The effects of astaxanthin treatment on a rat model of Alzheimer's disease.

Alzheimer's disease (AD), a progressive neurodegenerative disorder characterized by memory loss and dementia, could be a consequence of the abnormalities of cortical milieu, such as oxidative stress, inflammation, and/or accompanied with the aggregation of ß-amyloid. The majority of AD patients are sporadic, late-onset AD, which predominantly occurs over 65 years of age. Our results revealed that the ferrous amyloid buthionine (FAB)-infused sporadic AD-like model showed deficits in spatial learning and memory and with apparent loss of choline acetyltransferase (ChAT) expression in medial septal (MS) nucleus. In hippocampal CA1 region, the loss of pyramidal neurons was accompanied with cholinergic fiber loss and neuroinflammatory responses including glial reaction and enhanced expression of inducible nitric oxide synthase (iNOS). Surviving hippocampal CA1 pyramidal neurons showed the reduction of dendritic spines as well. Astaxanthin (ATX), a potent antioxidant, reported to improve the outcome of oxidative-stress-related diseases. The ATX treatment in FAB-infused rats decreased neuroinflammation and restored the ChAT + fibers in hippocampal CA1 region and the ChAT expression in MS nucleus. It also partly recovered the spine loss on hippocampal CA1 pyramidal neurons and ameliorated the behavioral deficits in AD-like rats. From these data, we believed that the ATX can be a potential option for slowing the progression of Alzheimer's disease.

Cortical compression rapidly trimmed transcallosal projections and altered axonal anterograde transport machinery.

Trauma and tumor compressing the brain distort underlying cortical neurons. Compressed cortical neurons remodel their dendrites instantly. The effects on axons however remain unclear. Using a rat epidural bead implantation model, we studied the effects of unilateral somatosensory cortical compression on its transcallosal projection and the reversibility of the changes following decompression. Compression reduced the density, branching profuseness and boutons of the projection axons in the contralateral homotopic cortex 1week and 1month post-compression. Projection fiber density was higher 1-month than 1-week post-compression, suggesting adaptive temporal changes. Compression reduced contralateral cortical synaptophysin, vesicular glutamate transporter 1 (VGLUT1) and postsynaptic density protein-95 (PSD95) expressions in a week and the first two marker proteins further by 1month. ßIII-tubulin and kinesin light chain (KLC) expressions in the corpus callosum (CC) where transcallosal axons traveled were also decreased. Kinesin heavy chain (KHC) level in CC was temporarily increased 1week after compression. Decompression increased transcallosal axon density and branching profuseness to higher than sham while bouton density returned to sham levels. This was accompanied by restoration of synaptophysin, VGLUT1 and PSD95 expressions in the contralateral cortex of the 1-week, but not the 1-month, compression rats. Decompression restored ßIII-tubulin, but not KLC and KHC expressions in CC. However, KLC and KHC expressions in the cell bodies of the layer II/III pyramidal neurons partially recovered. Our results show cerebral compression compromised cortical axonal outputs and reduced transcallosal projection. Some of these changes did not recover in long-term decompression.

Hydrocephalus compacted cortex and hippocampus and altered their output neurons in association with spatial learning and memory deficits in rats.

Hydrocephalus is a common neurological disorder in children characterized by abnormal dilation of cerebral ventricles as a result of the impairment of cerebrospinal fluid flow or absorption. Clinical presentation of hydrocephalus varies with chronicity and often shows cognitive dysfunction. Here we used a kaolin-induction method in rats and studied the effects of hydrocephalus on cerebral cortex and hippocampus, the two regions highly related to cognition. Hydrocephalus impaired rats' performance in Morris water maze task. Serial three-dimensional reconstruction from sections of the whole brain freshly froze in situ with skull shows that the volumes of both structures were reduced. Morphologically, pyramidal neurons of the somatosensory cortex and hippocampus appear to be distorted. Intracellular dye injection and subsequent three-dimensional reconstruction and analyses revealed that the dendritic arbors of layer III and V cortical pyramid neurons were reduced. The total dendritic length of CA1, but not CA3, pyramidal neurons was also reduced. Dendritic spine densities on both cortical and hippocampal pyramidal neurons were decreased, consistent with our concomitant findings that the expressions of both synaptophysin and postsynaptic density protein 95 were reduced. These cortical and hippocampal changes suggest reductions of excitatory connectivity, which could underlie the learning and memory deficits in hydrocephalus.

