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Axonal regeneration after injury to the CNS is hampered by myelin-derived inhibitors, such as Nogo-A. Natural products, such as green tea, which are neuroprotective and safe for long-term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor-differentiated neuronal-like Neuroscreen-1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin-3-gallate (EGCG), prevent both the neurite outgrowth-inhibiting activity and growth cone-collapsing activity of Nogo-66 (C-terminal domain of Nogo-A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67-kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N-acetylcysteine and cell-permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H2 O2 in this process. Accordingly, exogenous sublethal concentrations of H2 O2 , added as a bolus dose (5 μM) or more effectively through a steady-state generation (1-2 μM), mimicked GTPP in counteracting the action of Nogo-66. Exogenous H2 O2 mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H2 O2 , inhibit the antineuritogenic action of Nogo-A. Currently, several agents are being evaluated for overcoming axonal growth inhibitors to promote functional recovery after stroke and spinal cord injury. Epigallocatechin-3-gallate (EGCG), present in green tea polyphenol mixture (GTPP), prevents antineuritogenic activity of Nogo-A, a myelin-derived axonal growth inhibitor. The preventive action of EGCG involves the cell-surface-associated 67-kDa laminin receptor and H2 O2 . GTPP may complement ongoing efforts to treat neuronal injuries.>, (© 2014 International Society for Neurochemistry.)

Delivery of optimal amounts of brain-derived neurotrophic factor (BDNF) to regions of the brain affected by neurodegenerative diseases is a daunting task. Using natural products with neuroprotective properties, such as green tea polyphenols, would be a highly useful complementary approach for inexpensive long-term treatment of these diseases. In this study, we used PC12(TrkB) cells which ectopically express TrkB, a high affinity receptor for BDNF. They differentiate and induce neurite outgrowth in response to BDNF. Using this model, we show for the first time that treatment with extremely low concentrations (<0.1 μg/ml) of unfractionated green tea polyphenols (GTPP) and low concentrations (<0.5 μM) of their active ingredient, epigallocatechin-3-gallate (EGCG), potentiated the neuritogenic ability of a low concentration (2 ng/ml) of BDNF. A synergistic interaction was observed between GTPP constituents, where epigallocatechin and epicatechin, both individually lacking this activity, promoted the action of EGCG. GTPP-induced potentiation of BDNF action required the cell-surface associated 67 kDa laminin receptor (67LR) to which EGCG binds with high affinity. A cell-permeable catalase abolished GTPP/EGCG-induced potentiation of BDNF action, suggesting the possible involvement of H2O2 in the potentiation. Consistently, exogenous sublethal concentrations of H2O2, added as a bolus dose (5 μM) or more effectively through a steady-state generation (1 μM), potentiated BDNF action. Collectively, these results suggest that EGCG, dependent on 67 LR and H2O2, potentiates the neuritogenic action of BDNF. Intriguingly, this effect requires only submicromolar concentrations of EGCG. This is significant as extremely low concentrations of polyphenols are believed to reach the brain after drinking green tea., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The sensorimotor striatum is critical for the acquisition and consolidation of skilled learning-related motor sequences. Excitatory corticostriatal synapses undergo neuroplastic changes that impact signal transmission efficacy. Modification of N-methyl d-aspartate (NMDA) and α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit composition and phosphorylation is critical for bidirectional experience-driven plasticity observed at these synapses. Metaplastic regulation of the ratio of NR2A to NR2B subunits of the NMDA receptor controls the threshold for the induction of subsequent plasticity. However, little is known about how repeated practice effects the differential regulation of glutamate receptors during the acquisition of a unilateral motor skill. Using immunoblot analysis, we assessed changes in NMDA and AMPA receptors during the associative stage of skill acquisition in synaptoneurosome preparations from the rat sensorimotor striatum. We found that the NR2A/B subunit ratio in the striatum contralateral to the trained limb decreased during skill acquisition optimizing the threshold for inducing subsequent synaptic plasticity during learning of the lateralized motor skill. In contrast, there was a significant increase in the NR2A/B subunit ratio in the ipsilateral striatum making the induction of subsequent plasticity more difficult. In addition, there was a selective decrease in AMPAR phosphorylation levels at serine site 831 but not 845 on the GluR1 subunit ipsilaterally with a trend toward a decrease contralaterally. These findings suggest that the successful acquisition of a lateralized motor skill necessitates the integration of motor programs in both striata, each of which reflects unique changes in the NR2A/B ratio that modulate the different task demands on the associated limb., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The protein kinase C (PKC) family of isoenzymes may be a crucial player in transducing H2O2-induced signaling in a wide variety of physiological and pathophysiological processes. PKCs contain unique structural features that make them highly susceptible to oxidative modification. Depending on the site of oxidation and the extent to which it is modified, PKC can be either activated or inactivated by H2O2. The N-terminal regulatory domain contains zinc-binding, cysteine-rich motifs that are readily oxidized by H2O2. When oxidized, the autoinhibitory function of the regulatory domain is compromised, and as a result, PKC is activated in a lipid cofactor-independent manner. The C-terminal catalytic domain contains several reactive cysteine residues, which when oxidized with a higher concentration of H2O2 leads to an inactivation of PKC. Here, we describe the methods used to induce oxidative modification of purified PKC isoenzymes by H2O2 and the methods to assess the extent of this modification. Protocols are given for isolating oxidatively activated PKC isoenzymes from cells treated with H2O2. Furthermore, we describe the methods used to assess indirect regulation of PKC isoenzymes by determining their cytosol to membrane or mitochondrial translocation and tyrosine phosphorylation of PKCδ in response to sublethal levels of H2O2. Finally, as an example, we describe the methods used to demonstrate the role of H2O2-mediated cell signaling of PKCɛ in green tea polyphenol-induced preconditioning against neuronal cell death caused by oxygen-glucose deprivation and reoxygenation, an in vitro model for cerebral ischemic/reperfusion injury., (Copyright © 2013 Elsevier Inc. All rights reserved.)

As the development of synthetic drugs for the prevention of stroke has proven challenging, utilization of natural products capable of preconditioning neuronal cells against ischemia-induced cell death would be a highly useful complementary approach. In this study using an oxygen-glucose deprivation and reoxygenation (OGD/R) model in PC12 cells, we show that 2-day pretreatment with green tea polyphenols (GTPP) and their active ingredient, epigallocatechin-3-gallate (EGCG), protects cells from subsequent OGD/R-induced cell death. A synergistic interaction was observed between GTPP constituents, with unfractionated GTPP more potently preconditioning cells than EGCG. GTPP-induced preconditioning required the 67-kDa laminin receptor (67LR), to which EGCG binds with high affinity. 67LR also mediated the generation of reactive oxygen species (ROS) via activation of NADPH oxidase. An exogenous ROS-generating system bypassed 67LR to induce preconditioning, suggesting that sublethal levels of ROS are indeed an important mediator in GTPP-induced preconditioning. This role for ROS was further supported by the fact that antioxidants blocked GTPP-induced preconditioning. Additionally, ROS induced an activation and translocation of protein kinase C (PKC), particularly PKCε from the cytosol to the membrane/mitochondria, which was also blocked by antioxidants. The crucial role of PKC in GTPP-induced preconditioning was supported by use of its specific inhibitors. Preconditioning was increased by conditional overexpression of PKCε and decreased by its knock-out with siRNA. Collectively, these results suggest that GTPP stimulates 67LR and thereby induces NADPH oxidase-dependent generation of ROS, which in turn induces activation of PKC, particularly prosurvival isoenzyme PKCε, resulting in preconditioning against cell death induced by OGD/R.

