Sound‐specific plasticity in the primary auditory cortex as induced by the cholinergic pedunculopontine tegmental nucleus

Brain cholinergic modulation is essential for learning‐induced plasticity of the auditory cortex. The pedunculopontine tegmental nucleus (PPTg) is an important cholinergic nucleus in the brainstem, and appears to be involved in learning and subcortical plasticity. This study confirms the involvement of the PPTg in the plasticity of the auditory cortex in mice. We show here that electrical stimulation of the PPTg paired with a tone induced drastic changes in the frequency tunings of auditory cortical neurons. Importantly, the changes in frequency tuning were highly specific to the frequency of the paired tone; the best frequency of auditory cortical neurons shifted towards the frequency of the paired tone. We further demonstrated that such frequency‐specific plasticity was largely eliminated by either thalamic or cortical application of the muscarinic acetylcholine receptor antagonist atropine. Our finding suggests that the PPTg significantly contributes to auditory cortical plasticity via the auditory thalamus and cholinergic basal forebrain.     

Variability in interval production is due to timing‐dependent deficits in Huntington's disease

In Huntington's disease (HD), increased variability is seen in performance of motor tasks that require implicit control of timing. We examined whether timing variability was also evident in an explicit interval‐timing task. Sixty subjects (21 controls, 19 manifest HD, and 20 pre‐manifest HD) performed a single‐interval production task with three target intervals (1.1 s, 2.2 s, 3.3 s). We analyzed accuracy (proportional error) and precision (standard deviation) across groups and intervals. No differences were seen in accuracy across groups or intervals. Precision was significantly lower in manifest (P = 0.0001) and pre‐manifest HD (P = 0.04) compared with controls. This was particularly true for pre‐manifest subjects close to diagnosis (based on probability of diagnosis in 5 years). Precision was correlated with proximity to diagnosis (r² = 0.3, P < 0.01). To examine the source of reduced precision, we conducted linear regression of standard deviation with interval duration. Slope of the regression was significantly higher in manifest HD (P = 0.02) and in pre‐manifest HD close to diagnosis (P = 0.04) compared with controls and pre‐manifest participants far from diagnosis. Timing precision is impaired before clinical diagnosis in Huntington's disease. Slope analysis suggests that timing variability (decreased precision) was attributable to deficits in timing‐dependent processes. Our results provide additional support for the proposal that the basal ganglia are implicated in central timekeeping functions. Because the single interval production task was sensitive to deficits in pre‐manifest HD, temporal precision may be a useful outcome measure in future clinical trials. © 2014 International Parkinson and Movement Disorder Society     

Neonatal capsaicin treatment modulates experience‐dependent plasticity in the rat barrel cortex

Previous studies have reported that capsaicin‐induced C‐fiber depletion results in expansion of low threshold somatosensory mechanoreceptive fields. Here we used this paradigm to investigate its effect on experience‐dependent plasticity in the barrel cortex of rats. All but the D2 vibrissa were first plucked on postnatal day 0 (P0), P5, or P8, and kept plucked for a period of 30 days before being allowed to regrow for 7–9 days prior to the recording session. To assess receptive field characteristics the spared D2 principal whisker (PW) and the deprived D1 adjacent whisker (AW) were moved either singly or in concert, neuronal responses being recorded in layers IV and V of the D2 barrel. In vehicle‐treated rats, PW‐evoked ON responses (layer IV) were increased only in those animals that first had their vibrissae plucked on P0, whereas AW‐evoked ON responses (layers IV and V) were decreased in the P0, P5, and P8 groups. In the capsaicin‐treated animals, PW‐evoked ON responses (layer IV) were increased in all three groups, but no decrease was recorded in the AW‐evoked ON (layers IV and V) responses. In the vehicle‐ and capsaicin‐treated animals, the greatest decrease in inhibitory interactions was observed in the P5 and P0 groups, respectively. These findings indicate that, following the induction of experience‐dependent plasticity, the resultant changes in excitatory and integrative circuits can be further influenced by C‐fiber depletion. J. Comp. Neurol. 518:3427–3438, 2010. © 2010 Wiley‐Liss, Inc.     

