present study, we determined the colocalization of SST and SSTRs with DARPP-32 in rat brain cortical and striatal regions using immunofluorescence immunohistochemistry. We also determined the expression of DARPP-32 in SSTR1-5 immunoprecipitate prepared from cortex and striatum. SST-positive neurons in cortex and striatum are devoid of colocalization with DARPP-32. However, in cortical and striatal brain regions, three different neuronal populations either expressing SSTRs and DARPP-32 alone or displaying colocalization were identified. Quantitative analysis reveals that in cortex and striatum, SSTR1 and 5 are most predominant receptor subtypes colocalized with DARPP-32 followed by SSTR4, 2, and 3 in cortex whereas SSTR2, 4, and 3 in striatum. Importantly, DARPP-32 is expressed in SSTR1-5 immunoprecipitate prepared from cortex and striatum. Taken together, these results provide the first evidence that the SSTR-positive neurons lacking colocalization with DARPP-32 might be spared in excitotoxicity.     

Regional,developmental, and cell cycle-dependent differences in μ, δ, and κ-opioid receptor expression among cultured mouse astrocytes

The diversity of opioid receptor expression was examined in astrocytes in low-density and non-dividing (confluent) cultures from the cerebral cortex, hippocampus, cerebellum, and striatum of 1-day-old mice. μ, δ, and κ opioid receptor expression was assessed in individual cells immunocytochemically, by using flow cytometry, and functionally by examining agonist-induced changes in intracellular calcium ([Ca²⁺]_i). Significant spatial and temporal differences were evident in the pattern of expression of μ, δ, and κ receptors among astrocytes. In low-density cultures, greater proportions of astrocytes expressed μ-opioid receptor immunoreactivity in the cerebral cortex and hippocampus (26–34%) than in the cerebellum or striatum (7–12%). At confluence, a greater percentage of astrocytes in cerebellar (26%) and striatal (30%) cultures expressed μ-immunoreactivity. Fewer astrocytes possessed δ-immunoreactivity in low-density striatal cultures (8%) compared to other regions (16–22%). The proportion of δ receptor-expressing astrocytes declined in the cerebellum but increased in the hippocampus. κ-opioid receptors were uniformly expressed by 27–34% of astrocytes from all regions, except in cortical cultures, where the proportion of κ expressing cells was 38% at low-density and decreased to 22% at confluence. Selective μ (PLO 17; H-Tyr-Pro-Phe (N-Me) -D-Pro-NH₂, δ ([D-Pen², D-Pen⁵] enkephalin), or κ (U50,488H; trans-(±)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeneacetamide methanesulfonate) opioid receptor agonists increased [Ca²⁺]_i in subpopulations of astrocytes indicating the presence of functional receptors. Lastly, opioid receptor immunofluorescence varied during the cell division cycle. A greater proportion of astrocytes in the G₂/M phase of the cell cycle were μ or δ receptor immunofluorescent than at G₀/G₁. When astrocytes were reversibly arrested in G₁, significantly fewer cells expressed δ receptor immunofluorescence; however, upon reentry into the cell cycle immunofluorescent cells reappeared. In conclusion, opioid phenotype varies considerably among individual cultured astrocytes, and this diversity was determined by regional and developmental (age and cell cycle dependent) differences in the brain. These in vitro findings suggest astroglia contribute to regional and developmental idiosyncrasies in opioid function within the brain. GLIA 22:249–259, 1998. © 1998 Wiley-Liss, Inc.     