Exogenously administered nerve growth factor (NGF) repairs injured axons, but it does not cross the blood-brain barrier. Thus, agents that could potentiate the neuritogenic ability of endogenous NGF would be of great utility in treating neurological injuries. Using the PC12 cell model, we show here that unfractionated green tea polyphenols (GTPP) at low concentrations (0.1 μg/ml) potentiate the ability of low concentrations of NGF (2 ng/ml) to induce neuritogenesis at a level comparable to that induced by optimally high concentrations of NGF (50 ng/ml) alone. In our experiments, GTPP by itself did not induce neuritogenesis or increase immunofluorescent staining for β-tubulin III; however, it increased expression of mRNA and proteins for the neuronal markers neurofilament-L and GAP-43. Among the polyphenols present in GTPP, epigallocatechin-3-gallate (EGCG) alone appreciably potentiated NGF-induced neurite outgrowth. Although other polyphenols present in GTPP, particularly epigallocatechin and epicatechin, lack this activity, they synergistically promoted this action of EGCG. GTPP also induced an activation of extracellular signal-regulated kinases (ERKs). PD98059, an inhibitor of the ERK pathway, blocked the expression of GAP-43. K252a, an inhibitor of TrkA-associated tyrosine kinase, partially blocked the expression of these genes and ERK activation. Antioxidants, catalase (cell-permeable form), and N-acetylcysteine (both L and D-forms) inhibited these events and abolished the GTPP potentiation of NGF-induced neuritogenesis. Taken together, these results show for the first time that GTPP potentiates NGF-induced neuritogenesis, likely through the involvement of sublethal levels of reactive oxygen species, and suggest that unfractionated GTPP is more effective in this respect than its fractionated polyphenols., (Copyright © 2010 Wiley-Liss, Inc.)

Inbred strains of mice differ in their susceptibility to excitotoxin-induced cell death, but the genetic basis of individual variation in differential susceptibility is unknown. Previously, we identified a highly significant quantitative trait locus (QTL) on chromosome 18 that influenced susceptibility to kainic acid-induced cell death (Sicd1). Comparison of susceptibility to seizure-induced cell death between reciprocal congenic lines for Sicd1 and parental background mice indicates that genes influencing this trait were captured in both strains. Two positional gene candidates, Galr1 and Mbp, map to 55 cM, where the Sicd1 QTL had been previously mapped. Thus, this study was undertaken to determine if Galr1 and/or Mbp could be considered as candidate genes. Genomic sequence comparison of these two functional candidate genes from the C57BL/6J (resistant at Sicd1) and the FVB/NJ (susceptible at Sicd1) strains showed no single-nucleotide polymorphisms. However, expression studies confirmed that Galr1 shows significant differential expression in the congenic and parental inbred strains. Galr1 expression was downregulated in the hippocampus of C57BL/6J mice and FVB.B6-Sicd1 congenic mice when compared with FVB/NJ or B6.FVB-Sicd1 congenic mice. A survey of Galr1 expression among other inbred strains showed a significant effect such that 'susceptible' strains showed a reduction in Galr1 expression as compared with 'resistant' strains. In contrast, no differences in Mbp expression were observed. In summary, these results suggest that differential expression of Galr1 may contribute to the differences in susceptibility to seizure-induced cell death between cell death-resistant and cell death-susceptible strains.

In this study, we have used the PC12 cell model to elucidate the mechanisms by which sublethal doses of oxidants induce neuritogenesis. The xanthine/xanthine oxidase (X/XO) system was used for the steady state generation of superoxide, and CoCl(2) was used as a representative transition metal redox catalyst. Upon treatment of purified protein kinase C (PKC) with these oxidants, there was an increase in its cofactor-independent activation. Redox-active cobalt competed with the redoxinert zinc present in the zinc-thiolates of the PKC regulatory domain and induced the oxidation of these cysteine-rich regions. Both CoCl(2) and X/XO induced neurite outgrowth in PC12 cells, as determined by an overexpression of neuronal marker genes. Furthermore, these oxidants induced a translocation of PKC from cytosol to membrane and subsequent conversion of PKC to a cofactor-independent form. Isoenzyme-specific PKC inhibitors demonstrated that PKCepsilon plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKCepsilon and decreased with its knock-out by small interfering RNA. Parallel with PKC activation, an increase in phosphorylation of the growth-associated neuronal protein GAP-43 at Ser(41) was observed. Additionally, there was a sustained activation of extracellular signal-regulated kinases 1 and 2, which was correlated with activating phosphorylation (Ser(133)) of cAMP-responsive element-binding protein. All of these signaling events that are causally linked to neuritogenesis were blocked by antioxidant N-acetylcysteine (both L and D-forms) and by a variety of PKC-specific inhibitors. Taken together, these results strongly suggest that sublethal doses of oxidants induce neuritogenesis via a direct redox activation of PKCepsilon.

Motor cortex lesions in rats partially denervate the striatum, producing behavioral deficits and inducing reactive neuroplasticity. Plastic responses include changes in growth-associated protein marker expression and anatomical restructuring. Corticostriatal plasticity is dependent on dopamine at the striatal target, where D1 receptor signaling reinforces behaviorally relevant neural activity. To determine whether striatal dopamine D1 receptor signaling is important for the growth-associated protein responses and behavioral recovery that follow unilateral motor cortex aspiration, the dopamine D1 receptor antagonist SCH23390 was intrastriatally infused in cortically lesioned animals. After a cortical aspiration lesion in Long Evans rats, the growth-associated proteins SCG10 and GAP-43 were upregulated in the cortex contralateral to the lesion at 30 days post-lesion. However, continuous unilateral intrastriatal infusion of SCH23390 prevented this aspiration-induced upregulation. Furthermore, lesioned rats demonstrated spontaneous sensorimotor improvement, in terms of limb-use symmetry, about 1 month post-lesion. This improvement was prevented with chronic intrastriatal SCH23390 infusion. The D1 receptor influence may be important to normalize corticostriatal activity (and observable behavior), either in a long-term manner or temporarily until other more permanent means of synaptic regulation, such as sprouting or synaptogenesis, may be implemented.

