Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

The nucleus tractus solitarius (NTS) plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs) in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 µM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM) amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV). The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration.

Quantitative evidence for neurofilament heavy subunit aggregation in motor neurons of spinal cords of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of unknown etiology, affects motor neurons leading to atrophy of skeletal muscles, paralysis and death. There is evidence for the accumulation of neurofilaments (NF) in motor neurons of the spinal cord in ALS cases. NF are major structural elements of the neuronal cytoskeleton. They play an important role in cell architecture and differentiation and in the determination and maintenance of fiber caliber. They are composed of three different polypeptides: light (NF-L), medium (NF-M) and heavy (NF-H) subunits. In the present study, we performed a morphological and quantitative immunohistochemical analysis to evaluate the accumulation of NF and the presence of each subunit in control and ALS cases. Spinal cords from patients without neurological disease and from ALS patients were obtained at autopsy. In all ALS cases there was a marked loss of motor neurons, besides atrophic neurons and preserved neurons with cytoplasmic inclusions, and extensive gliosis. In control cases, the immunoreaction in the cytoplasm of neurons was weak for phosphorylated NF-H, strong for NF-M and weak for NF-L. In ALS cases, anterior horn neurons showed intense immunoreactivity in focal regions of neuronal perikarya for all subunits, although the difference in the integrated optical density was statistically significant only for NF-H. Furthermore, we also observed dilated axons (spheroids), which were immunopositive for NF-H but negative for NF-M and NF-L. In conclusion, we present qualitative and quantitative evidence of NF-H subunit accumulation in neuronal perikarya and spheroids, which suggests a possible role of this subunit in the pathogenesis of ALS.

Lesion of the subthalamic nucleus reverses motor deficits but not death of nigrostriatal dopaminergic neurons in a rat 6-hydroxydopamine-lesion model of Parkinson's disease

The objective of the present study was to determine whether lesion of the subthalamic nucleus (STN) promoted by N-methyl-D-aspartate (NMDA) would rescue nigrostriatal dopaminergic neurons after unilateral 6-hydroxydopamine (6-OHDA) injection into the medial forebrain bundle (MFB). Initially, 16 mg 6-OHDA (6-OHDA group) or vehicle (artificial cerebrospinal fluid - aCSF; Sham group) was infused into the right MFB of adult male Wistar rats. Fifteen days after surgery, the 6-OHDA and SHAM groups were randomly subdivided and received ipsilateral injection of either 60 mM NMDA or aCSF in the right STN. Additionally, a control group was not submitted to stereotaxic surgery. Five groups of rats were studied: 6-OHDA/NMDA, 6-OHDA/Sham, Sham/NMDA, Sham/Sham, and Control. Fourteen days after injection of 6-OHDA, rats were submitted to the rotational test induced by apomorphine (0.1 mg/kg, ip) and to the open-field test. The same tests were performed again 14 days after NMDA-induced lesion of the STN. The STN lesion reduced the contralateral turns induced by apomorphine and blocked the progression of motor impairment in the open-field test in 6-OHDA-treated rats. However, lesion of the STN did not prevent the reduction of striatal concentrations of dopamine and metabolites or the number of nigrostriatal dopaminergic neurons after 6-OHDA lesion. Therefore, STN lesion is able to reverse motor deficits after severe 6-OHDA-induced lesion of the nigrostriatal pathway, but does not protect or rescue dopaminergic neurons in the substantia nigra pars compacta.

Hypoxic-ischemic injury decreases anxiety-like behavior in rats when associated with loss of tyrosine-hydroxylase immunoreactive neurons of the substantia nigra

Neonatal Sprague-Dawley rats were randomly divided into normal control, mild hypoxia-ischemia (HI), and severe HI groups (N = 10 in each group at each time) on postnatal day 7 (P7) to study the effect of mild and severe HI on anxiety-like behavior and the expression of tyrosine hydroxylase (TH) in the substantia nigra (SN). The mild and severe HI groups were exposed to hypoxia (8% O2/92% N2) for 90 and 150 min, respectively. The elevated plus-maze (EPM) test was performed to assess anxiety-like behavior by measuring time spent in the open arms (OAT) and OAT%, and immunohistochemistry was used to determine the expression of TH in the SN at P14, P21, and P28. OAT and OAT% in the EPM were significantly increased in both the mild (1.88-, 1.99-, and 2.04-fold, and 1.94-, 1.51-, and 1.46-fold) and severe HI groups (1.69-, 1.68-, and 1.87-fold, and 1.83-, 1.43-, and 1.39-fold, respectively; P < 0.05). The percent of TH-positive cells occupying the SN area was significantly and similarly decreased in both the mild (17.7, 40.2, and 47.2%) and severe HI groups (16.3, 32.2, and 43.8%, respectively; P < 0.05). The decrease in the number of TH-positive cells in the SN and the level of protein expression were closely associated (Pearson correlation analysis: r = 0.991, P = 0.000 in the mild HI group and r = 0.974, P = 0.000 in the severe HI group) with the impaired anxiety-like behaviors. We conclude that neonatal HI results in decreased anxiety-like behavior during the juvenile period of Sprague-Dawley rats, which is associated with the decreased activity of TH in the SN. The impairment of anxiety and the expression of TH are not likely to be dependent on the severity of HI.