Anandamide and WIN 55212-2 inhibit cyclic AMP formation through G-protein-coupled receptors distinct from CB1 cannabinoid receptors in cultured astrocytes

The effects of anandamide and the cannabinoid receptor agonists WIN 55212-2 and CP 55940 on the evoked formation of cyclic AMP were compared in cultured neurons and astrocytes from the cerebral cortex and striatum of mouse embryos. The three compounds inhibited the isoproterenol-induced accumulation of cyclic AMP in neuronal cells, and these responses were blocked by the selective CB1 receptor antagonist SR 141716A. The three agonists were more potent in cortical than striatal neurons. Interestingly, WIN 55212-2, CP 55940 and anandamide also inhibited the isoproterenol-evoked accumulation of cyclic AMP in astrocytes but, in contrast to WIN 55212-2 and CP 55940, anandamide was much more potent in striatal than cortical astrocytes. Inhibition was prevented by pertussis toxin pretreatment, but not blocked by SR 141716A. Therefore, G-protein-coupled receptors, distinct from CB1 receptors, are involved in these astrocytic responses. Moreover, specific binding sites for [3H]-SR 141716A were found in neurons but not astrocytes. Furthermore, using a polyclonal CB1 receptor antibody, staining was observed in striatal and cortical neurons, but not in striatal and cortical astrocytes. Taken together, these results suggest that glial cells possess G-protein-coupled receptors activated by cannabinoids distinct from the neuronal CB1 receptor, and that glial cells responses must be taken into account when assessing central effects of cannabinoids.     

Regional distribution of type II Ca2+/calmodulin-dependent protein kinase in rat brain

The distribution of type II Ca2+/calmodulin-dependent protein kinase has been mapped in rat brain by immunochemical and immunohistochemical methods using an antibody against its alpha-subunit. The concentration of the kinase, measured by radioimmunoassay, varies markedly in different brain regions. It is most highly concentrated in the telencephalon where it comprises approximately 2% of the total hippocampal protein, 1.3% of cortical protein, and 0.7% of striatal protein. It is less concentrated in lower brain structures, ranging from about 0.3% of hypothalamic protein to 0.1% of protein in the pons/medulla. The gradient of staining intensity observed in brain sections by immunohistochemistry corroborates this distribution. Neurons and neuropil of the hippocampus are densely stained, whereas little staining is observed in lower brain regions such as the superior colliculus. Within the diencephalon and midbrain, dense staining is observed only in thalamic nuclei and the substantia nigra. The skewed distribution of alpha-subunit appears to be due in part to the occurrence in the cerebellum and pons/medulla of forms of the kinase with a high ratio of beta- to alpha-subunits. However, most of the variation is due to the extremely high concentration of the kinase in particular neurons, especially those of the hippocampus, cortex and striatum. The unusually high expression of the kinase in these neurons is likely to confer upon them specialized responses to calcium ion that are different from those of neurons in lower brain regions.     

Regional sensitivity to neuroinflammation: in vivo and in vitro studies

Background: Neuroinflammation is involved in several acute‐onset neuropathologies such as meningitis, encephalitis, stroke, and traumatic brain injury as well as in neurodegenerative diseases. All of these patholologies are associated with cognitive deficits. Using a model of pure neuroinflammation (intracisternal injection of endotoxin in mice), we tested the hypothesis that brain regions involved in cognition are the most vulnerable to inflammatory insults, and this vulnerability is an inherent property of neocortical neurons. Methods: Mice (n = 10/group) injected with endotoxin (LPS) or saline in the cisterna magna underwent neurobehavioral and cognitive testing followed by quantitative autoradiographic assessment of regional neuroinflammation with [3H]PK11195, an established marker of microgliosis. In parallel, cocultures of cortical and striatal neurons taken from embryonic day 19 rat embryos or postnatal day 1 mice expressing green fluorescent protein were exposed for 24 h to the proinflammatory cytokine TNFalpha, glutamate, or a combination of the two agents. Results: LPS‐treated mice exhibited significant deficits in memory and significant increases in specific PK11195 binding in cortical and hippocampal regions, but not in striatum. Cultured neurons of cortical origin showed significantly lower survival rate relative to striatal neurons in response to TNFalpha, glutamate, or a combination of the two agents. Furthermore, TNFalpha exerted neuroprotective rather than neurotoxic effects in the striatal but not in the cortical neurons. Conclusions: These results suggest that the cortex is inherently more sensitive than the striatum to the deleterious effects of neuroinflammation, and may offer an explanation for the preponderance of cognitive deficits in neuropathologies with a neuroinflammatory component. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Trophic and tropic effects of striatal astrocytes on cografted mesencephalic dopamine neurons and their axons

