Lysosomal dysfunction results in lamina-specific meganeurite formation but not apoptosis in frontal cortex.

An inhibitor of cathepsins B and L was used to test if lysosomal dysfunction in cultured slices of rat frontal cortex induces pathological features that develop in the human cortex during aging and Alzheimer's disease (AD). Incubation for 6 days with N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD) resulted in a massive proliferation of endosomes-lysosomes in all cortical layers. Slices additionally exposed to a washout of 4 days had numerous meganeurites, blister-like structures in the region of the axon hillock, in layer III but not in other cortical laminae. Meganeurites are a characteristic feature of the human frontal cortex after age 50 and are largely restricted to layer III. Tests for apoptosis were carried out at two intervals following meganeurite formation. TUNEL-labeled neurons were confined to layers II/III on the surface of the slices but there was no evidence for a ZPAD effect. In all, 6 days of lysosomal dysfunction reproduces characteristic effects of normal aging in neocortex without generating some key features of AD.

Experimentally induced lysosomal dysfunction disrupts processing of hypothalamic releasing factors.

Previous studies have shown that experimentally induced lysosomal dysfunction elicits various features of aging in the cortical telencephalon. The present study used cultured slices to test if: (1) it causes similar changes in the hypothalamus, and/or (2) modifies the processing of two releasing factors important to aging. A 2-day exposure to N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), a selective inhibitor of cathepsins B and L, triggered a pronounced increase in the numbers of lysosomes in the ventromedial and dorsomedial nuclei, and in lateral hypothalamus. Continued incubation with the inhibitor for 3-12 days resulted in the spread of endosomes-lysosomes into dendrites and, in the lateral hypothalamus, the formation of massive, lysosome-filled expansions of neuronal processes (meganeurites). These effects did not occur in the arcuate nucleus, making it the first region so far examined in which lysosomal proliferation is not initiated by hydrolase inhibitors. Despite this, a dense plexus of axons and terminals in the median eminence was partially depleted of growth hormone releasing hormone (GHRH) within 48 hours after addition of ZPAD. Moreover, the inhibitor caused axonal GHRH to become collected into large puncta, an effect highly suggestive of a partial failure in axonal transport. GHRH mRNA levels were not greatly affected by 6 days of ZPAD exposure, indicating that reduced expression did not play a major role in the peptide changes seen at 48 hours. Similar but less pronounced immunocytochemical changes were recorded for the somatostatin system in the arcuate and periventricular nucleus. It is concluded that lysosome dysfunction: (1) has different consequences for the arcuate nucleus than other brain regions, and (2) disrupts transport of hypothalamic releasing factors. The potential significance of the results to endocrine senescence is discussed.

Neonatal treatment with 192 IgG-saporin produces long-term forebrain cholinergic deficits and reduces dendritic branching and spine density of neocortical pyramidal neurons.

The role of basal forebrain-derived cholinergic afferents in the development of neocortex was studied in postnatal rats. Newborn rat pups received intraventricular injections of 192 IgG-saporin. Following survival periods ranging from 2 days to 6 months, the brains were processed to document the cholinergic lesion and to examine morphological consequences. Immunocytochemistry for choline acetyltransferase (ChAT) and in situ hybridization for ChAT mRNA demonstrate a loss of approximately 75% of the cholinergic neurons in the medial septum and nucleus of the diagonal band of Broca in the basal forebrain. In situ hybridization for glutamic acid decarboxylase mRNA reveals no loss of basal forebrain GABAergic neurons. Acetylcholinesterase histochemistry demonstrates a marked reduction of the cholinergic axons in neocortex. Cholinergic axons are reduced throughout the cortical layers; this reduction is more marked in medial than in lateral cortical areas. The thickness of neocortex is reduced by approximately 10%. Retrograde labeling of layer V cortico-collicular pyramidal cells reveals a reduction in cell body size and also a reduction in numbers of branches of apical dendrites. Spine densities on apical dendrites are reduced by approximately 20-25% in 192 IgG-saporin-treated cases; no change was detected in number of spines on basal dendrites. These results indicate a developmental or maintenance role for cholinergic afferents to cerebral cortical neurons.

In situ hybridization for c-fos mRNA reveals the involvement of the superior colliculus in the propagation of seizure activity in genetically epilepsy-prone rats.

Previous work showed that bilateral lesions made between the inferior and superior colliculi reduced the severity of audiogenic seizures in genetically epilepsy-prone rats (GEPR-9s), and indicated that the connections between these two structures are vital for the propagation of seizure activity. To determine the involvement of the superior colliculus (SC) in seizure propagation, GEPR-9s were given four audiogenic seizures within 1 h by ringing a loud bell, and their brains were processed 30 min later for in situ hybridization for c-fos mRNA. Brain sections from such rats showed dense labeling in both the dorsal cortex and external nucleus of the inferior colliculus. Labeling continued rostrally into the intermediate and deep layers of the SC and the periaqueductal gray region. In addition, other brain regions such as the amygdala, piriform cortex and dorsal endopiriform nucleus showed dense labeling for c-fos mRNA. Comparable increases were not observed in the brains of Sprague-Dawley (SD) rats receiving auditory stimulation or in unstimulated GEPR-9s and SD rats, thereby indicating that increases in stimulated GEPR-9s are seizure-specific. This study provides further evidence that the SC is involved in the propagation of seizure activity in GEPR-9s, and also demonstrates the activation of other brain regions by audiogenic seizures.

Cellular localization of NGF and NT-3 mRNAs in postnatal rat forebrain.