Investigation of the elements underlying synapse replacement after brain injury is essential for predicting the neural compensation that can be achieved after various types of damage. The growth-associated proteins superior cervical ganglion-10 and growth-associated protein-43 have previously been linked with structural changes in the corticostriatal system in response to unilateral deafferentation. To examine the regulation of this response, unilateral cortical aspiration lesion was carried out in combination with ipsilateral 6-hydroxydopamine lesion of the substantia nigra, and the time course of the contralateral cortical molecular response was followed. Unilateral cortical aspiration lesion in rats corresponds with an upregulation of superior cervical ganglion-10 mRNA at 3 and 10 days post-lesion, and protein, sustained from three to at least 27 days following lesion. With the addition of substantia nigra lesion, the response shifts to an upregulation of growth-associated protein-43 mRNA at 3 and 10 days post-lesion, and protein after 10 days. Nigral lesion alone does not alter contralateral expression of either gene. Likewise, motor function assessment using the rotorod test revealed no significant long-term deficits in animals that sustained only nigrostriatal damage, but cortical lesion was associated with a temporary deficit which was sustained when nigrostriatal input was also removed. Growth-associated protein-43 and superior cervical ganglion-10, two presynaptic genes that are postulated to play roles in lesion-induced sprouting, are differentially upregulated in corticostriatal neurons after cortical versus combined cortical/nigral lesions. The shift in contralateral gene response from superior cervical ganglion-10 to growth-associated protein-43 upregulation and associated behavioral deficit following combined cortical and nigral denervation suggest that nigrostriatal afferents regulate cortical lesion-induced gene expression and ultimate functional outcome.

Defining the selective pattern of synapse replacement that occurs in different areas of the damaged brain is essential for predicting the limits of functional compensation that can be achieved after various types of brain injury. Here we describe the time course of dendritic reorganization, spine loss and recovery, and synapse replacement in the striatum following a unilateral cortex ablation. We found that the time course for the transient loss and recovery of dendritic spines on medium spiny I (MSI) neurons, the primary postsynaptic target for corticostriatal axons, paralleled the time course for the removal of degenerating axon terminals from the neuropil and the formation of new synapses on MSI neurons. Reinnervation of the deafferented striatum occurred chiefly by axon terminals that formed asymmetric synapses with dendritic spines of MSI neurons, and the mean density of asymmetric synapses recovered to 86% of the sham-operated rat value by 30 days postlesion. In addition, the synaptic circuitry of the reconstructed striatum was characterized by an increase in the number of multiple synaptic boutons (MSBs), i.e., presynaptic axon terminals that make contact with more than one dendritic spine. Whether the postsynaptic contacts of MSBs are formed with the dendritic spines of the same or a different parent dendrite in the striatum is unknown. Nevertheless, these data suggest that the formation of MSBs is an essential part of the compensatory response to the loss of input from the ipsilateral cortex following the aspiration lesion and may serve to modulate activity-dependent adaptive changes in the reconstructed striatum that can lead to functional recovery., (Copyright 2003 Wiley-Liss, Inc.)

Ultrastructural changes within the ipsilateral dorsolateral striatum were investigated 1 month following a unilateral ablation of the rat frontal cortex (CTX), removing corticostriatal input, or injection of the neurotoxin, 6-hydroxydopamine (6-OHDA), into the substantia nigra pars compacta, removing nigrostriatal input. In addition, a combined ipsilateral cortical and 6-OHDA lesion (CTX/6-OHDA) was carried out. We find that following a CTX, 6-OHDA, or CTX/6-OHDA lesion, there was a significant decrease in the density of striatal nerve terminal glutamate immunoreactivity compared to the control group. There was also a significant increase in all three lesion groups in the mean percentage of asymmetrical synapses associated with a perforated postsynaptic density. There was a large increase within the CTX/6-OHDA-lesioned group and a smaller but still significant increase in the CTX-lesioned group in the percentage of terminals or boutons with multiple synaptic contacts (i.e., multiple synaptic boutons, MSBs), compared to either the 6-OHDA or the control group. There was no change in any of these measurements within the contralateral striatum. There was a significant decrease in the number of apomorphine-induced contralateral rotations in the CTX/6-OHDA versus the 6-OHDA-lesioned group. Animals receiving just the single CTX or 6-OHDA lesion recovered in motor function compared to the control group as measured by the Rotorod test, while the CTX/6-ODA-lesioned group recovered to less than 50% of the control level. The data suggest that following a CTX and/or 6-OHDA lesion, there is an increase in striatal glutamatergic function. The large increase in the percentage of MSBs in the combined lesion group suggests that dopamine or other factors released by the dopamine terminals assist in regulating synapse formation., (Copyright 2000 Academic Press.)

Synapse replacement after brain injury has been widely documented by anatomical studies in various parts of both the developing and adult nervous system. However, the molecular events that define the specificity of the empirically derived rules of reactive synaptogenesis in different regions of the adult brain remain unclear. In this study we examined the differential regulation of the lesion-induced response of the two growth-associated proteins, superior cervical ganglia-10 and growth-associated protein-43, after unilateral cortex ablation, and determined a hierarchical order for the lesion response from remaining afferent projection neurons originating from the contralateral cortex, ipsilateral thalamus and substantia nigra. We report that in response to unilateral cortex ablation both messenger RNA, by northern blot, and protein, by western blot, for superior cervical ganglia-10 but not growth-associated protein-43 was increased in the homologous area of the contralateral cortex but not the ipsilateral thalamus or substantia nigra. In addition, the specificity of the superior cervical ganglia-10 response, assessed by combined in situ hybridization and retrograde FluoroGold labeling of striatal afferent neurons, found that superior cervical ganglia-10 messenger RNA was increased prominently in layer V pyramidal neurons of the contralateral corticostriatal pathway but was unchanged in afferent projection neurons from the thalamus and substantia nigra. Furthermore, the increase in both superior cervical ganglia-10 messenger RNA and protein seen at three days postlesion in contralateral corticostriatal neurons coincides in time with the initiation of neurite outgrowth in the deafferented striatum by contralateral corticostriatal axons described in our previous ultrastructural study. However, if cortical input to the striatum was removed bilaterally the lesion-induced response for superior cervical ganglia-10 messenger RNA shifted secondarily to thalamostriatal neurons in the ipsilateral thalamus. These data provide evidence that superior cervical ganglia-10 and growth-associated protein-43 are differentially regulated in neurons of the contralateral corticostriatal pathway in response to unilateral cortex ablation and suggests that superior cervical ganglia-10 plays a role in the regulation of neurite outgrowth in the adult striatum after brain injury. However, the specific role that superior cervical ganglia-10 may play in reactive synaptogenesis remains unclear. In addition, our data suggest that a hierarchical order exists for the reinnervation of deafferented striatal neurons after unilateral cortex ablation with preference given to homologous axons from the contralateral cortex.

Synaptic replacement in rat striatum following a unilateral cortical lesion was investigated using electron microscopy and the anterograde tracer, biotinylated dextrin amine (BDA). In the deafferented striatum evidence of axon sprouting and synapse replacement was seen at 20 days after the lesion and most newly-formed axon terminals were labeled with BDA injected previously into the contralateral cortex. In addition, BDA-labeled fibers from the contralateral cortex formed multiple asymmetric axospinous synapses with deafferented striatal neurons, a morphological feature rarely seen in unlesioned rats. These data suggest that in response to a unilateral cortex lesion axons from the contralateral cortex sprout and reinnervated the deafferented striatal neurons and that reinnervation by 'like' afferents maybe crucial for the establishment of functional recovery after the unilateral cortex lesion.