Astrocyte‐restricted disruption of connexin‐43 impairs neuronal plasticity in mouse barrel cortex

There is intensive gap‐junctional coupling between glial processes, but their significance in sensory functions remains unknown. Connexin‐43 (Cx43), a major component of astrocytic gap‐junction channels, is abundantly expressed in astrocytes. To investigate the role of Cx43‐mediated gap junctions between astrocytes in sensory functions, we generated Cx43 knockout (KO) mice with a mouse line carrying loxP sites flanking exon 2 of the Cx43 gene and the transgenic line expressing Cre recombinase under control of the glial fibrillary acidic protein promoter, which exhibited a significant loss of Cx43 in astrocytes in the barrel cortex. Although Cx43 expression between the astrocytes measured by immunohistochemistry was virtually abolished in Cx43 KO mice, they had normal architecture in the barrel cortex but the intensity of cytochrome oxide histochemistry decreased significantly. In vivo electrophysiological analysis revealed that the long‐term potentiation of the vibrissal evoked responses in the barrel cortex evoked by high‐frequency rhythmic vibrissal stimuli (100 Hz, 1 s) was abolished in Cx43 KO mice. Current source density analysis also revealed that astrocytic Cx43 was important to the flow of excitation within the laminar connections in barrel cortex. Behavioral tests showed that the ability of Cx43 KO mice to sense the environment with their whiskers decreased. Even so, the jump‐stand experiment showed that they could still discriminate rough from smooth surfaces. Our findings suggest that Cx43‐mediated gap‐junctional coupling between astrocytes is important in the neuron–glia interactions required for whisker‐related sensory functions and plasticity.     

Interaction of inhibition and triplets of excitatory spikes modulates the NMDA‐R‐mediated synaptic plasticity in a computational model of spike timing‐dependent plasticity

Spike timing‐dependent plasticity (STDP) experiments have shown that a synapse is strengthened when a presynaptic spike precedes a postsynaptic one and depressed vice versa. The canonical form of STDP has been shown to have an asymmetric shape with the peak long‐term potentiation at +6 ms and the peak long‐term depression at ?5 ms. Experiments in hippocampal cultures with more complex stimuli such as triplets (one presynaptic spike combined with two postsynaptic spikes or one postsynaptic spike with two presynaptic spikes) have shown that pre–post–pre spike triplets result in no change in synaptic strength, whereas post–pre–post spike triplets lead to significant potentiation. The sign and magnitude of STDP have also been experimentally hypothesized to be modulated by inhibition. Recently, a computational study showed that the asymmetrical form of STDP in the CA1 pyramidal cell dendrite when two spikes interact switches to a symmetrical one in the presence of inhibition under certain conditions. In the present study, I investigate computationally how inhibition modulates STDP in the CA1 pyramidal neuron dendrite when it is driven by triplets. The model uses calcium as the postsynaptic signaling agent for STDP and is shown to be consistent with the experimental triplet observations in the absence of inhibition: simulated pre–post–pre spike triplets result in no change in synaptic strength, whereas simulated post–pre–post spike triplets lead to significant potentiation. When inhibition is bounded by the onset and offset of the triplet stimulation, then the strength of the synapse is decreased as the strength of inhibition increases. When inhibition arrives either few milliseconds before or at the onset of the last spike in the pre–post–pre triplet stimulation, then the synapse is potentiated. Variability in the frequency of inhibition (50 vs. 100 Hz) produces no change in synaptic strength. Finally, a 5% variation in model's calcium parameters (calcium thresholds) proves that the model's performance is robust. © 2012 Wiley Periodicals, Inc.     