Reproductive experience modified dendritic spines on cortical pyramidal neurons to enhance sensory perception and spatial learning in rats.

Behavioral adaptations during motherhood are aimed at increasing reproductive success. Alterations of hormones during motherhood could trigger brain morphological changes to underlie behavioral alterations. Here we investigated whether motherhood changes a rat's sensory perception and spatial memory in conjunction with cortical neuronal structural changes. Female rats of different statuses, including virgin, pregnant, lactating, and primiparous rats were studied. Behavioral test showed that the lactating rats were most sensitive to heat, while rats with motherhood and reproduction experience outperformed virgin rats in a water maze task. By intracellular dye injection and computer-assisted 3-dimensional reconstruction, the dendritic arbors and spines of the layer III and V pyramidal neurons of the somatosensory cortex and CA1 hippocampal pyramidal neurons were revealed for closer analysis. The results showed that motherhood and reproductive experience increased dendritic spines but not arbors or the lengths of the layer III and V pyramidal neurons of the somatosensory cortex and CA1 hippocampal pyramidal neurons. In addition, lactating rats had a higher incidence of spines than pregnant or primiparous rats. The increase of dendritic spines was coupled with increased expression of the glutamatergic postsynaptic marker protein (PSD-95), especially in lactating rats. On the basis of the present results, it is concluded that motherhood enhanced rat sensory perception and spatial memory and was accompanied by increases in dendritic spines on output neurons of the somatosensory cortex and CA1 hippocampus. The effect was sustained for at least 6 weeks after the weaning of the pups.

Rostral Intralaminar Thalamic Deep Brain Stimulation Triggered Cortical and Hippocampal Structural Plasticity and Enhanced Spatial Memory.

BACKGROUND/AIMS: Rostral intralaminar thalamic nucleus (ILN) has been shown to modulate cognition through indirect connection with the hippocampus and prefrontal cortex. We explored the effects of deep brain stimulation (DBS) to the rostral ILN on spatial memory acquisition, brain neuronal activation and cortical and hippocampal synaptic changes in rats. METHODS: The Morris water maze (MWM) task was used to evaluate the spatial memory of the rats. The expression of c-fos, an immediate early gene, was used to identify neural activation in the cerebral cortex and hippocampus. Synaptic changes in the somatosensory cortical and hippocampal neurons were explored with dendritic spine analysis following Golgi-Cox staining. RESULTS: Our results showed that a barrage of DBS to the rostral ILN of normal rats significantly shortened their escape latency in MWM compared with sham-stimulated and untreated control rats. Rats with enhanced spatial memory had more c-fos immunoreactive cells in layer IV of the somatosensory cortex. Layer III cortical and CA1 hippocampal pyramidal neurons showed over 50% increase of dendritic spines, while only the proximal apical dendrites of layer V cortical pyramidal neurons had more dendritic spines. CONCLUSIONS: Rostral ILN-DBS activated neurons in the cerebral cortex and triggered cortical and hippocampal structural plasticity in association with spatial memory enhancement.

NMDA receptor triggered molecular cascade underlies compression-induced rapid dendritic spine plasticity in cortical neurons.

Compression causes the reduction of dendritic spines of underlying adult cortical pyramidal neurons but the mechanisms remain at large. Using a rat epidural cerebral compression model, dendritic spines on the more superficial-lying layer III pyramidal neurons were found quickly reduced in 12h, while those on the deep-located layer V pyramidal neurons were reduced slightly later, starting 1day following compression. No change in the synaptic vesicle markers synaptophysin and vesicular glutamate transporter 1 suggest no change in afferents. Postsynaptically, N-methyl-d-aspartate (NMDA) receptor trafficking to synaptic membrane was detected in 10min and lasting to 1day after compression. Translocation of calcineurin to synapses and enhancement of its enzymatic activity were detected within 10min as well. These suggest that compression rapidly activated NMDA receptors to increase postsynaptic calcium, which then activated the phosphatase calcineurin. In line with this, dephosphorylation and activation of the actin severing protein cofilin, and the consequent depolymerization of actin were all identified in the compressed cortex within matching time frames. Antagonizing NMDA receptors with MK801 before compression prevented this cascade of events, including NR1 mobilization, calcineurin activation and actin depolymerization, in the affected cortex. Morphologically, MK801 pretreatment prevented the loss of dendritic spines on the compressed cortical pyramidal neurons as well. In short, we demonstrated, for the first time, mechanisms underlying the rapid compression-induced cortical neuronal dendritic spine plasticity. In addition, the mechanical force of compression appears to activate NMDA receptors to initiate a rapid postsynaptic molecular cascade to trim dendritic spines on the compressed cortical pyramidal neurons within half a day.