Relationships between dendritic morphology, spatial distribution and firing patterns in rat layer 1 neurons

The cortical layer 1 contains mainly small interneurons, which have traditionally been classified according to their axonal morphology. The dendritic morphology of these cells, however, has received little attention and remains ill defined. Very little is known about how the dendritic morphology and spatial distribution of these cells may relate to functional neuronal properties. We used biocytin labeling and whole cell patch clamp recordings, associated with digital reconstruction and quantitative morphological analysis, to assess correlations between dendritic morphology, spatial distribution and membrane properties of rat layer 1 neurons. A total of 106 cells were recorded, labeled and subjected to morphological analysis. Based on the quantitative patterns of their dendritic arbor, cells were divided into four major morphotypes: horizontal, radial, ascendant, and descendant cells. Descendant cells exhibited a highly distinct spatial distribution in relation to other morphotypes, suggesting that they may have a distinct function in these cortical circuits. A significant difference was also found in the distribution of firing patterns between each morphotype and between the neuronal populations of each sublayer. Passive membrane properties were, however, statistically homogeneous among all subgroups. We speculate that the differences observed in active membrane properties might be related to differences in the synaptic input of specific types of afferent fibers and to differences in the computational roles of each morphotype in layer 1 circuits. Our findings provide new insights into dendritic morphology and neuronal spatial distribution in layer 1 circuits, indicating that variations in these properties may be correlated with distinct physiological functions.

GABA-induced inactivation of Cebus apella V2 neurons: effects on orientation tuning and direction selectivity

We investigated the GABA-induced inactivation of V2 neurons and terminals on the receptive field properties of this area in an anesthetized and paralyzedCebus apella monkey. Extracellular single-unit activity was recorded using tungsten microelectrodes in a monkey before and after pressure-injection of a 0.25 or 0.5 M GABA solution. The visual stimulus consisted of a bar moving in 8 possible directions. In total, 24 V2 neurons were studied before and after blocker injections in 4 experimental sessions following GABA injection into area V2. A group of 10 neurons were studied over a short period. An additional 6 neurons were investigated over a long period after the GABA injection. A third group of 8 neurons were studied over a very long period. Overall, these 24 neurons displayed an early (1-20 min) significant general decrease in excitability with concomitant changes in orientation or direction selectivity. GABA inactivation in area V2 produced robust inhibition in 80% and a significant change in directional selectivity in 60% of the neurons examined. These GABA projections are capable of modulating not only levels of spontaneous and driven activity of V2 neurons but also receptive field properties such as direction selectivity.

Effects of nitric oxide on magnocellular neurons of the supraoptic nucleus involve multiple mechanisms

Physiological evidence indicates that the supraoptic nucleus (SON) is an important region for integrating information related to homeostasis of body fluids. Located bilaterally to the optic chiasm, this nucleus is composed of magnocellular neurosecretory cells (MNCs) responsible for the synthesis and release of vasopressin and oxytocin to the neurohypophysis. At the cellular level, the control of vasopressin and oxytocin release is directly linked to the firing frequency of MNCs. In general, we can say that the excitability of these cells can be controlled via two distinct mechanisms: 1) the intrinsic membrane properties of the MNCs themselves and 2) synaptic input from circumventricular organs that contain osmosensitive neurons. It has also been demonstrated that MNCs are sensitive to osmotic stimuli in the physiological range. Therefore, the study of their intrinsic membrane properties became imperative to explain the osmosensitivity of MNCs. In addition to this, the discovery that several neurotransmitters and neuropeptides can modulate their electrical activity greatly increased our knowledge about the role played by the MNCs in fluid homeostasis. In particular, nitric oxide (NO) may be an important player in fluid balance homeostasis, because it has been demonstrated that the enzyme responsible for its production has an increased activity following a hypertonic stimulation of the system. At the cellular level, NO has been shown to change the electrical excitability of MNCs. Therefore, in this review, we focus on some important points concerning nitrergic modulation of the neuroendocrine system, particularly the effects of NO on the SON.