Fluoxetine‐induced plasticity in the visual cortex outlasts the duration of the naturally occurring critical period

Heightened neuronal plasticity expressed during early postnatal life has been thought to permanently decline once critical periods have ended. For example, monocular deprivation is able to shift ocular dominance in the mouse visual cortex during the first months of life, but this effect is lost later in life. However, various treatments, such as the antidepressant fluoxetine, can reactivate a critical period‐like plasticity in the adult brain. When monocular deprivation is supplemented with chronic fluoxetine administration, a major shift in ocular dominance is produced after the critical period has ended. In the current study, we characterized the temporal patterns of fluoxetine‐induced plasticity in the adult mouse visual cortex, using in vivo optical imaging. We found that artificially induced plasticity in ocular dominance extended beyond the duration of the naturally occurring critical period and continued as long as fluoxetine was administered. However, this fluoxetine‐induced plasticity period ended as soon as the drug was not given. These features of antidepressant‐induced plasticity may be useful when designing treatment strategies involving long‐term antidepressant treatment in humans.     

Involvement of cannabinoid‐1 and cannabinoid‐2 receptors in septic ileus

Background Cannabinoid (CB) receptors are involved in the regulation of gastrointestinal (GI) motility under physiological and pathophysiological conditions. We aimed to characterize the possible influence of CB₁ and CB₂ receptors on motility impairment in a model of septic ileus. Methods Lipopolysaccharide (LPS) injections were used to mimic pathophysiological features of septic ileus. Spontaneous jejunal myoelectrical activity was measured in rats in vivo, and upper GI transit was measured in vivo by gavaging of a charcoal marker into the stomach of mice, in absence or presence of LPS, and CB₁ and CB₂ receptor agonists and antagonists. Tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 levels were measured using enzyme‐linked immunosorbent assay. Histology was performed with haematoxylin–eosin staining. Key Results Lipopolysaccharide treatment significantly reduced amplitude and frequency of myoelectric spiking activity and GI transit in vivo in a dose‐dependent manner. TNF‐α and IL‐6 were increased in LPS‐treated animals and histology showed oedema and cell infiltration. Both, the CB₁ agonist HU210 and the CB₂ agonist JWH133 reduced myoelectrical activity whereas the CB₁ antagonist AM251 caused an increase of myoelectrical activity. Pretreatment with AM251 or AM630 prevented against LPS‐induced reduction of myoelectrical activity, and also against the delay of GI transit during septic ileus in vivo. Conclusions & Inferences The LPS model of septic ileus impairs jejunal myoelectrical activity and delays GI transit in vivo. Antagonists at the CB₁ receptor or the CB₂ receptor prevent the delay of GI transit and thus may be powerful tools in the future treatment of septic ileus.     

Differential synaptic vesicle protein expression in the barrel field of developing cortex

The distribution of four proteins associated with synaptic vesicles, SV2, synaptophysin, synapsin I, and rab3a, was investigated during postnatal development of the posteromedial barrel subfield (PMBSF) in the rat somatosensory cortex. A distinct progression in the appearance of the different synaptic vesicle proteins within the PMBSF was observed. SV2, synapsin I, and synaptophysin revealed the organization of the barrel field in the neonate. This early demarcation of the cortical representation of the vibrissal array coincides with the earliest known age for the emergence of the cytoarchitectonic organization of this region. In contrast, rab3a did not delimit the barrels until the end of the 1st postnatal week, coincident with the known onset of adult-like physiological activity and the loss of plasticity in afferents to this region. In addition, the appearance of the different synaptic vesicle proteins occurred earlier within the PMBSF than in the adjacent extra-barrel regions of the cortex. These results show that the molecular differentiation of synaptic fields across the cortex is not a homogeneous and synchronous process in terms of synaptic vesicle protein expression. Because these proteins act together in mature synapses to ensure the regulated release of neurotransmitters, our results suggest that this temporo-spatial asynchrony may underlie different potentials for synaptic activity and thus contribute to the development of cortical maps. © 1996 Wiley-Liss, Inc.     