The VGF gene encodes a neuronal secretory-peptide precursor that is rapidly induced by neurotrophic growth factors and by depolarization in vitro. VGF expression in the animal peaks during critical periods in the developing peripheral and central nervous systems. To gain insight into the possible functions and regulation of VGF in vivo, we have used in situ hybridization to examine the regulation of VGF messenger RNA by experimental manipulations, and have found it to be regulated in the CNS by paradigms that affect electrical activity and by lesion. Inhibition of retinal electrical activity during the critical period of visual development rapidly repressed VGF messenger RNA in the dorsal lateral geniculate nucleus of the thalamus. In the adult, kainate-induced seizures transiently induced VGF messenger RNA in neurons of the dentate gyrus, hippocampus, and cerebral cortex within hours. Cortical lesion strongly induced VGF messenger RNA in ipsilateral cortex within hours, and strongly repressed expression in ipsilateral striatum. Ten days postlesion there was a delayed induction of VGF messenger RNA in a portion of deafferented striatum where compensatory cortical sprouting has been detected. Expression of the neuronal secretory-peptide precursor VGF is therefore modulated in vivo by monocular deprivation, seizure, and cortical lesion, paradigms which lead to neurotrophin induction, synaptic remodeling and axonal sprouting.

This study evaluates the time course of expression of three astrocytic mRNAs, glial fibrillary acidic protein (GFAP), apolipoprotein E (ApoE), and clusterin, in the rat striatum (ST) following a unilateral lesion of either the cortex (CX) or the substantia nigra (SN), using Northern blot and in situ hybridization analyses. We found that while there was a time-dependent increase in astrocytic GFAP mRNA in the deafferented ST following both the CX and the SN lesions, the time course of the response was different between the two lesion paradigms. Specifically, the increase in GFAP mRNA in striatal astrocytes after the SN lesion was rapid and transient returning to control levels by 10 days postlesion, while the response was long lasting and remained increased until at least 27 days after the CX lesion. In addition, the mRNA response for both ApoE and clusterin was differentially regulated in response to the two lesions. Specifically, both clusterin and ApoE mRNAs were rapidly increased in the ST following the CX lesion while both mRNAs remained unchanged following the SN lesion. Data from this study extend information derived from previous investigations on the multifunctional role of astrocytes in the response to brain injury. Specifically, our data support the notion that while the time course of the GFAP response in striatal astrocytes may vary between lesion paradigms, the upregulation of GFAP is part of a generalized response of reactive astrocytes to diverse brain injuries. By comparison, upregulation of the mRNAs for the lipoproteins clusterin and ApoE are lesion specific and may play a role in the transport of recycled myelin lipids from dying axons to actively growing axons and dendrites in reactive synaptogenesis.

Stathmin (p19) is developmentally regulated as a neural-enriched phosphoprotein associated with neurite outgrowth and synaptic formation during cell proliferation and differentiation, and remains highly abundant in adult rat brain. Whether stathmin is involved in injury-induced reactive synaptogenesis in adult rat was examined in this study. Following unilateral cortical lesion, a significant increase in stathmin mRNA expression was found in the cells of contralateral homotypic cortex and in the subventricular zone of the lateral ventricle. This increase coincided in time with the corticostriatal axon sprouting and synaptic remodeling previously found in denervated striatum. Our data suggest that stathmin plays an important role in regulation of reactive synaptogenesis in adult brain.

Golgi-Cox method and morphometric analyses were used to study the plasticity of striatal medium spiny I neurons in 6-month-old C57BL/6N mice after unilateral or bilateral lesion of the cerebral cortex or combined lesions of the ipsilateral cerebral cortex and intralaminar thalamus. In adult mouse, unilateral lesions of the cerebral cortex did not result in a net gain or loss of linear dendritic length in a randomly selected population of striatal medium spiny I neurons. In addition, there was a well-defined time course of striatal spine loss and replacement occurring after a unilateral cortical lesion. By day 3 postlesion the average 20-microm dendritic segment had lost 30% of the unlesioned control spine value, reached its nadir, lost 45.5%, at 10 days postlesion, and recovered to 80% of unlesioned control levels by 20 days postlesion. The recovery of spines was blocked by a secondary lesion on the contralateral cortex but not on the ipsilateral intralaminar thalamus. These data suggest that striatal medium spiny I neurons of adult mice have a remarkable capacity for plasticity and reactive synaptogenesis following a decortication. The recovery of spine density is primarily induced by axonal sprouting of survival homologous afferent fibers from the contralateral cortex.

Aging disrupts the expression of synaptic plasticity in many central nervous system (CNS) structures including the striatum. We found age differences in paired-pulse plasticity to persist at excitatory striatal synapses following block of gamma aminobutyric acid (GABA)A and GABA(B) receptors, a property that was independent of the number of afferents activated. High Mg2+/low Ca2+ artificial cerebral spinal fluid (ACSF) reduced release probability and consequently the size of the evoked excitatory post-synaptic potential (EPSP). High Mg2+/low Ca2+ ACSF also increased the expression of paired-pulse facilitation and eliminated the age difference seen previously in normal ACSF. These data suggest that age differences in paired-pulse plasticity reflect an alteration in release probability at excitatory striatal synapses. In support of this hypothesis, we found age differences in another presynaptic form of plasticity referred to as synaptic augmentation. Examination of the synaptic depression that developed during the conditioning tetanus also revealed an age-related increase in synaptic depression. These data indicate that age-related changes in facilitation may be due in part to a reduction in the readily releasable pool of synaptic vesicles. Dendritic structure (spine density and dendritic length) was correlated with short-term synaptic plasticity, but these relationships depended upon the variance associated with age (hierarchical regression). Post-hoc within-age group regressions demonstrated relationship between spine density and paired-pulse plasticity. No other age-specific correlations were found. These findings imply an age-dependent association between altered dendritic morphology and changes in synaptic plasticity.

This study analyzed the effects of acute systemic treatment with buthionine sulfoximine (BSO), a synthesis inhibitor of the antioxidant reduced glutathione (GSH), on dopaminergic neurons of the murine nigrostriatal pathway. Part 1 of the study established a dose-response curve and the temporal pattern of GSH loss and recovery in the substantia nigra and striatum following acute BSO treatment. Part 2 of the study determined the effect of acute BSO treatment on the morphology and biochemistry of nigrostriatal neurons. We found that decreases in GSH levels had profound morphological effects, including decreased catecholamine fluorescence per cell, increased levels of lipid peroxidation and lipofuscin accumulation, and increased numbers of dystrophic axons in dopaminergic neurons of the nigrostriatal pathway. However, no measurable effects were observed in biochemical levels of either dopamine or its metabolites. These changes mimic those that have been reported to occur in the nigrostriatal system of rodents with advancing age. Our data suggest that reduction of GSH via BSO treatment results in the same types of nigrostriatal degenerative effects that occur during the aging process and consequently is a good model system for examining the role of GSH in protecting this area of the brain against the harmful effects of age-related oxidative stress.