Exogenous dehydroisoandrosterone sulfate reverses the dendritic changes of the central neurons in aging male rats.

Sex hormones are known to help maintaining the cognitive ability in male and female rats. Hypogonadism results in the reduction of the dendritic spines of central neurons which is believed to undermine memory and cognition and cause fatigue and poor concentration. In our previous studies, we have reported age-related regression in dendrite arbors along with loss of dendritic spines in the primary somatosensory cortical neurons in female rats. Furthermore, castration caused a reduction of dendritic spines in adult male rats. In light of this, it was surmised that dendritic structures might change in normal aging male rats with advancing age. Recently, dehydroepiandrosterone sulfate (DHEAS) has been reported to have memory-enhancing properties in aged rodents. In this study, normal aging male rats, with a reduced plasma testosterone level of 75-80%, were used to explore the changes in behavioral performance of neuronal dendritic arbor and spine density. Aging rats performed poorer in spatial learning memory (Morris water maze). Concomitantly, these rats showed regressed dendritic arbors and spine loss on the primary somatosensory cortical and hippocampal CA1 pyramidal neurons. Exogenous DHEAS and testosterone treatment reversed the behavioral deficits and partially restored the spine loss of cortical neurons in aging male rats but had no effects on the dendritic arbor shrinkage of the affected neurons. It is concluded therefore that DHEAS, has the efficacy as testosterone, and that it can exert its effects on the central neuron level to effectively ameliorate aging symptoms.

Genistein partly eases aging and estropause-induced primary cortical neuronal changes in rats.

Gonadal hormones can modulate brain morphology and behavior. Recent studies have shown that hypogonadism could result in cortical function deficits. To this end, hormone therapy has been used to ease associated symptoms but the risk may outweigh the benefits. Here we explored whether genistein, a phytoestrogen, is effective in restoring the cognitive and central neuronal changes in late middle age and surgically estropause female rats. Both animal groups showed poorer spatial learning than young adults. The dendritic arbors and spines of the somatosensory cortical and CA1 hippocampal pyramidal neurons were revealed with intracellular dye injection and analyzed. The results showed that dendritic spines on these neurons were significantly decreased. Remarkably, genistein treatment rescued spatial learning deficits and restored the spine density on all neurons in the surgically estropause young females. In late middle age females, genistein was as effective as estradiol in restoring spines; however, the recovery was less thorough than on young OHE rats. Neither genistein nor estradiol rectified the shortened dendritic arbors of the aging cortical pyramidal neurons suggesting that dendritic arbors and spines are differently modulated. Thus, genistein could work at central level to restore excitatory connectivity and appears to be potent alternative to estradiol for easing aging and menopausal syndromes.

Morphological changes of cortical pyramidal neurons in hepatic encephalopathy.

BACKGROUND: Hepatic encephalopathy (HE) is a reversible neuropsychiatric syndrome associated with acute and chronic liver diseases. It includes a number of neuropsychiatric disturbances including impaired motor activity and coordination, intellectual and cognitive function. RESULTS: In the present study, we used a chronic rat HE model by ligation of the bile duct (BDL) for 4 weeks. These rats showed increased plasma ammonia level, bile duct hyperplasia and impaired spatial learning memory and motor coordination when tested with Rota-rod and Morris water maze tests, respectively. By immunohistochemistry, the cerebral cortex showed swelling of astrocytes and microglia activation. To gain a better understanding of the effect of HE on the brain, the dendritic arbors of layer V cortical pyramidal neurons and hippocampal CA1 pyramidal neurons were revealed by an intracellular dye injection combined with a 3-dimensional reconstruction. Although the dendritic arbors remained unaltered, the dendritic spine density on these neurons was significantly reduced. It was suggested that the reduction of dendritic spines may be the underlying cause for increased motor evoked potential threshold and prolonged central motor conduction time in clinical finding in cirrhosis. CONCLUSIONS: We found that HE perturbs CNS functions by altering the dendritic morphology of cortical and hippocampal pyramidal neurons, which may be the underlying cause for the motor and intellectual impairments associated with HE patients.