Microglia and neurons in the hippocampus of migratory sandpipers

The semipalmated sandpiper Calidris pusilla and the spotted sandpiper Actitis macularia are long- and short-distance migrants, respectively. C. pusilla breeds in the sub-arctic and mid-arctic tundra of Canada and Alaska and winters on the north and east coasts of South America. A. macularia breeds in a broad distribution across most of North America from the treeline to the southern United States. It winters in the southern United States, and Central and South America. The autumn migration route of C. pusilla includes a non-stop flight over the Atlantic Ocean, whereas autumn route of A. macularia is largely over land. Because of this difference in their migratory paths and the visuo-spatial recognition tasks involved, we hypothesized that hippocampal volume and neuronal and glial numbers would differ between these two species. A. macularia did not differ from C. pusilla in the total number of hippocampal neurons, but the species had a larger hippocampal formation and more hippocampal microglia. It remains to be investigated whether these differences indicate interspecies differences or neural specializations associated with different strategies of orientation and navigation.

Activity of brainstem cholinergic neurons during 22 kHz ultrasonic vocalization in rats /

Adult rats emit 22 kHz ultrasonic alann calls in aversive situations. This type of call IS a component of defensive behaviour and it functions predominantly to warn conspecifics about predators. Production of these calls is dependent on the central cholinergic system. The laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) contain largely cholinergic neurons, which create a continuous column in the brainstem. The LDT projects to structures in the forebrain, and it has been implicated in the initiation of 22 kHz alarm calls. It was hypothesized that release of acetylcholine from the ascending LDT terminals in mesencephalic and diencephalic areas initiates 22 kHz alarm vocalization. Therefore, the tegmental cholinergic neurons should be more active during emission of alarm calls. The aim of this study was to demonstrate increased activity of LDT cholinergic neurons during emission of 22 kHz calls induced by air puff stimuli. Immunohistochemical staining of the enzyme choline acetyltransferase identified cell bodies of cholinergic neurons, and c-Fos immunolabeling identified active cells. Double labeled cells were regarded as active cholinergic cells. There were significantly more (pneurons in the LDT of vocalizing animals than in the non-vocalizing controls. Such a difference between vocalizing and control animals was not found in the neighbouring PPT nucleus. Results suggest that there are cholinergic and non-cholinergic cells, which are selectively active in the LDT during emission of 22 kHz alarm calls.

A possible role for intrinsic calcium buffers in the long- term adaptation of crayfish neurons

Increasing the impulse activity of neurons in vivo over 3 or more days causes a reduction in transmitter release that persists for days or weeks (eg. Mercier and Atwood, 1989). This effect is usually accompanied by decreased synaptic fatigue. These two changes involve presynaptic mechanisms and indicate "long-term adaptation" (LTA) of nerve terminals. Previous experiments have shown that LTA requires extracellular calcium and protein synthesis (eg. Hong and Lnenicka, Soc. Neurosci. Abstr. 17:1322) and appears to involve communication between the cell body and the nerve terminals. The present study examines the possibility that the reduction in transmitter release is caused by an -increase in the calcium buffering ability within the nerve terminals. It examines the responses of adapted and control nerve terminals to exogenously applied calcium buffer, BAPTA-AM, which decreases transmitter release (Robitialle and Charlton, 1992). If LTA increases intrinsic Ca2+-buffering, the membrane permeant form of BAPTA should have less effect on adapted nerve terminals than on controls. Experiments are performed on the phasic abdominal extensor motor neurons of the crayfish, Procambarns clarkii. BAPTA-AM decreases excitatory postsynaptic potentials (EPSP's) of the phasic extensor muscles in a dosedependent manner between 5 and 50 JLM. LTA is elicited by in vivo stimulation at 2.5 Hz for 2-4 h per day over 3 days, which reduces EPSP's by over 50%. Experiments indicate that BAPTA-AM produces no significant change in EPSP reduction in adapted neurons when compared to controls. These results do not support the hypothesis that increased daily activity alters rapid intrinsic calcium buffers, that are able to reduce transmitter output in the same manner as BAPTA.