1. The influence of age on striatal neuron Ca2+ physiology was studied through an analysis of intracellularly recorded Ca(2+)-mediated plateau potentials. In vitro brain slices from young and aged rats were treated with the K+ channel blocker tetraethylammonium (30 mM) to facilitate the expression of plateau potentials. A sample of neurons was also filled with biocytin and post hoc correlations were performed between morphology and physiology. 2. Testing of sampling parameters in neurons from young rats revealed that tetrodotoxin did not affect the amplitude or duration of plateau potentials. The membrane potential induced during plateau testing and the rate of plateau potential generation, however, had to be held constant because these variables affected plateau potential duration. 3. A significant age-related decrease was found in the duration of Ca(2+)-mediated plateau potentials that could not be explained by alterations in the activation or inactivation properties of the plateau potential. Investigation into relationships between cell morphology and plateau potential duration revealed a number of correlations. Soma size and dendritic length were correlated with plateau potential duration, independent of age (hierarchical regression), and an age-related decrease in dendritic length but not in soma size was found. Spine density and plateau potential duration were also correlated, but the significance depended on the variance associated with age. These data indicate that the extent of somadendritic membrane (including spines) affects plateau potential duration in striatal neurons and that dendrite and spine loss in aged animals may contribute to age-related decreases in plateau potential duration. 4. The response to replacement of Ca2+ with Ba2+ was age dependent, with Ba2+ causing a greater increase in the duration of plateau potentials in young neurons. These data rule out an increase in Ca(2+)-mediated inactivation of Ca2+ channels as a primary cause for the shortening of plateau potentials in aged neurons. Our morphological findings suggest that dendritic regression in aged neurons may have reduced the number of Ca2+ channels participating in plateau potential generation, but other mechanisms related to changes in the type of Ca2+ channel expressed and possible differences in their inactivation kinetics may also contribute to the age-related change in plateau potential duration.

This study examined the time course of dendritic reorganization of dentate granule neurons of the hippocampus following the loss of input from both the fimbria fornix (FF) and the entorhinal cortex (EC). We used the Golgi-Cox stain to assess the morphology of dentate granule neurons at six postlesion time points (4, 8, 14, 30, 45, and 60 days) and dendritic measures included total dendritic length, number of segments, number of branch points, and spine density. We found that as early as 4 days postlesion, total dendritic length and number of segments were significantly decreased with the greatest change occurring in the distal parts of the dendritic arbor located in the outer molecular layer of the dentate gyrus. Dendritic measures related to segment number and dendritic length returned to 70% of intact values by 30 days postlesion and were not significantly different from unlesioned rats at 45 and 60 days postlesion. In contrast, the recovery of spine density was transient. Spine density in the outer molecular layer of the dentate gyrus decreased by 60% at 4 days postlesion and returned to 87% of intact values by 30 days postlesion. However, there was a second loss of dendritic spines along the distal portion of the dendrite between 30 and 60 days postlesion. These data provide evidence that the ability of granule neurons to recover a dendritic morphology similar to that of unlesioned rats is impaired following the combined EC/FF lesion and that the "secondary loss" of dendritic spine density on granule neurons may significantly limit the chances of the hippocampus reforming a synaptic circuitry that could lead to functional recovery after the EC/FF lesion.

The receptor protein tyrosine phosphatase (RPTP) zeta/beta and a major isoform, phosphacan, a chondroitin sulfate proteoglycan that contains the RPTP zeta/beta extracellular domain but not the transmembrane and intracellular phosphatase domains, are expressed abundantly in the nervous system, primarily by astroglia. Because of similarities in the expression patterns of RPTP zeta/beta and the receptor tyrosine kinase TrkB, we investigated whether RNAs encoding these proteins were co-localized during development, which would suggest that these molecules might functionally interact in vivo. By in-situ hybridization, we noted extensive areas of overlap in the expression of trkB and RPTP zeta/beta mRNAs in the developing peripheral and central nervous systems. Analysis with a probe specific for the catalytic TrkB isoform suggested that RPTP zeta/beta and non-catalytic trkB mRNAs were co-expressed in particular regions of the nervous system while the catalytic trkB and RPTP zeta/beta transcripts were also, but to a lesser extent. RPTP zeta/beta and phosphacan expression were extremely similar, differing particularly in the level of expression in the ventricular and subventricular zones, hippocampus, and ependyma. Furthermore, both RPTP zeta/beta and phosphacan mRNAs were found in several subsets of neurons as well as astrocytes. Following CNS injury, we observed robust induction of RPTP zeta/beta mRNA in areas of axonal sprouting, and of both RPTP zeta/beta and phosphacan mRNAs in areas of glial scarring, implying that the encoded proteins and the cell adhesion molecules and extracellular matrix proteins to which they bind may contribute to recovery from injury and perhaps regulation of axonal regrowth in the nervous system.

From previous lesion studies of the hippocampus it has been reported that axons of the commissural/associational pathway expand their termination zone in the molecular layer of the dentate gyrus by 20-25% in response to loss of input from the entorhinal cortex. However, although much is known about the response of the commissural/associational pathway with regard to extent, latency, and speed of the reinnervation response following an entorhinal cortex lesion, little is known about how the loss of additional afferent systems might modulate this response. To address this issue, we examined at 14, 30, and 45 days postlesion, the sprouting of commissural/associational afferents following either a unilateral fimbria fornix transection, a unilateral entorhinal cortex lesion, or combined lesions of both the entorhinal cortex and the fimbria fornix. Loss of septal innervation to the hippocampus was assessed using the cholinesterase stain, whereas sprouting from the commissural/associational pathway was determined from Holmes fiber-stained sections. In addition, the Timms stain was used to examine the time course of the loss of terminal fields of the various zinc-containing afferent systems within the hippocampus. Following the removal of input to the hippocampus via the fimbria fornix transection, there was no evidence of sprouting of the commissural/associational fibers into the deafferented portion of the dentate gyrus. In contrast, rats receiving an entorhinal cortex lesion showed a significant increase (28%) in the width of the commissural/associational fiber plexus that was present by 14 days postlesion. By comparison, the magnitude of the expansion of the commissural/associational fiber plexus was significantly larger after lesioning both the entorhinal cortex and the fimbria than after the entorhinal cortex lesion alone (45% vs. 28%). In addition, the expansion of the commissural/associational fiber plexus was not increased at 14 days postlesion but was significantly increased at 30 days postlesion. The delay in the sprouting of the commissural/associational pathway coincided with the time course of loss of zinc-containing fibers in the outer molecular layer of the dentate gyrus as assessed with the Timms stain. These results suggest that the magnitude and time course for the sprouting of axons from the commissural/associational pathway into the partially deafferented hippocampus of the adult rat is lesion dependent and that the effect of the loss of input from the entorhinal cortex can be modulated and enhanced by the concomitant depletion of input from the fimbria fornix.

Glial fibrillary acidic protein (GFAP) increases during proestrus in astrocytes of the hypothalamic arcuate nucleus (ARC). These changes are associated with altered astrocyte-neuron contacts and synaptic remodelling, during preparation for the preovulatory gonadotrophin surge. This study of young C57BL/6J mice showed transient elevations of GFAP mRNA on proestrus in the ARC by in situ hybridization. Basal GFAP mRNA was regained within 18 h. We hypothesize that changes in astrocytic GFAP on proestrus result from elevations of GFAP mRNA that are, in turn, driven by ovarian secretions of estradiol.

Age-related increases of the astrocyte marker, glial fibrillary acidic protein (GFAP), were further resolved by in situ hybridization and immunocytochemistry in female C57BL/6J mice. The age groups represented the major stages of reproductive aging: young (5 months), middle-age (18 months), and old (23 and 26 months). GFAP mRNA and protein showed generalized increases in old mice. Major white fiber tracts, such as the corpus callosum, fimbria, stria terminalis, and optic tract, showed increased GFAP immunostaining and mRNA. Gray matter showed robust > or = twofold increases in GFAP mRNA with age, especially in the thalamus and hypothalamus, areas that expressed little GFAP in the young. These generalized age-related increases of GFAP in many brain regions imply the existence of a widespread stimulus for increased activity of astrocytes during aging.