Methylcobalamin facilitates collateral sprouting of donor axons and innervation of recipient muscle in end-to-side neurorrhaphy in rats.

Using ulnar nerve as donor and musculocutaneous nerve as recipient we found earlier that end-to-side neurorrhaphy resulted in weak functional reinnervation after lengthy survival. End-to-side neurorrhaphy however is the sole choice of nerve repair at times and has the advantage of conserving donor nerve function. Here, we investigated whether myelination-enhancing agent methylcobalamin and motoneuron trophic factor pleiotrophin enhances the recovery after end-to-side neurorrhaphy. Methylcobalamin significantly increased the expression of growth associated protein 43 and S100 protein and ßIII tubulin in musculocutaneous nerve 1 month after neurorrhaphy suggesting the ingrowth of ulnar axonal sprouts in reactive Schwann cell environment. Upper limb functional test, compound muscle action potential measurements, motor end plate counts, and axon and myelin analyses showed that methylcobalamin treatment alone or with pleiotrophin improved the recovery significantly, 3 and 6 months post-surgery. There were fewer axons, closer in number to that of the intact recipient nerve, found in the distal repaired nerve of the methylcobalamin-treated than that of the vehicle control, suggesting that methylcobalamin facilitates axonal maturation and eliminates supernumerary sprouts. In conclusion, our results showed that methylcobalamin does indeed enhance the recovery of peripheral nerve repaired in end-to-side configuration.

Testosterone modulation of dendritic spines of somatosensory cortical pyramidal neurons.

Brain structures and functions are increasingly recognized to be directly affected by gonadal hormones, which classically determine reproductive functions and sexual phenotypes. In this regard, we found recently that ovariectomy trimmed the dendritic spines of female rat primary somatosensory cortical neurons and estradiol supplement reversed it. Here, we investigated whether in the male androgen also has a cortical modulatory effect. The dendritic arbors and spines of rat somatosensory cortical pyramidal neurons were studied following intracellular dye injection and three-dimensional reconstruction. Dendritic spines, but not length, of the layers III and V pyramidal neurons were found reduced at 2 weeks and rebounded slightly at 4 weeks and further at 8 and 24 weeks following castration, which, however, remained significantly fewer than those of the intact animals. Two weeks of osmotic pump-delivered testosterone treatment to animals castrated for 4 weeks replenished serum testosterone and reversed the densities of dendritic spines on these neurons to control animal levels. Androgen receptor appears to mediate this effect as its antagonist flutamide reduced the dendritic spines of normal adult rats while causing a mild feedback surge of serum testosterone. On the other hand, blocking the conversion of testosterone to estrogen with the aromatase inhibitor anastrozole failed to alter the dendritic spine densities in male adult rats. In conclusion, these results support our hypothesis that testosterone acts directly on the androgen receptor in males to modulate the dendritic spines of somatosensory cortical output neurons.

Active endocytosis and microtubule remodeling restore compressed pyramidal neuron morphology in rat cerebral cortex.

Previous studies have shown that compression alone reduced the thickness of rat cerebral cortex and apical dendritic lengths of pyramidal neurons without apparent cell death. Besides, decompression restored dendritic lengths at different degrees depending on duration of compression. To understand the mechanisms regulating dendritic shortening and lengthening upon compression and decompression, we applied transmission electron microscopy to examine microtubule and membrane structure of pyramidal neurons in rat sensorimotor cortex subjected to compression and decompression. Microtubule densities within apical dendritic trunks decreased significantly and arranged irregularly following compression for a period from 30 min to 24 h. In addition, apical dendritic trunks showed twisted contour. Two reasons are accounted for the decrease of microtubule density within this period. First, microtubule depolymerized and resulted in lower number of microtubules. Second, the twisted membrane widened the diameters of apical dendritic trunks, which also caused a decrease in microtubule density. Interestingly, these compression-induced changes were quickly reversed to control level following decompression, suggesting that these changes were accomplished passively. Furthermore, microtubule densities were restored to control level and the number of endocytotic vesicles significantly increased along the apical dendritic membrane in neurons subjected to 36 h or longer period of compression. However, decompression did not make significant changes on dendrites compressed for 36 h, for they had already shown straight appearance before decompression. These results suggest that active membrane endocytosis and microtubule remodeling occur in this adaptive stage to make the apical dendritic trunks regain their smooth contour and regular microtubule arrangement, similar to that of the normal control neurons.

