Differential regulation of the growth-associated proteins GAP-43 and superior cervical ganglion 10 in response to lesions of the cortex and substantia nigra in the adult rat.

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.

Alterations in rat striatal glutamate synapses following a lesion of the cortico- and/or nigrostriatal pathway.

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.

Differential regulation of astrocytic mRNAs in the rat striatum after lesions of the cortex or substantia nigra.

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.

Retrospective demonstration of endogenous peroxidase activity in plastic-embedded tissues conventionally prepared for electron microscopy.

A procedure is described that permits retrospective demonstration of intracellular endogenous peroxidase activity in tissue conventionally prepared for electron microscopy, i.e., doubly fixed with aldehydes and osmium tetroxide, "stained" in block with uranyl acetate, and embedded in epoxy resins. Using sodium ethoxide, plastic was removed from 1 micrometer sections; subsequently, the sections were incubated for 20 min in diaminobenzidine solution (44 mg/100 ml) made in acetate-citric acid buffer, pH 5.6, with 0.01% hydrogen peroxide. After this treatment, the sections were rinsed, dehydrated, and mounted. Cell types known to have endogenous peroxidase activity (red blood cells, macrophages, and retinal pigment epithelium cells in our preparations) show positive granules in their cytoplasm--control sections were uniformly negative. This method, which could also be used prospectively, cytochemically demonstrates endogenous peroxidase activity upon optical microscopical examination of the treated tissues; correlative electron microscopic studies may be performed on the same tissue block, or even adjacent sections.