This ultrastructural and light microscopic immunocytochemical study describes the time course of anatomical changes that occur in striatal astrocytes in response to neuronal deafferentation in young adult rats and the coordinate distribution of two astrocytic proteins involved in reactive synaptogenesis, glial fibrillary acidic protein and clusterin. We found that following a unilateral lesion of the cerebral cortex, striatal astrocytes undergo a rapid ultrastructural transformation from a protoplasmic to a reactive type of astroglia and are the primary cells involved in the removal of degenerating axon terminals, but not axons of passage, from the neuropil. In addition, at 10 and 27 days postlesion, processes of reactive astrocytes are also seen to occupy vacant postsynaptic spines after degenerating presynaptic terminals are removed, suggesting that they may also participate in the reinnervation of the deafferented neurons. By immunocytochemistry, reactive astrocytes were characterized by a significant increase in the intensity of glial fibrillary acidic protein staining beginning at three days postlesion and lasting for at least 27 days postlesion. Reactive astrocytes were characterized by cellular hypertrophy and an increase in the density of immunoreactive processes distributed throughout the deafferented striatum. However, our analysis of astrocyte cell number found no evidence of astrocyte proliferation in response to the deafferentation lesion. Although previous in situ hybridization studies have reported elevated clusterin messenger RNA in reactive astrocytes after decortication, clusterin immunoreactivity was not seen in the cell soma of reactive astrocytes but was distributed as punctate deposits, ranging from 1 to 2 microns in diameter, within the neuropil of the deafferented striatum. At 10 days postlesion, the distribution of clusterin staining appeared as large aggregates of immunoreactive deposits adjacent to neurons. However, by 27 days postlesion, the aggregates of clusterin reaction product were replaced by a fine scattering of individual punctate deposits distributed evenly over the dorsal part of the deafferented striatum. These data support the notion that reactive astrocytes serve multiple, time-dependent roles in response to brain injury and are involved in both the removal of degenerative debris from the lesion site as well as in reforming the synaptic circuitry of the damaged brain. Our data suggest that, in response to decortication, reactive astrocytes are the primary cells responsible for removing degenerating axon terminals, but not axons of passage, from the deafferented striatum and that the coordinate increase in glial fibrillary acidic protein may serve to stabilize the extension of reactive astrocytic processes during phagocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)

SCG10 is a developmentally regulated, growth-associated protein (GAP) that was isolated as a neuronal marker of the neural crest. It was recently found that SCG10 shares an amino acid sequence similarity with a phosphoprotein named stathmin or p19 of which phosphorylation is induced by nerve growth factor and vasoactive intestinal peptide in PC12 cells and striatal neurons, respectively. While expression of SCG10 messenger RNA dramatically decreases during postnatal development, significant levels of expression still persist into adulthood. To examine possible roles of SCG10 in the adult brain, we examined the distribution of messenger RNAs encoding SCG10 and p19/stathmin as well as GAP-43 in adult rat brain sections by northern blot, RNase protection and in situ hybridization. SCG10 transcripts are found at high levels in long-distance projecting neurons and neurons with extensive dendritic arbors, while p19/stathmin messenger RNA was weakly distributed over most brain areas. Both messenger RNAs are expressed in neuronal subpopulations but not in glia, although the overall distribution of the transcripts of these two structurally related genes is distinct. The spatial and temporal expression profiles of SCG10 messenger RNA is comparable to that of GAP-43, another neuronal GAP, in the developing nervous system, however the expression of SCG10 messenger RNA in the adult brain is distinct from that of GAP-43, especially in the hippocampus and brain stem, where the dentate granule cells and sensory and motor neurons of brainstem express SCG10 but not GAP-43. These results suggest that SCG10 may have a unique role in the neuronal growth-response of subsets of mature neurons, and that SCG10 plays a stathmin-like function at nerve terminals, to which it may be rapidly transported by means of membrane attachment due to a hydrophobic domain present in SCG10 but not in p19/stathmin. This suggests that SCG10 may play a role in structural plasticity in the adult brain.

This study demonstrates that gonadal steroids (estradiol, testosterone, dihydrotestosterone) can regulate the expression of glial fibrillary acidic protein in the adult male rat brain. Previously, we showed that castration of adult male rats increased glial fibrillary acidic protein messenger RNA in the hippocampus and that this increase was additive with the increase induced by deafferenting entorhinal cortex lesions [Day et al. (1990) Molec. Endocr. 4, 1995-2002 . We extended these effects of castration and entorhinal cortex lesion to glial fibrillary acidic protein, using immunoassays. Furthermore, we found regional differences in responses to castration and inhibited by sex steroids. In contrast, hypothalamic glial fibrillary acidic protein expression was inhibited by castration. Similar regional differences were also shown for astrocyte glial fibrillary acidic protein distribution by immunocytochemistry. The regional specificity of glial fibrillary acidic protein expression after castration and sex steroid replacement is pertinent to the role of astrocytes in synaptic plasticity in unlesioned adults as well as in responses to lesions where the steroid milieu has been shown to influence sprouting.

This investigation describes the schedule and regional distribution of astrocytic responses in striatum following deafferentation by unilateral frontal cortex ablation. In the ipsilateral deafferented striatum, glial fibrillary acidic protein and clusterin (sulfated glycoprotein-2) messengerRNA showed peak elevations by 10 days postlesioning (Northern blots). Vimentin messengerRNA responded faster, with a transient elevation by three days postlesioning. The messengerRNA for glial fibrillary acidic protein, clusterin and vimentin returned toward control levels by 27 days postlesioning. However, the neuronal marker growth-associated protein messengerRNA, was decreased at all postlesion times. By in situ hybridization, the increased glial fibrillary acidic protein messengerRNA and clusterin messengerRNA signals were localized mainly to the dorsal half of the ipsilateral deafferented striatum and followed the same schedule as found by Northern blots. Glial fibrillary acidic protein messengerRNA was widely diffused in the dorsal striatum and was excluded from fascicles of the internal capsule; a similar distribution was found for glial fibrillary acidic protein-immunopositive astrocytes. While clusterin messengerRNA signal showed a distinct clustering, its immunoreactivity appeared as deposits in the deafferented striatal neuropil; Western blots confirmed the immunocytochemical results. By in situ hybridization, vimentin messengerRNA was mostly localized to the cortical wound cavity dorsal to the deafferented striatum and overlapped the distribution of vimentin-immunopositive cells. These findings suggest a coordination of striatal astrocytic messengerRNA responses with the degeneration of corticostriatal afferents. We also compared these same parameters with those from published reports on the hippocampus after deafferenting lesions. Certain astrocyte molecular responses to deafferentation are detected about five days earlier in the hippocampus than in the striatum. This different schedule in response to decortication may pertain to differences in synaptic remodeling in the hippocampus vs striatum.