Properties of 4 Hz stimulation‐induced parallel fiber–Purkinje cell presynaptic long‐term plasticity in mouse cerebellar cortex in vivo

Cerebellar parallel fiber–Purkinje cell (PF–PC) long‐term synaptic plasticity is important for the formation and stability of cerebellar neuronal circuits, and provides substrates for motor learning and memory. We previously reported both presynaptic long‐term potentiation (LTP) and long‐term depression (LTD) in cerebellar PF–PC synapses in vitro. However, the expression and mechanisms of cerebellar PF–PC synaptic plasticity in the cerebellar cortex in vivo are poorly understood. In the present study, we studied the properties of 4 Hz stimulation‐induced PF–PC presynaptic long‐term plasticity using in vivo the whole‐cell patch‐clamp recording technique and pharmacological methods in urethane‐anesthetised mice. Our results demonstrated that 4 Hz PF stimulation induced presynaptic LTD of PF–PC synaptic transmission in the intact cerebellar cortex in living mice. The PF–PC presynaptic LTD was attenuated by either the N‐methyl‐D‐aspartate receptor antagonist, D‐aminophosphonovaleric acid, or the group 1 metabotropic glutamate receptor antagonist, JNJ16259685, and was abolished by combined D‐aminophosphonovaleric acid and JNJ16259685, but enhanced by inhibition of nitric oxide synthase. Blockade of cannabinoid type 1 receptor activity abolished the PF–PC LTD and revealed a presynaptic PF–PC LTP. These data indicate that both endocannabinoids and nitric oxide synthase are involved in the 4 Hz stimulation‐induced PF–PC presynaptic plasticity, but the endocannabinoid‐dependent PF–PC presynaptic LTD masked the nitric oxide‐mediated PF–PC presynaptic LTP in the cerebellar cortex in urethane‐anesthetised mice.     

Self‐organization of repetitive spike patterns in developing neuronal networks in vitro

The appearance of spontaneous correlated activity is a fundamental feature of developing neuronal networks in vivo and in vitro. To elucidate whether the ontogeny of correlated activity is paralleled by the appearance of specific spike patterns we used a template‐matching algorithm to detect repetitive spike patterns in multi‐electrode array recordings from cultures of dissociated mouse neocortical neurons between 6 and 15 days in vitro (div). These experiments demonstrated that the number of spiking neurons increased significantly between 6 and 15 div, while a significantly synchronized network activity appeared at 9 div and became the main discharge pattern in the subsequent div. Repetitive spike patterns with a low complexity were first observed at 8 div. The number of repetitive spike patterns in each dataset as well as their complexity and recurrence increased during development in vitro. The number of links between neurons implicated in repetitive spike patterns, as well as their strength, showed a gradual increase during development. About 8% of the spike sequences contributed to more than one repetitive spike patterns and were classified as core patterns. These results demonstrate for the first time that defined neuronal assemblies, as represented by repetitive spike patterns, appear quite early during development in vitro, around the time synchronized network burst become the dominant network pattern. In summary, these findings suggest that dissociated neurons can self‐organize into complex neuronal networks that allow reliable flow and processing of neuronal information already during early phases of development.     

Transmembrane signaling through phospholipase C-beta in the developing human prefrontal cortex

To investigate changes in muscarinic receptor-stimulated phospholipase C-beta (PLC-beta) activity during brain development, we examined the functional coupling of each of the three major protein components of the phosphoinositide system (M1, M3, and M5 muscarinic receptor subtypes; Gq/11 proteins; PLC-beta1-4 isoforms) in membrane preparations from post-mortem human prefrontal cerebral cortex collected at several stages of prenatal and postnatal development. In human prenatal brain membranes, PLC was found to be present and could be activated by calcium, but the ability of guanosine-5'-o-3 thiotriphosphate (GTPgammaS) or carbachol (in the presence of GTPgammaS) to modulate prenatal PLC-beta was significantly weaker than that associated with postnatal PLC-beta. Western blot analysis revealed that the levels of Galphaq/11 did not change significantly during development. In contrast, dramatically higher levels of expression of PLC-beta1-4 isoforms and of M1, M3, and M5 muscarinic receptors were detected in the child vs. the fetal brain, a finding that might underlie the observed increased activity of PLC. Thus, inositol phosphate production may be more efficiently regulated by altering the amount of effectors (PLC-beta1-4) and receptors (M1,3,5 subtypes) than by altering the level of Galphaq/11 subunits. These results demonstrate that different PLC isoforms are expressed in the prefrontal cortex of the developing human brain in an age-specific manner, suggesting specific roles not only in synaptic transmission but also in the differentiation and maturation of neurons in the developing brain.     