Astrocytes from the ventral mesencephalon and from the striatum respectively promote the dendritic and axonal arborization of dopamine (DA) neurons in vitro. To test this response in vivo, astrocytes in primary cultures from the neonatal cerebral cortex, ventral mesencephalon, or striatum were coimplanted with fetal ventral mesencephalic tissue into the intact or DA-denervated striatum of adult rats and these cografts examined after 3–6 months by tyrosine hydroxylase (TH) immunohistochemistry (intact recipients) or after 5–6 months by in vitro [H]DA-uptake autoradiography (DA-denervated recipients). In contrast with single ventral mesencephalic grafts, all types of cograft displayed a rather uniform distribution of TH-immunoreactive perikarya. The average size of TH-immunoreactive cell bodies was not significantly different in cografts containing cortical or mesencephalic astrocytes and in single ventral mesencephalic grafts, but it was significantly larger in cografts containing striatal astrocytes. Nevertheless, the number of [H]DA-labeled terminals in the DA-lesioned host striatum was clearly smaller with cografts of striatal astrocytes than with single mesencephalic grafts or with cografts containing cortical astrocytes. On the other hand, cografts of striatal astrocytes contained much higher numbers of [H]DA-labeled terminals than the other types of graft or cograft. Thus, while cografted astrocytes in general influence the distribution of DA neurons within the graft, astrocytes from the neonatal striatum have a trophic effect on DA perikarya and a tropic effect on DA axons, keeping the latter within the graft.     

Differentiation of the RN33B cell line into forebrain projection neurons after transplantation into the neonatal rat brain

The rat neural cell line RN33B has a remarkable ability to undergo region-specific neuronal differentiation after transplantation into the CNS. To further study its neurogenic properties in vivo, we used a recombinant lentiviral vector to genetically label the cells with the Green Fluorescent Protein (GFP) gene before implantation into the striatum/cortex, hippocampus, or mesencephalon of newborn rats. Three weeks after implantation, about 1-2% of the GFP-expressing cells had developed morphologies typical of neurons, astrocytes, or oligodendrocytes, the rest remained as either immature or undifferentiated nestin-positive cells. At 15-17 weeks postgrafting, the immature cells had disappeared in most graft recipients and only cells with neuronal or glial morphologies remained in similar numbers as at 3 weeks. The GFP distributed throughout the expressing cells, revealing fine morphological details, including dendrites with spines and extensive axonal projections. In all forebrain regions, the grafted cells differentiated into neurons with morphologies characteristic for each site, including large numbers of pyramidal-like cells in the cortex and the hippocampus, giving rise to dense projections to normal cortical target regions and to the contralateral hippocampus, respectively. In lower numbers, it was also possible to identify GFP-positive granulelike cells in the hippocampus, as well as densely spiny neurons in the striatum. In the mesencephalon by contrast, cells with astrocytic features predominated. The ability of the grafted RN33B cells to undergo region-specific differentiation into highly specialized types of forebrain projection neurons and establish connections with appropriate targets suggests that cues present in the microenvironment of the neonatal rat brain can effectively guide the development of immature progenitors, also in the absence of ongoing neurogenesis.     

Structural and functional damage sustained by mitochondria after traumatic brain injury in the rat: evidence for differentially sensitive populations in the cortex and hippocampus.