Ascorbic acid and α-tocopherol supplement starting prenatally enhances the resistance of nucleus tractus solitarius neurons to hypobaric hypoxic challenge.

Hypobaric hypoxia, encountered at high altitude, could result in severe consequences. Ascorbic acid (AA) and α-tocopherol (αTC), the two readily available over-the-counter antioxidants, are known to protect nervous tissue against oxidative stress. Here we study whether AA or αTC supplement starting prenatally protects animals against hypobaric hypoxic challenge at adulthood. Expressions of c-fos and the NR1 subunit of the N-methyl-D-aspartate receptors in the nucleus tractus solitarius (NTS) subserving cardiorespiratory functions were investigated. AA and αTC supplement reduced the number of c-fos immunoreactive neurons and intensity of NR1 expression in young and adult animals under normoxia. The treatment, in addition, attenuated the activation of NTS neurons, in terms of c-fos and NR1 expressions, and reduced the anxiety behaviors of adult rats subjected to hypobaric hypoxic challenge. Reduction of c-fos immunoreactive neurons was found concentrated in the chemoreceptor, baroreceptor, and tracheobronchial tree NTS subnuclei that receive corresponding afferents. The protective effect was not found in normal adult animals supplemented with AA or αTC a week before hypobaric hypoxic challenge. In short, prenatal and sustained AA or αTC supplement altered NTS substrate and ameliorated animals' reactions to hypobaric hypoxic insult, suggesting that this may be considered to protect animals from hypoxic insults from young to adult.

Compression alters kinase and phosphatase activity and tau and MAP2 phosphorylation transiently while inducing the fast adaptive dendritic remodeling of underlying cortical neurons.

In traumatic brain injury (TBI) there is often compression of the cerebral cortex. Using a rat epidural bead implantation model we found that mechanical compression distorted the dendrites of underlying cortical pyramidal neurons, and that the deformed dendrites regained straight morphology in 3 days. This was accompanied by a transient increase in the phosphorylation of microtubule-associated proteins (MAPs) at sites known to destabilize microtubules, including MAP2 from 30 min to 1 h, and tau from 10 min to 12 h following compression. Immunostaining confirmed that phosphorylated MAPs were concentrated at the somata and dendrites of compressed cortical pyramidal neurons. Enzymes regulating MAP phosphorylation were found to be simultaneously altered, including downregulation of protein phosphatase 2A, but not 2B, activity from 10 min to 1 day, and transient excitatory phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38/mitogen-activated protein kinase. The temporal coincidence of these events suggests that alterations of phosphatase and kinase activity underlie MAP2 and tau phosphorylation, thus causing the compressed cortical neurons to remodel their dendrites, including the proximal segments. The rapid onset of these molecular changes demonstrates that compression causes cortical neurons to undergo active changes much early than expected. The large-scale structural changes that result can alter cortical function for an extended period of time.

The efficacy of end-to-end and end-to-side nerve repair (neurorrhaphy) in the rat brachial plexus.

Proximal nerve injury often requires nerve transfer to restore function. Here we evaluated the efficacy of end-to-end and end-to-side neurorrhaphy of rat musculocutaneous nerve, the recipient, to ulnar nerve, the donor. The donor was transected for end-to-end, while an epineurial window was exposed for end-to-side neurorrhaphy. Retrograde tracing showed that 70% donor motor and sensory neurons grew into the recipient 3 months following end-to-end neurorrhaphy compared to 40-50% at 6 months following end-to-side neurorrhaphy. In end-to-end neurorrhaphy, regenerating axons appeared as thick fibers which regained diameters comparable to those of controls in 3-4 months. However, end-to-side neurorrhaphy induced slow sprouting fibers of mostly thin collaterals that barely approached control diameters by 6 months. The motor end plates regained their control density at 4 months following end-to-end but remained low 6 months following end-to-side neurorrhaphy. The short-latency compound muscle action potential, typical of that of control, was readily restored following end-to-end neurorrhaphy. End-to-side neurorrhaphy had low amplitude and wide-ranging latency at 4 months and failed to regain control sizes by 6 months. Grooming test recovered successfully at 3 and 6 months following end-to-end and end-to-side neurorrhaphy, respectively, suggesting that powerful muscle was not required. In short, both neurorrhaphies resulted in functional recovery but end-to-end neurorrhaphy was quicker and better, albeit at the expense of donor function. End-to-side neurorrhaphy supplemented with factors to overcome the slow collateral sprouting and weak motor recovery may warrant further exploration.