Perceptions of intergroup bias : the roles of social projection and meta-stereotypes

Underlying intergroup perceptions include processes of social projection (perceiving personal traitslbeliefs in others, see Krueger 1998) and meta-stereotyping (thinking about other groups' perceptions of one's own group, see Vorauer et aI., 1998). Two studies were conducted to investigate social projection and meta-stereotypes in the domain of White-Black racial relations. Study 1, a correlational study, examined the social projection of prejudice and 'prejudiced' meta-stereotypes among Whites. Results revealed that (a) Whites socially projected their intergroup attitudes onto other Whites (and Blacks) [i.e., Whites higher in prejudice against Blacks believed a large percentage of Whites (Blacks) are prejudiced against Blacks (Whites), whereas Whites low in prejudice believed a smaller percentage of Whites (Blacks) are prejudiced]; (b) Whites held the meta:..stereotype that their group (Whites) is viewed by Blacks to be prejudiced; and (c) prejudiced meta-stereotypes may be formed through the social projection of intergroup attitudes (result of path-model tests). Further, several correlates of social projection and meta-stereotypes were identified, including the finding that feeling negatively stereotyped by an outgroup predicted outgroup avoidance through heightened intergroup anxiety. Study 2 replicated and extended these findings, investigating the social projection of ingroup favouritism and meta- and other-stereotypes about ingroup favouritism. These processes were examined experimentally using an anticipated intergroup contact paradigm. The goal was to understand the experimental conditions under which people would display the strongest social projection of intergroup attitudes, and when experimentally induced meta-stereotypes (vs. other-stereotypes; beliefs about the group 11 preferences of one's outgroup) would be most damaging to intergroup contact. White participants were randomly assigned to one of six conditions and received (alleged) feedback from a previously completed computer-based test. Depending on condition, this information suggested that: (a) the participant favoured Whites over Blacks; (b) previous White participants favoured Whites over Blacks; (c) the participant's Black partner favoured Blacks over Whites; (d) previous Black participants favoured Blacks over Whites; (e) the participant's Black partner viewed the participant to favour Whites over Blacks; or (£) Black participants previously participating viewed Whites to favour Whites over Blacks. In a defensive reaction, Whites exhibited enhanced social projection of personal intergroup attitudes onto their ingroup under experimental manipulations characterized by self-concept threat (i.e., when the computer revealed that the participant favoured the ingroup or was viewed to favour the ingroup). Manipulated meta- and otherstereotype information that introduced intergroup contact threat, on the other hand, each exerted a strong negative impact on intergroup contact expectations (e.g., anxiety). Personal meta-stereotype manipulations (i.e., when the participant was informed that her/ his partner thinks s/he favours the ingroup) exerted an especially negative impact on intergroup behaviour, evidenced by increased avoidance of the upcoming interracial interaction. In contrast, personal self-stereotype manipulations (i.e., computer revealed that one favoured the ingroup) ironically improved upcoming intergroup contact expectations and intentions, likely due to an attempt to reduce the discomfort of holding negative intergroup attitudes. Implications and directions for future research are considered.

Mechanisms underlying Retinoic acid-induced chemoattraction in molluscan neurons

Retinoic acid, a derivative of vitamin A, is known to play diverse roles in development and regeneration. Previous research in the mollusc Lymnaea stagnalis has shown that a gradient of all-trans retinoic acid attracts the growth cones of cultured neurons. The present study investigates the sub-cellular mechanisms within the growth cones of Lymnaea pedal A neurons which mediate the attractive response to a gradient of alltrans retinoic acid. In this study, the mechanism of growth cone turning is shown to be local, as neurites mechanically isolated from their cell body retain the capacity to turn towards an exogenous gradient of all-trans retinoic acid. The turning response is dependent on the initiation of protein synthesis and calcium influx, but does not appear to involve signaling through protein kinase C (PKC). The retinoid X receptor (RXR), which classically functions as a transcription factor, was also shown to be involved in the turning response, functioning locally through a non-genomic pathway. These data show, for the first time in any species, that all-trans retinoic acid's chemotropic action involves a local mechanism involving non-genomic signaling through the RXR. As retinoic acid is known to playa role in regeneration, understanding the mechanisms underlying retinoic acid signaling may lead to further advances in regenerative neuroscience.