Age-related decline in estrous cycle frequency and impaired pre-ovulatory gonadotropin surges at mid-life are modelled in young C57BL/6J mice by chronic (3 months) oral administration of estradiol (E2). However, the cellular events that induce damage to the neuroendocrine center that regulate gonadotropins with age or following E2 treatment are unclear. To address this issue, possible neuron loss was examined in relation to the loss of estrous cyclicity in E2-treated mice, in particular neurons of the hypothalamic luteinizing hormone releasing hormone (LHRH) and/or tuberoinfundibular dopaminergic (TIDA) systems. By immunocytochemical methods, there was no change in the number of LHRH or TIDA neurons in mice that have become acyclic due to age or E2 treatment. We conclude that the onset of acyclicity at middle-age or following chronic E2 treatment is not associated with loss of LHRH or TIDA neurons and that other neuroendocrine changes must be considered for the cause of acyclicity, particularly those involved in the synaptic regulation of LHRH secretion.

Unilateral ablation of the frontal cortex induced 30%-50% decrease of dopamine (DA) concentration in the ipsilateral striatum at 10 and 27 days after lesioning. There were increased ratios of dihydroxyphenylacetic acid (DOPAC): DA and homovanillic acid (HVA): DA by 20%-60% at 10 days post-lesioning, which suggest compensatory increases of DA metabolism. While no change in total striatal tyrosine hydroxylase (TH) polypeptide concentration was found at any post-lesion time, TH catalytic activity was decreased slightly (-25%) at 10 days. Among individual rats, at 3, 10 and 27 days post-lesioning, striatal DA concentration was inversely related to striatal glial fibrillary acidic protein (GFAP) concentration, a marker of astrocytic activity. The loss of DA was observed whether or not DA was normalized to striatal protein, which suggests that DA loss cannot be simply attributed to increased astrocytic proteins. These data suggest reciprocal relationships between the extent of astrocytic reactions after cortical deafferentation and striatal DA loss, which could involve local remodelling without primary damage to the nigro-striatal terminals.

Thios study showed responses of sulfated glycoprotein-2 (SGP-2) in the rat hippocampus after deafferenting lesion. SGP-2 is a plasma protein that also occurs in many peripheral tissues. In some circumstances, elevations of SGP-2 mRNA are associated with cell degeneration and responses to injury. This study used entorhinal cortex lesions (ECL) to partially deafferent the hippocampus by damaging the perforant path and to induce synaptic remodeling. SGP-2 mRNA is increased in hippocampal astrocytes after ECL. Western blot analysis of soluble hippocampal proteins identified 3 major forms of rat SGP-2 protein: a precursor (61 kDa) and 2 reduced subunits at 39.5 and 35 kDa. These forms increased at 4 days post ECL ipsilaterally to the lesion. By immunocytochemistry (ICC), SGP-2 showed an increased immunoreactivity on the lesioned side by 2 days post ECL that continued through 14 days post ECL. Besides immunopositive astrocytes, punctate immunochemical reaction products occurred among the degenerating fibers of the perforant path. We conclude that changes of SGP-2 protein in the hippocampus after ECL occur roughly in parallel with increases of SGP-2 mRNA. The punctate immuno-deposits could represent secreted SGP-2 and may be useful as a marker for degenerating pathways.

Patients with Alzheimer's disease (AD) often show emotional, motivational, and memory disturbances which may have morphological substrates that include the amygdaloid complex. Neuropathological studies in other limbic areas have recently been enhanced by immunocytochemical studies with Alz 50 antibody. Therefore, we examined the distribution of Alz 50 immunoreactive (Alz 50-IR) neuritic plaques (NP) and neurofibrillary tangles (NFT) in the amygdala in AD cases, in one aged patient with Down's syndrome, and in controls of similar ages. In all AD cases numerous NP and variable numbers of NFT were observed and a distinctive subregional anatomical distribution of NP and Alz 50-IR neuropil in the amygdala existed, whereas no similar selective topography for NFT or Alz 50-IR neurons was found. A high density of NP was demonstrated in the ventromedial aspects of the basolateral and corticomedial nuclear regions. There was no correlation with the pattern of cholinergic innervation. There was, however, a correspondence between intraamygdaloid- and amygdaloid-hippocampal connections and regions of high NP density. Our findings support the concept that the disease process may occur along anatomically defined pathways, and the amygdala may be a central participant in this process.

This study extends the unexpected finding of Sloviter et al. (Science, 1989, 243: 535-538) that adrenalectomy (ADX) of young rats casues a loss of granule neurons in the dentate gyrus. In particular, we determined how the vulnerability of dentate granule neurons to the cytocidal effect of ADX is related to the completeness of the ADX and whether sulfated glycoprotein-2, a putative component of programmed cell death, is associated with the death of granule neurons after ADX. We report that 4 months after bilateral ADX of young (150-175 g) rats only ADX rats that had attenuated weight gain and less than 2 ng/ml of serum corticosterone lost granule neurons; whereas as little as 15 ng/ml of serum corticosterone was sufficient to protect granule neurons from cell death. In addition, by immunocytochemistry, SGP-2 was distributed as punctate deposits throughout the molecular layer of the dentate gyrus and in glial cells juxtaposed to surviving neurons in the dentate of ADX rats with a granule cell loss. However, immunoreactivity for SGP-2 was not found in granule neurons that exhibited morphological signs of cellular generation after ADX.

This study concerns effects of the testes on two macromolecules in the rat hippocampus that were previously not known to be responsive to this endocrine axis. Castration for 3 weeks elevated the expression of glial fibrillary acidic protein (GFAP) and sulfated glycoprotein-2 (SGP-2) in male rat hippocampus, as shown by Northern blots and immunocytochemistry. SGP-2 mRNA was colocalized with GFAP, implying increased prevalence in astrocytes after castration. During hippocampal responses to deafferentation by entorhinal cortex lesions that damage the perforant path and induce synaptic reorganization, both mRNA and protein for SGP-2 and GFAP increase. Moreover, prior castration had an additive effect with entorhinal cortex lesions in the increase in GFAP and SGP-2 mRNA. These data suggest that testicular hormones regulate hippocampal astrocyte activity in intact adult rats as well as during synaptic reorganization in response to deafferenting lesions.

Our study used quantitative morphometric analysis and Golgi staining methods to evaluate postnatal changes in the dendritic architecture of MSI neurons of the striatum between 1 and 30 months of age. Morphological changes and chronological age were also correlated with functional testing in order to identify subpopulations of aged mice with dendritic alterations that may be more characteristic of a motor deficit rather than the normal aging process. We found that the overall size of the dendritic arbor of MSI neurons in the rostral striatum remained stable with age, while caudal MSI neurons exhibited a significant elongation of terminal dendritic segments between 25 and 30 months of age. In addition, our correlation analysis of motor performance and chronological age found that neither striatal-motor deficits nor their associated anatomical correlates were inevitable consequences of senescence but were characteristic for a select subpopulation of aged mice with striatal-motor deficits. We found that mice that tested poorly on the balance rod had a significant increase in the number of MSI neurons with small dendritic arbors in various stages of atrophic degeneration. Conversely, 30-month-old mice that had no functional impairment showed no significant change in the number of neurons with atrophic dendrites. These data reinforce the premise that the correlation of structure and function plays an important role in the analysis of an aging population since data may vary based on the number of functionally impaired or unimpaired mice that make up an experimental group.