Gonadal hormones modulate the dendritic spine densities of primary cortical pyramidal neurons in adult female rat.

Adult dendritic arbors and spines can be modulated by environment and gonadal hormones that have been reported to affect also those of hippocampal and prefrontal cortical neurons. Here we investigated whether female gonadal hormones and estrous cycle alter the dendrites of primary cortical neurons. We employed intracellular dye injection in semifixed brain slices and 3-dimensional reconstruction to study the dendritic arbors and spines of the major cortical output cells, layer III and V pyramidal neurons, during different stages of the estrous cycle. Dendritic spines of both pyramidal neurons were more numerous during proestrus than estrus and diestrus, whereas dendritic arbors remained unaffected. Ovariohysterectomy (OHE) reduced dendritic spines by 24-30% in 2 weeks, whereas subcutaneous estrogen or progesterone supplement restored it to normal estrous/diestrous level in 14 days; neither treatment affected the dendritic arbors. Reduction of dendritic spines following OHE was associated with decrease of PSD-95 suggesting decrease of excitatory synapses. Thus, fluctuation of gonadal hormones during the female sex cycle is likely to modulate primary cortical functions and loss of gonadal hormones for instance following menopause might compromise cortical function, and the effect could be reversed by exogenous female sex hormones.

Proximity of lesioning determines response of facial motoneurons to peripheral axotomy.

We recently found that rubrospinal (RS) neurons, which typify central neurons projecting within the central nervous system (CNS), exhibited different neuronal and glial reactions to axotomy at proximal as opposed to distal sites. To determine whether distance also determines the reaction to axonal injury of central neurons projecting to the periphery, we studied the temporal expression of four free-radical-related enzymes as well as the severity of cell loss, perineuronal astrocytic and microglial reactions, and degeneration of the proximal central axons of facial motoneurons after axotomies performed at various sites on the brainstem surface and in the stylomastoid foramen, respectively. Distal lesions resulted in upregulation of these neurons' expression of nitric oxide synthase (NOS) and persistent downregulation of their expression of the NOS-activating enzyme calcineurin. It also led to transient upregulation of their expression of manganese-dependent superoxide dismutase (Mn-SOD), and resulted in a mild neuronal loss. Proximal axotomy led to an upregulation of NOS but a transient downregulation in the expression of calcineurin and Mn-SOD at 4 weeks after injury. This was accompanied by severe cell loss and swelling of mitochondria at 2-4 weeks postinjury. However, neither proximal nor distal axonal lesioning led to nuclear fragmentation or TUNEL staining of neurons. Proximal as opposed to distal axotomy produced an earlier transformation of glial morphology, including the hypertrophy of astrocytic processes and metamorphosis of ramified microglia to amoeboid cells. We unexpectedly found that unlike RS neurons, whose central axons degenerated slowly and in an anterograde manner only after the severe cell loss induced by proximal axotomy, the central axons of facial motoneurons degenerated rapidly and in a retrograde manner independently of the severity of loss of these neurons after axotomy. However, degeneration began sooner after proximal than after distal axotomy. Since the central axons of both rubrospinal neurons and facial motoneurons lie within the CNS, the differences in whether and how they degenerated after axotomy suggests that central neurons that project within and outside the CNS are inherently different. The significance of these and also the free radical environment regulation differences between these two types of neurons following close and distant axotomies remains to be explored.

Neonatal toluene exposure alters N-methyl-D-aspartate receptor subunit expression in the hippocampus and cerebellum in juvenile rats.