GluN2B‐containing N‐methyl‐D‐aspartate receptors compensate for the inhibitory control of synaptic plasticity during the early critical period in the rat visual cortex

In the visual cortex, synaptic plasticity is very high during the early developmental stage known as the critical period and declines with development after the critical period. Changes in the properties of N‐methyl‐D‐aspartate receptor (NMDAR) and γ‐aminobutyric acid type A receptor (GABA_AR) have been suggested to underlie the changes in the characteristics of plasticity. However, it is largely unknown how the changes in the two receptors interact to regulate synaptic plasticity. The present study investigates the changes in the properties of NMDAR and GABA_AR from 3 to 5 weeks of age in layer 2/3 pyramidal neurons of the rat visual cortex. The impact of these changes on the characteristics of long‐term potentiation (LTP) is also investigated. The amplitude and decay time constant of GABA_AR‐mediated currents increased during this period. However, the decay time constant of NMDAR‐mediated currents decreased as a result of the decrease in the proportion of the GluN2B subunit‐mediated component. Induction of NMDAR‐dependent LTP at 3 weeks depended on the GluN2B subunit, but LTP at 5 weeks did not. Enhancement of GABA_AR‐mediated inhibition suppressed the induction of LTP only at 5 weeks. However, partial inhibition of the GluN2B subunit with a low concentration of ifenprodil allowed the GABA_AR‐mediated suppression of LTP at 3 weeks. These results suggest that changes in the properties of NMDAR‐ and GABA_AR‐mediated synaptic transmission interact to determine the characteristics of synaptic plasticity during the critical period in the visual cortex. © 2015 Wiley Periodicals, Inc.     

ATP‐induced IL‐1β secretion is selectively impaired in microglia as compared to hematopoietic macrophages

Under stressful conditions nucleotides are released from dying cells into the extracellular space, where they can bind to purinergic P2X and P2Y receptors. High concentrations of extracellular ATP in particular induce P2X7‐mediated signaling, which leads to inflammasome activation. This in turn leads to the processing and secretion of pro‐inflammatory cytokines, like interleukin (IL)−1β. During neurodegenerative diseases, innate immune responses are shaped by microglia and we have previously identified microglia‐specific features of inflammasome‐mediated responses. Here, we compared ATP‐induced IL‐1β secretion in primary rhesus macaque microglia and bone marrow‐derived macrophages (BMDM). We assessed the full expression profile of P2 receptors and characterized the induction and modulation of IL‐1β secretion by extracellular nucleotides. Microglia secreted significantly lower levels of IL‐1β in response to ATP when compared to BMDM. We demonstrate that this is not due to differences in sensitivity, kinetics or expression of ATP‐processing enzymes, but rather to differences in purinergic receptor expression levels and usage. Using a combined approach of purinergic receptor agonists and antagonists, we demonstrate that ATP‐induced IL‐1β secretion in BMDM was fully dependent on P2X7 signaling, whereas in microglia multiple purinergic receptors were involved, including P2X7 and P2X4. These cell type‐specific features of conserved innate immune responses may reflect adaptations to the vulnerable CNS microenvironment. GLIA 2016;64:2231–2246     

The TIR‐domain‐containing adapter inducing interferon‐β‐dependent signaling cascade plays a crucial role in ischemia–reperfusion‐induced retinal injury,whereas the contribution of the myeloid differentiation primary response 88‐dependent signaling cascade is not as pivotal