The presence of transiently elevated levels of mRNA for nerve growth factor (NGF) and neurotrophin-3 (NT-3) in postnatal development of several brain areas suggests that these factors may be expressed by a greater number of cell types in the immature than in the adult brain. To evaluate this possibility, in situ hybridization was used to determine the cellular localization of NGF mRNA and NT-3 mRNA in hippocampus, cingulate cortex, posterolateral neocortex, thalamus, and cerebellum of postnatal rat. In areas expressing both neurotrophins (i.e., hippocampus, cingulate cortex, and anteroventral thalamus), NT-3 mRNA was detected at earlier ages than NGF mRNA. Patterns of hybridization in hippocampus and cerebellum indicate that NT-3 is expressed by neurons soon after leaving the mitotic cycle whereas NGF expression is a feature of more mature neurons. The exception to this pattern was NGF expression in the lateral geniculate nuclei which was present by Postnatal Day 1 and retained in the adult. Both neurotrophins were transiently expressed in several brain areas. The loss of expression with age was most striking in thalamus with transient expression of NT-3 mRNA by the majority of dorsal thalamic relay nuclei and of NGF mRNA by fewer nuclei including the posterior, anteroventral, ventrolateral, and ventromedial nuclei. NT-3 expression also was transient in caudal cingulate/retrosplenial cortex, hippocampal CA3 stratum pyramidale, and the granule cells of archicerebellum. In early postnatal cingulate and retrosplenial cortices there were reciprocal rostrocaudal gradients of NGF and NT-3 expression. These results suggest both distinct and overlapping functions for NT-3 and NGF in early developmental processes including involvement of NT-3 in cerebellar development and of NGF in the development and maintenance of visual afferents to thalamus. Patterns of neurotrophin expression in medial limbic cortex may establish trophic gradients which influence the topography of thalamic innervation.

Nerve growth factor mRNA-containing cells are distributed within regions of cholinergic neurons in the rat basal forebrain.

It has been proposed that nerve growth factor (NGF) provides critical trophic support for the cholinergic neurons of the basal forebrain and that it becomes available to these neurons by retrograde transport from distant forebrain targets. However, neurochemical studies have detected low levels of NGF mRNA within basal forebrain areas of normal and experimental animals, thus suggesting that some NGF synthesis may actually occur within the region of the responsive cholinergic cells. In the present study with in situ hybridization and immunohistochemical techniques, the distribution of cells containing NGF mRNA within basal forebrain was compared with the distribution of cholinergic perikarya. The localization o NGF mRNA was examined by using a 35S-labeled RNA probe complementary to rat preproNGF mRNA and emulsion autoradiography. Hybridization of the NGF cRNA labeled a large number of cells within the anterior olfactory nucleus and the piriform cortex as well as neurons in a continuous zone spanning the lateral aspects of both the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus. In the latter regions, large autoradiographic grain clusters labeled relatively large Nissl-pale nuclei; it did not appear that glial cells were autoradiographically labeled. Comparison of adjacent tissue sections processed for in situ hybridization to NGF mRNA and immunohistochemical localization of choline acetyltransferase (ChAT) demonstrated overlapping fields of cRNA-labeled neurons and ChAT immunoreactive perikarya in both the horizontal limb of the diagonal band and magnocellular preoptic regions. However, no hybridization of the cRNA probe was observed in other principal cholinergic regions including the medial septum, the vertical limb of the diagonal band, or the nucleus basalis of Meynert.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholecystokinin innervation of rat thalamus, including fibers to ventroposterolateral nucleus from dorsal column nuclei.

The distribution of cholecystokinin octapeptide immunoreactive fibers and puncta in the adult rat thalamus was studied using immunocytochemical methods. Small to moderate numbers of immunoreactive fibers were present in the lateral habenular nucleus, ventral lateral geniculate nucleus, zona incerta, parataenial, mediodorsal, medioventral, and submedial nuclei, the rhomboid, paracentral, central lateral and parafascicular nuclei, and in the medial geniculate and dorsal lateral geniculate nuclei. Moderate to large numbers of cholecystokinin (CCK)-positive fibers were present in the paraventricular nuclei, the reticular nucleus, the anteroventral, anteromedial, and central medial nuclei, and in the rostral extension of the internal medullary lamina between the parataenial and anteroventral nuclei. Dense concentrations of immunoreactive fibers were also found in a principal sensory relay nucleus, the ventroposterolateral nucleus (VPL), of the ventrobasal complex. The number of CCK-positive fibers in VPL showed a marked unilateral decrease in rats which had received lesions of the contralateral gracile and cuneate nuclei. The results of this study demonstrate that CCK-immunoreactive fibers and puncta are widely distributed in the rat thalamus, and that the source of these fibers in VPL is probably the dorsal column nuclei.

Proenkephalin is processed in a projection-specific manner in the rat central nervous system.

The biosynthesis and posttranslational proteolytic processing of proenkephalin was studied in three projection systems in the rat central nervous system--the caudate-putamen to the globus pallidus, the paraventricular nucleus of the hypothalamus to the median eminence, and the mossy fiber system of the granule cells of the hippocampus. By using the techniques of in vivo radiolabeling and sequential high-performance liquid chromatographic purification coupled with chemical modification, the biosynthesis of six radiolabeled [Met]enkephalin-containing peptides--[Met5]enkephalin, [Met5,Arg6,Gly7,Leu8]enkephalin, [Met5,Arg6,Phe7]enkephalin, metorphamide, peptide E, and BAM 18P--was followed. In each projection system, radiolabeled enkephalins were purified to constant radiochemical specific activity. However, the posttranslational processing of proenkephalin was found to differ between these three systems, as judged by the relative ratio of these peptides. These findings imply that specific, different physiologies and behaviors may be elicited by the enkephalins based upon the specific [Met]enkephalin-containing peptides that are cleaved from proenkephalin and released in synaptic terminal fields.