The cellular and molecular pathways initiated by traumatic brain injury (TBI) may compromise the function and structural integrity of mitochondria, thereby contributing to cerebral metabolic dysfunction and cell death. The extent to which TBI affects regional mitochondrial populations with respect to structure, function, and swelling was assessed 3 hours and 24 hours after lateral fluid-percussion brain injury in the rat. Significantly less mitochondrial protein was isolated from the injured compared with uninjured parietotemporal cortex, whereas comparable yields were obtained from the hippocampus. After injury, cortical and hippocampal tissue ATP concentrations declined significantly to 60% and 40% of control, respectively, in the absence of respiratory deficits in isolated mitochondria. Mitochondria with ultrastructural morphologic damage comprised a significantly greater percent of the population isolated from injured than uninjured brain. As determined by photon correlation spectroscopy, the mean mitochondrial radius decreased significantly in injured cortical populations (361 +/- 40 nm at 24 hours) and increased significantly in injured hippocampal populations (442 +/- 36 at 3 hours) compared with uninjured populations (Ctx: 418 +/- 44; Hipp: 393 +/- 24). Calcium-induced deenergized swelling rates of isolated mitochondrial populations were significantly slower in injured compared with uninjured samples, suggesting that injury alters the kinetics of mitochondrial permeability transition (MPT) pore activation. Cyclosporin A (CsA)-insensitive swelling was reduced in the cortex, and CsA-sensitive and CsA-insensitive swelling both were reduced in the hippocampus, demonstrating that regulated MPT pores remain in mitochondria isolated from injured brain. A proposed mitochondrial population model synthesizes these data and suggests that cortical mitochondria may be depleted after TBI, with a physically smaller, MPT-regulated population remaining. Hippocampal mitochondria may sustain damage associated with ballooned membranes and reduced MPT pore calcium sensitivity. The heterogeneous mitochondrial response to TBI may underlie posttraumatic metabolic dysfunction and contribute to the pathophysiology of TBI.     

Temporal and spatial profile of Bid cleavage after experimental traumatic brain injury.

Apoptosis plays an essential role in the cascade of CNS cell degeneration after traumatic brain injury. However, the underlying mechanisms are poorly understood. The authors examined the temporal profile and cell subtype distribution of the proapoptotic protein Bid from 6 hours to 7 days after cortical impact injury in the rat. Increased protein levels of tBid were seen in the cortex ipsilateral to the injury site from 6 hours to 3 days after trauma. Immunohistologic examinations revealed expression of tBid in neurons, astrocytes, and oligodendrocytes from 6 hours to 3 days after impact injury, and concurrent assessment of DNA damage using TUNEL identified tBid-immunopositive cells with apoptoticlike morphology in the traumatized cortex. Moreover, Bid cleavage and activation of caspase-8 and caspase-9 occurred at similar time points and in similar brain regions (i.e., cortical layers 2 to 5) after impact injury. In contrast, there was no evidence of caspase-8 or caspase-9 processing or Bid cleavage in the ipsilateral hippocampus, contralateral cortex, and hippocampus up to 7 days after the injury. The results provide the first evidence of Bid cleavage in the traumatized cortex after experimental traumatic brain injury in vivo, and demonstrate that tBid is expressed in neurons and glial cells. Further, findings indicate that cleavage of Bid may be associated with the activation of the initiator caspase-8 and caspase-9. Finally, these data support the hypothesis that cleavage of Bid contributes to the apoptotic degeneration of different CNS cells in the injured cortex.     

Local injection of kainic acid causes widespread degeneration of NADPH-d neurons and induction of NADPH-d in neurons, endothelial cells and reactive astrocytes

Nitric oxide (NO), a diffusable gas, is a messenger molecule that mediates vascular dilatation and neural transmission. The enzyme nitric oxide synthase (NOS) present in neurons is activated by Ca²⁺ influx associated with activation of glutamate receptors. Cultured cortical neurons containing NOS are selectively vulnerable to injury by kainic acid (KA). However, the relationship between NOS neurons and excitotoxicity under in vivo condition is not entirely clear. In the present study, we examined the time course and spatial distribution of changes in NOS neurons caused by an intracortical microinjection of KA in adult rats. NADPH-diaphorase (NADPH-d) histochemistry was used as a marker for NOS and the neuronal changes were correlated with changes in glial cells and endothelial cells. We demonstrated a rapid loss of NADPH-d neurons in the lesion center and degeneration of NADPH-d neurons and nerve terminals throughout ipsilateral cortex and hippocampus; the striatal neurons appeared to be unaffected. Subsequent to cortical neuronal degeneration, new NADPH-d activity appeared in proliferative reactive astrocytes and in endothelial cells at lesion periphery, and in neuronal groups at lesion periphery, in ipsilateral entorhinal cortex and bilateral hippocampus. These findings indicate that neurons expressing NADPH-d in cerebral cortex and hippocampus are selectively vulnerable to KA toxicity in vivo. The subsequent induction of NOS in neural and non-neural cells may be regarded as an adaptive response to the kainate-induced brain lesion.     