Toll‐like receptor 4 (Tlr4) plays an important role in ischemia–reperfusion (IR)‐induced retinal inflammation and damage. However, the role of two Tlr4‐dependent signaling cascades, myeloid differentiation primary response 88 (Myd88) and TIR‐domain‐containing adapter inducing interferon‐β (Trif), in retinal IR injury is poorly understood. In this study, we investigated the contribution of the Myd88‐dependent and Trif‐dependent signaling cascades in retinal damage and inflammation triggered by IR, by using Myd88 knockout (Myd88KO) and Trif knockout (TrifKO) mice. Retinal IR injury was induced by unilateral elevation of intraocular pressure for 45 min by direct corneal cannulation. To study IR‐induced retinal ganglion cell (RGC) death in vitro, we used an oxygen and glucose deprivation (OGD) model. Our data suggested that Myd88 was present in many retinal layers of sham‐operated and ischemic mice, whereas Trif was mainly present in the ganglion cell layer (GCL). The level of Myd88 was increased in the retina after IR. We found that retinas of TrifKO mice had a significantly reduced neurotoxic pro‐inflammatory response and significantly increased survival of the GCL neurons after IR. Although Myd88KO mice had relatively low levels of inflammation in ischemic retinas, their levels of IR‐induced retinal damage were notably higher than those of TrifKO mice. We also found that Trif‐deficient RGCs were more resistant to death induced by OGD than were RGCs isolated from Myd88KO mice. These data suggested that, as compared with the Myd88‐dependent signaling cascade, Trif signaling contributes significantly to retinal damage after IR.     

Whisker experience‐dependent mGluR signaling maintains synaptic strength in the mouse adolescent cortex

Sensory experience‐dependent plasticity in the somatosensory cortex is a fundamental mechanism of adaptation to the changing environment not only early in the development but also in adolescence and adulthood. Although the mechanisms underlying experience‐dependent plasticity during early development have been well documented, the corresponding understanding in the mature cortex is less complete. Here, we investigated the mechanism underlying whisker deprivation‐induced synaptic plasticity in the barrel cortex in adolescent mice. Layer 4 (L4) to L2/3 excitatory synapses play a crucial role for whisker experience‐dependent plasticity in rodent barrel cortex and whisker deprivation is known to depress synaptic strength at L4–L2/3 synapses in adolescent and adult animals. We found that whisker deprivation for 5 days or longer decreased the presynaptic glutamate release probability at L4–L2/3 synapses in the barrel cortex in adolescent mice. This whisker deprivation‐induced depression was restored by daily administration of a positive allosteric modulator of the type 5 metabotropic glutamate receptor (mGluR5). On the other hand, the administration of mGluR5 antagonists reproduced the effect of whisker deprivation in whisker‐intact mice. Furthermore, chronic and selective suppression of inositol 1,4,5‐trisphosphate (IP₃) signaling in postsynaptic L2/3 neurons decreased the presynaptic release probability at L4–L2/3 synapses. These findings represent a previously unidentified mechanism of cortical plasticity, namely that whisker experience‐dependent mGluR5‐IP₃ signaling in the postsynaptic neurons maintains presynaptic function in the adolescent barrel cortex.     

Ca2+/calmodulin‐dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ‐aminobutyric acid type a receptor inhibition

The fast inhibitory synaptic transmission mediated by the γ‐aminobutyric acid type A receptor (GABA_AR) within spinal dorsal horn exerts a gating control over the synaptic conveyance of nociceptive information from the periphery to higher brain regions. Although a large body of evidence has demonstrated that the impairment of GABAergic inhibition alone is sufficient to elicit pain hypersensitivity in intact animals, the underlying mechanisms remain to be characterized. The present study shows that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is an important signaling protein downstream of reduced GABAergic inhibition. We found that pharmacological removal of inhibition by intrathecal application of the GABA_AR antagonist bicuculline significantly enhanced the autophosphorylation of CaMKII at Thr286 in spinal dorsal horn of mice. In addition to increased CaMKII activity, bicuculline also promoted CaMKII interaction with N‐methyl‐D‐aspartate (NMDA)‐subtype glutamate receptors and induced the translocation of CaMKII from cytosolic compartments to the synaptosomal membrane fraction. Immunoblotting analysis revealed that the phosphorylation levels of NMDA receptor NR2B subunit at Ser1303 and of AMPA‐subtype glutamate receptor GluR1 subunit at Ser831, two important CaMKII phosphorylation sites, were substantially enhanced after bicuculline application. Behavioral tests illustrated that intrathecal administration of the CaMKII inhibitor KN‐93, NMDA receptor antagonist D‐APV, or AMPA receptor antagonist GYKI 52466 effectively ameliorated the mechanical allodynia evoked by bicuculline. These data thus indicate that CaMKII signaling is critical for the reduced inhibition to evoke spinal sensitization. © 2013 Wiley Periodicals, Inc.     