Projection-Based Statistical Inference in Linear Structural Models with Possibly Weak Instruments

It is well known that standard asymptotic theory is not valid or is extremely unreliable in models with identification problems or weak instruments [Dufour (1997, Econometrica), Staiger and Stock (1997, Econometrica), Wang and Zivot (1998, Econometrica), Stock and Wright (2000, Econometrica), Dufour and Jasiak (2001, International Economic Review)]. One possible way out consists here in using a variant of the Anderson-Rubin (1949, Ann. Math. Stat.) procedure. The latter, however, allows one to build exact tests and confidence sets only for the full vector of the coefficients of the endogenous explanatory variables in a structural equation, which in general does not allow for individual coefficients. This problem may in principle be overcome by using projection techniques [Dufour (1997, Econometrica), Dufour and Jasiak (2001, International Economic Review)]. AR-types are emphasized because they are robust to both weak instruments and instrument exclusion. However, these techniques can be implemented only by using costly numerical techniques. In this paper, we provide a complete analytic solution to the problem of building projection-based confidence sets from Anderson-Rubin-type confidence sets. The latter involves the geometric properties of “quadrics” and can be viewed as an extension of usual confidence intervals and ellipsoids. Only least squares techniques are required for building the confidence intervals. We also study by simulation how “conservative” projection-based confidence sets are. Finally, we illustrate the methods proposed by applying them to three different examples: the relationship between trade and growth in a cross-section of countries, returns to education, and a study of production functions in the U.S. economy.

Src family kinases are required for limb trajectory selection by spinal motor axons

Signal relay by guidance receptors at the axonal growth cone is a process essential for the assembly of a functional nervous system. We investigated the in vivo function of Src family kinases (SFKs) as growth cone guidance signaling intermediates in the context of spinal lateral motor column (LMC) motor axon projection toward the ventral or dorsal limb mesenchyme. Using in situ mRNA detection we determined that Src and Fyn are expressed in LMC motor neurons of chick and mouse embryos at the time of limb trajectory selection. Inhibition of SFK activity by C-terminal Src kinase (Csk) overexpression in chickLMCaxons using in ovo electroporation resulted inLMC axons selecting the inappropriate dorsoventral trajectory within the limb mesenchyme, with medial LMC axon projecting into the dorsal and ventral limb nerve with apparently random incidence. We also detected LMC axon trajectory choice errors in Src mutant mice demonstrating a nonredundant role for Src in motor axon guidance in agreement with gain and loss of Src function in chickLMCneurons which led to the redirection ofLMCaxons. Finally, Csk-mediated SFK inhibition attenuated the retargeting ofLMCaxons caused by EphA or EphB over-expression, implying the participation of SFKs in Eph-mediated LMC motor axon guidance. In summary, our findings demonstrate that SFKs are essential for motor axon guidance and suggest that they play an important role in relaying ephrin:Eph signals that mediate the selection of motor axon trajectory in the limb.

Implication du récepteur aux cannabinoïdes CB2 dans le développement de la voie rétinothalamique

Durant le développement du système visuel, les cellules ganglionnaires de la rétine (CGRs) envoient des axones qui seront influencés par divers signaux guidant leur cône de croissance, permettant ainsi la navigation des axones vers leurs cibles terminales. Les endocannabinoïdes, des dérivés lipidiques activant les récepteurs aux cannabinoides (CB1 et CB2), sont présents de manière importante au cours du développement. Nous avons démontré que le récepteur CB2 est exprimé à différents points du tractus visuel durant le développement du hamster. L’injection d’agonistes et d’agonistes inverses pour le récepteur CB2 a modifié l’aire du cône de croissance et le nombre de filopodes présents à sa surface. De plus, l’injection d’un gradient d’agoniste du récepteur CB2 produit la répulsion du cône de croissance tandis qu’un analogue de l’AMPc (db-AMPc) produit son attraction. Les effets du récepteur CB2 sur le cône de croissance sont produits en modulant l’activité de la protéine kinase A(PKA), influençant la présence à la membrane cellulaire d’un récepteur à la nétrine-1 nommé Deleted in Colorectal Cancer (DCC). Notamment, pour que le récepteur CB2 puisse moduler le guidage du cône de croissance, la présence fonctionnelle du récepteur DCC est essentielle.. Suite à une injection intra-occulaire d’un agoniste inverse du récepteur CB2, nous avons remarqué une augmentation de la longueur des branches collatérales des axones rétiniens au niveau du LTN (noyau lateral terminal). Nous avons également remarqué une diminution de la ségrégation des projections ganglionnaires au niveau du dLGN, le noyau genouillé lateral dorsal, chez les animaux transgéniques cnr2-/-, ayant le gène codant pour le récepteur CB2 inactif. Nos données suggèrent l’implication des endocannabinoïdes et de leur récepteur CB2 dans la modulation des processus de navigation axonale et de ségrégation lors du développement du système visuel.