Progression of brain atrophy in multiple system atrophy

In this study, we aimed to determine the progression of brain atrophy in the parkinson variant of multiple system atrophy (MSA-P). Voxel-based morphometry was applied to two consecutive high resolution MR images of 14 patients with probable MSA-P in comparison to 14 patients with Parkinson's disease (PD). The time interval between baseline and follow-up investigations (1.0 +/- 0.5 SD years in MSAP and 1.4 +/- 0.6 SD years in PD patients) was introduced as covariate in the statistical analysis. Additionally, correlation analyses were performed between the progression maps and clinical data. We observed marked progression of brain atrophy in the MSA-P cohort, the regions including striatum, mesencephalon, thalamus and cerebellum, but also cortical regions such as the primary sensorimotor cortex, supplementary motor area, lateral premotor cortex, medial frontal gyrus, middle frontal gyrus, orbito-frontal cortex,insula, posterior parietal cortex and hippocampus. Short disease duration was correlated with progression of atrophy in the striatum whereas longer disease duration was correlated with increasing atrophy in the cortical areas and cerebellar hemispheres. The UPDRS-III score was not significantly correlated with any brain region. Our data suggest that cortical atrophy is prominent in MSA-P and early degeneration of the basal ganglia drives late onset cortical atrophy.     

Effect of basic fibroblast growth factor on neurons cultured from various regions of postnatal rat brain

Neurons from various brain regions of postnatal (15 days after birth) and fetal (16 days gestation) rats were cultured in the presence of basic fibroblast growth factor (bFGF). bFGF increased the survival of neurons from postnatal septum, striatum, midbrain, and hippocampus. Fetal neurons derived from cerebral cortex, septum, striatum, midbrain, thalamus, and colliculus were far more dependent on bFGF for survival in comparison with postnatal neurons. In contrast, cerebellum neurons of postnatal and fetal rat brain did not respond to bFGF. The increase of postnatal and fetal neuronal survival with bFGF treatment (0.01–10 ng/ml) was dose-dependent and reached 2–4-fold and 5–10-fold more than the control, respectively. Fetal cortical neurons showed almost complete dependence on bFGF since almost all neurons died in control cultures. Nerve growth factor was slightly effective only on postnatal septal and striatal neurons, being ineffective on the other neurons tested. These results indicate that bFGF can function as a neurotrophic factor not only on fetal but also on postnatal neurons of the central nervous system, and that bFGF has great potential for application in vivo.     

Decreased parvalbumin immunoreactivity in the cortex and striatum of mice lacking the CB1 receptor

Cortical and striatal regions of the brain contain high levels of the cannabinoid-1 (CB1) receptor, the central neuronal mediator of activity-dependent synaptic plasticity evoked by endocannabinoids. The expression levels of parvalbumin, a calcium-binding protein found in fast-spiking interneurons of both regions, may be controlled in part by synaptic activity during critical periods of development. However, there is currently no evidence that CB1 receptor expression affects parvalbumin levels in either cortical or striatal interneurons. To assess this possibility, we examined parvalbumin immunoreactivity in the dorsolateral striatum, primary motor cortex (M1), and prefrontal cortex (PFC) of CB1 knockout and wild-type C57/BL6 mice. Quantitative densitometry showed a significant decrease in parvalbumin immunoreactivity within individual neurons in each of these regions of CB1 knockout mice relative to controls. A significantly lower density (number of cells per unit area) of parvalbumin-labeled neurons was observed in the striatum, but not the cortical regions of CB1 knockout mice. These findings suggest that CB1 receptor deletion may elicit a compensatory mechanism for network homeostasis affecting parvalbumin-containing cortical and striatal interneurons.