Developmental study of dendritic bundles in layer 1 of the rat granular retrosplenial cortex with special reference to a cell adhesion molecule, OCAM

In the granular retrosplenial cortex (GRS) of adult rats, callosally projecting pyramidal neurons in layer 2 form dendritic bundles, 30-100 micro m wide, in layer 1. The distinctness of these bundles makes the GRS an attractive model system for investigating the developmental, microcircuitry, and basic organizational features related to dendritic modularity. In this report, we investigate the developmental time course of the dendritic bundles, visualized by immunohistochemistry for microtubule-associated protein 2 (MAP2) and glutamate receptor subunits 2/3 (GluR2/3). Bundles in layer 1 are apparent as early as postnatal day 5, first with GluR2/3 and then, from postnatal day 14, with MAP2. As a step toward understanding the mechanisms of dendritic aggregation, we further investigated the ontogeny of expression of the cell adhesion molecule OCAM. OCAM exhibits a patchy distribution in layer 1 from postnatal day 3 to adult, and the regions of weak OCAM immunoreactivity selectively correspond to the dendritic bundles (in both GluR2/3 and MAP2). The periodic geometry of OCAM-immunoreactive regions, the time course of their appearance and the distinct localization complementary to the bundles support the possibility that this molecule is one contributor to the establishment and maintenance of dendritic modules. The interdigitating relationship between regions of high OCAM immunoreactivity and the dendritic bundles in layer 1 suggests that OCAM may have a repellent influence on the formation of these bundles.     

Manganese‐induced downregulation of astroglial glutamine transporter SNAT3 involves ubiquitin‐mediated proteolytic system

SNAT3 is a major facilitator of glutamine (Gln) efflux from astrocytes, supplying Gln to neurons for neurotransmitter synthesis. Our previous investigations have shown that, in primary cortical astrocyte cultures, SNAT3 protein is degraded after exposure to manganese (Mn²⁺). The present studies were performed to identify the processes responsible for this effect. One of the well‐established mechanisms for protein‐level regulation is posttranslational modification via ubiquitination, which leads to the rapid degradation of proteins by the 26S proteasome pathway. Here, we show that astrocytic SNAT3 directly interacts with the ubiquitin ligase, Nedd4‐2 (neural precursor cells expressed developmentally downregulated 4‐2), and that Mn²⁺ increases both Nedd4‐2 mRNA and protein levels. Additionally, we have found that Mn²⁺ exposure elevates astrocytic ubiquitin B mRNA expression, free ubiquitin protein levels, and total protein ubiquitination. Furthermore, Mn²⁺ effectively decreases astrocytic mRNA expression and the phosphorylation of serum and glucocorticoid‐inducible kinase, a regulatory protein, which, in the active phosphorylated form, is responsible for the phosphorylation and subsequent inactivation of Nedd4‐2. Additional findings establish that Mn²⁺ increases astrocytic caspase‐like proteolytic proteasome activity and that the Mn²⁺‐dependent degradation of SNAT3 protein is blocked by the proteasome inhibitors, N‐acetyl‐leu‐leu‐norleucinal and lactacystin. Combined, these results demonstrate that Mn²⁺‐induced SNAT3 protein degradation and the dysregulation of Gln homeostasis in primary astrocyte cultures proceeds through the ubiquitin‐mediated proteolytic system. © 2010 Wiley‐Liss, Inc.