The present study was carried out to examine the extrapyramidal motor system of C57BL/6N mice for age-related cell loss in cholinergic neurons of the striatum (ST) and dopaminergic (DA) neurons of the substantia nigra (SN). Immunocytochemistry using antibodies against tyrosine hydroxylase (TH) or choline acetyltransferase (CAT) were used to identify DA or cholinergic neurons of the SN and ST in 6 age groups of young (3 months), middle (6, 10, 20 months) and old (25, 30 months) aged mice. We found that while there was a small decline (11%) in the total number of DA neurons of the SN with age, this decrease did not reach statistical significance. In contrast, the total number of cholinergic neurons of the ST significantly decreased between 25 and 30 months of age with the largest cell loss (38%) found in the rostral ST. In addition, the loss of cholinergic neurons in 30-month-old mice was paralleled by a decline in the mean cross-sectional area of the cell soma and nucleus of remaining cholinergic neurons. These data suggest that advancing age has a differential effect on neurotransmitter neurons of the SN and ST and supports the notion that cell loss is not an inevitable characteristic of senescence but is brain region- and cell type-specific. In addition, these data are consistent with the hypothesis that the proliferation of striatal dendrites described previously in aged C57BL/6N mice may result, in part, from a compensatory growth of these processes secondary to age-related cell loss of striatal neurons.

Alterations in neurotransmitter systems of the basal ganglia have been postulated to contribute to the disruption of motor function and balance associated with aging. This study examined nigrostriatal (A9) and mesolimbic (A10) dopamine neurons for qualitative age-correlated changes using fluorescence histochemistry for catecholamines and immunocytochemical techniques for the catecholamine-synthesizing enzyme, tyrosine hydroxylase. Results from this study suggest that age-correlated morphological changes in A9 but not all A10 neurons in the midbrain are present in mature adult (10-month) C57BL/6NNia mice and show a progressive increase in severity until at least 30 months of age. These changes are characterized by a progressive accumulation of lipofuscin in dopamine-containing perikarya, a markedly reduced dopamine content per cell as determined visually by histofluorescence, and an increase in the number of large, fluorescent axonal dilations in dopamine-containing fibers of the mesolimbic and nigrostriatal systems. These data suggest that heterogeneous morphological aging patterns exist within dopamine-containing neurons of the midbrain and that based upon their terminal projection sites, various regions of the striatum and cortex may be differentially affected in the aged brain. In addition, these findings support the belief that age-related changes in neural structure are not generalized to an entire brain nucleus or cell type but are selective for individual cells within an affected area.

A technique for the simultaneous visualization of monoamines and neuropeptides is described. This technique provides for the correlative examination of monoamine and neuropeptide interrelationships either simultaneously using two adjacent sections of freeze-dried tissue or sequentially using a single freeze-dried tissue section for both histofluorescence and immunocytochemistry. Both techniques provide for the investigation of precise anatomical interrelationships and used together provide confirmation and complementary information concerning neuropeptide-monoamine interactions. Histological comparisons between freeze-dried and Bouin's fixed tissue revealed three differences: First, the cytoplasm of neurons in freeze-dried tissue retained a reticulated appearance; secondly, neurons in freeze-dried tissue demonstrated a greater affinity for chemical stains; and thirdly, tissue fixed in Bouin's solution appeared shrunken when compared to tissue which had been freeze-dried. Application of immunocytochemistry to freeze-dried tissue proved successful with all antisera tested and the quality of the immunostaining was similar in both freeze-dried and Bouin's fixed tissue. These observations suggest that freeze-drying fixation provides a suitable tissue preparation for immunocytochemistry and a technical means for coupling immunocytochemical staining of neuropeptides and fluorescence histochemical methods for monoamines in a single tissue block.

Loss of dopaminergic neurons from the pars compacta of the substantia nigra is the pathological hallmark of Parkinson's disease (PD) and results in a partial deafferentation to the striatum. Since deafferentation is known to induce transynaptic atrophy of postsynaptic cells, we examined by Golgi impregnation the morphology of medium spiny I (MSI) striatal neurons, the principal target population for both nigrostriatal and corticostriatal fibers. Our quantitative data indicate that the dendritic arbor of MSI neurons in the putamen is significantly reduced in both length and number and MSI neurons are morphologically characterized by truncated dendrites with few dendritic spines and irregular, bulbous swellings. These data provide morphological evidence for the atrophy of striatal dendrites in PD and may explain, in part, the declining efficacy of chronic L-DOPA replacement therapy in advanced PD.

Processes of magnocellular neurosecretory cells in the rat supraoptic nucleus which project along the pial surface in the ventral glial lamina were investigated using immunocytochemistry, Golgi stains and electron microscopy. Immunocytochemical studies revealed that although both oxytocin- and vasopressin-containing processes were evident in the ventral glial lamina, vasopressin-containing processes predominated. Ventral processes were thicker and of a different morphology than dorsal axon-like processes which joined the hypothalamo-neurohypophysial tract and exhibited large varicosities along their length or at their apparent termination. Golgi stains revealed that classically defined dendrites of supraoptic neurons projected primarily ventrally and often invaded the ventral glial lamina. No axons were traced to the lamina. Ultrastructurally, processes within the ventral glial lamina characterized as dendrites could be stained immunocytochemically for neurophysin and were post-synaptic to a variety of presynaptic elements. The results suggest that many dendrites from magnocellular neurosecretory cells in the supraoptic nucleus project to the ventral glial lamina and form a restricted, receptive plexus. The previously demonstrated coexistence of catecholamine-containing varicosities and other axon types with these processes in the lamina indicates an important role for supraoptic dendrites in integrating a wide variety of information relevant to neurohypophysial hormone release.

This study examined the amygdaloid complex in Alzheimer's disease (AD). We compared the distribution and morphology of somatostatin (SOM-) and neuropeptide Y-immunoreactive (NPY-IR) neurons in the amygdala with the distribution of neuritic plaques (NP) and acetylcholinesterase (AChE) staining patterns in various subnuclei. We found that in AD, there was an increase in the number of small, atrophic neurons for both SOM and NPY, and subregional analysis revealed similar size reductions in all subnuclei. In contrast, the highest density of NP was found in the corticomedial nuclei and densest staining for AChE in the basal nucleus. Although NPY- and SOM-IR fibers were occasionally associated with NP, a dense, morphologically preserved peptidergic fiber-network was found in all areas including subnuclei with high numbers of NP. Our study indicates that atrophic SOM- and NPY-IR neurons are not correlated with the subregional distribution of NP or cholinesterase staining pattern of the amygdala, and suggests that alterations in SOM and NPY neurons are not characteristics of the primary pathogenic process that underlie the formation of NP or cholinergic cell loss in AD.

A correlative fluorescence-immunocytochemical technique for the localization of monoamines and neurophysin on sections of freeze-dried tissues is described. An extensive network of monoamine-containing perikarya and terminals was found throughout the hypothalamus and median eminence. Immunocytochemical localization of antisera for neurophysin was found in the supraoptic and paraventricular nuclei of the hypothalamus and in both the zona interna and zona externa of the median eminence. This correlative demonstration of both catecholamines and neuropeptides within the same tissue provides a new approach to the study of neurotransmitters and neurohormones and their role in the regulation of the peripheral endocrine system.