Recent evidence indicates that toluene is a non-competitive inhibitor of N-methyl-d-aspartate (NMDA) receptor-mediated synaptic currents. The NMDA receptor plays a major role in neuronal development and differentiation. The present study characterized the long-term effects of toluene exposure during synaptogenesis on the expression of NMDA receptor subunits (NR1, NR2A and NR2B). Neonatal rats were administered toluene (500 mg/kg, ip) daily over postnatal days (PN) 4-9. The expression of NMDA receptor subunits in rat brain was measured on PN 30. Western blot analysis demonstrated that toluene exposure significantly increased NR2A expression in the hippocampus and cerebellum. Immunohistochemical results indicated that the increased NR2A expression is mainly in hippocampal CA1-stratum oriens, CA1-stratum radiatum, CA1-lacunosm molecular, CA2- stratum oriens, and dentate gyrus-molecular layer and the cerebellar Purkinje cell layer, respectively. In contrast, the levels of NR2B in the toluene-exposed rats were decreased in the molecular layer. These results suggest that the region-specific changes in the expression of NMDA receptor subunits may be related to the neurobehavioral dysfunction following toluene exposure during synaptogenesis.

Spinal axonal injury transiently elevates the level of metabotropic glutamate receptor 5, but not 1, in cord-projection central neurons.

In investigating the effect of spinal injury on cord-projection central neurons, we found that rat rubrospinal neurons retained glutamatergic afferents and, in general, ionotropic glutamate receptor expression following spinal axotomy. Since glutamate also acts on second-messenger-coupled metabotropic receptors, the expression of group I metabotropic glutamate receptors, mGluR1 and mGluR5, was examined following similar treatment. mGluR1 expression began to decline in the perikarya 2 days postlesion and a day later in the neuropil. The decline slowed down by the fifth day and recovered in both the perikarya and neuropil 1 week postlesion. However, expression in both the perikarya and neuropil declined again and persisted up to 2 years postlesion. Similarly, the mGluR5 displayed an early transient decrease and returned to normal levels by 7 days post-lesion. However, rather than progressing to a secondary decline, the expression of mGluR5 increased to levels dramatically higher than those of control nuclei at 2-4 weeks postlesion, subsiding again by 8 weeks, and remaining low up to 2 years postinjury. Although mGluR5 has been shown to save cultured neurons from excitotoxic cell death, its elevated expression in the present model corresponds in time to an increased input/output relationship and excitability of the injured neurons as well as a period of maximal somatic shrinkage and cell loss. In addition to the cell bodies and dendrites, axon-like profiles also contain mGluR1. Their decrease following rubrospinal axotomy suggests that axonal injury may also compromise the presynaptic regulation of afferent activities onto injured cord-projection central neurons.

Spinal axonal injury induces brief downregulation of ionotropic glutamate receptors and no stripping of synapses in cord-projection central neurons.

Spinal cord injury often damages the axons of cord-projecting central neurons. To determine whether their excitatory inputs are altered following axonal injury, we used rat rubrospinal neurons as a model and examined their excitatory input following upper cervical axotomy. Anterograde tracing showed that the primary afferents from the cerebellum terminated in a pattern similar to that of control animals. Ultrastructurally, neurons in the injured nucleus were contacted by excitatory synapses of normal appearance, with no sign of glial stripping. Since cerebellar fibers are glutamatergic, we examined the expression of ionotropic receptor subunits GluR1-4 and NR1 for AMPA and NMDA receptors, respectively, in control and injured neurons using immunolabeling methods. In control neurons, GluR2 appeared to be low as compared to GluR1, GluR3, and GluR4, while NR1 labeling was intense. Following unilateral tractotomy, the levels of expression of each subunit in axotomized neurons appeared to be normal, with the exception that they were lower than those of control neurons of the nonlesioned side at 2-6 days postinjury. These findings suggest that axotomized neurons are only temporarily protected from excitotoxicity. This is in sharp contrast to the responses of central neurons that innervate peripheral targets, in which both synaptic stripping and reduction of their ionotropic glutamate receptor subunits persist following axotomy. The absence of an injury-induced trimming of afferents and stripping of synapses and the lack of a persistent downregulation of postsynaptic receptors might enable injured cord-projection neurons to continue to control their supraspinal targets during most of their postinjury survival. Although this may support neurons by providing trophic influences, it nevertheless may subject them to excitotoxicity and ultimately lead to their degenerative fate.