Possible involvement of programmed cell death pathways in the neuroprotective action of polyphenols.

One of the hallmarks of Alzheimer's disease is the accumulation of senile plaques composed of extra-cellular aggregates of beta-amyloid (Aß) peptides. It is well established that at least in vitro, Aß triggers apoptotic cell death via the activation of caspase-dependent and -independent cell death effectors, namely caspase-3 and apoptosis inducing factor (AIF), respectively. Epidemiological studies have reported that elderly people have a lower risk (up to 50%) of developing dementia if they regularly eat fruits and vegetables and drink tea and red wine (in moderation). Numerous studies indicate that polyphenols derived from these foods and beverages account for the observed neuroprotective effects. In particular, we have reported that polyphenols extracted from green tea (i.e. epigallocatechin gallate or EGCG) and red wine (i.e. resveratrol) block Aß-induced hippocampal cell death, by at least partially inhibiting Aß fibrillisation. It has been shown that polyphenols may also modulate caspase-dependent and -independent programmed cell death (PCD) pathways. Indeed, polyphenols including resveratrol, EGCG and luteolin significantly inhibit the activation of the key apoptotic executioner, caspase-3 and are able to modulate mitogen-activated protein kinases known to play an important role in neuronal apoptosis. Moreover, it has been reported that polyphenols may exert their anti-apoptotic action by inhibiting AIF release from mitochondria, thus providing new mechanism of action for polyphenols. This review aims to update the current knowledge regarding the differential effects of polyphenols on PCD pathways and discuss their putative neuroprotective action resulting from their capacity to modulate these pathways.

Localization and modulation of calcitonin gene-related peptide-receptor component protein-immunoreactive cells in the rat central and peripheral nervous systems.

Calcitonin gene-related peptide (CGRP) is widely distributed in the central and peripheral nervous system. Its highly diverse biological activities are mediated via the G protein-coupled receptor that uniquely requires two accessory proteins for optimal function. CGRP receptor component protein (RCP) is a coupling protein necessary for CGRP-receptor signaling. In this study, we established the anatomical distribution of RCP in the rat central and peripheral nervous system and its relationship to CGRP immunoreactivity. RCP-immunoreactive (IR) perikarya are widely and selectively distributed in the cerebral cortex, septal nuclei, hippocampus, various hypothalamic nuclei, amygdala, nucleus colliculus, periaqueductal gray, parabrachial nuclei, locus coeruleus, cochlear nuclei, dorsal raphe nuclei, the solitary tractus nucleus and gracile nucleus, cerebellar cortex, various brainstem motor nuclei, the spinal dorsal and ventral horns. A sub-population of neurons in the dorsal root ganglia (DRG) and trigeminal ganglia were strongly RCP-IR. Overall, the localization of RCP-IR closely matched with that of CGRP-IR. We also determined whether RCP in DRG and dorsal horn neurons can be modulated by CGRP receptor blockade and pain-related pathological stimuli. The intrathecal injection of the antagonist CGRP(8-37) markedly increased RCP expression in the lumbar DRG and spinal dorsal horn. Carrageenan-induced plantar inflammation produced a dramatic bilateral increase in RCP expression in the dorsal horn while a partial sciatic nerve ligation reduced RCP expression in the ipsilateral superficial dorsal horn. Our data suggest that the distribution of RCP immunoreactivity is closely matched with CGRP immunoreactivity in most of central and peripheral nervous systems. The co-localization of RCP and CGRP in motoneurons and primary sensory neurons suggests that CGRP has an autocrine or paracrine effect on these neurons. Moreover, our data also suggest that RCP expression in DRG and spinal cord can be modulated during CGRP receptor blockade, inflammation or neuropathic pain and this CGRP receptor-associated protein is dynamically regulated.

Expression of calcitonin gene-related peptide, substance P and protein kinase C in cultured dorsal root ganglion neurons following chronic exposure to mu, delta and kappa opiates.

The mechanisms involved in morphine tolerance are poorly understood. It was reported by our group that calcitonin gene-related peptide (CGRP)-like immunoreactivity (IR) was increased in the spinal dorsal horn during morphine tolerance [Ménard et al. (1996) J. Neurosci. 16, 2342-2351]. More recently, we observed that it was possible to mimic these results in cultured dorsal root ganglion (DRG) neurons allowing for more detailed mechanistic studies [Ma et al. (2000) Neuroscience 99, 529-539]. The aim of the present series of experiments was to further validate the DRG cell culture model by establishing which subtypes of opioid receptors are involved in the induction of CGRP in cultured rat DRG neurons, and to examine the signaling pathway possibly involved in the induction of CGRP-like IR following repeated opiate treatments. Other neuropeptides known to be expressed in DRG neurons, such as substance P (SP), neuropeptide Y (NPY) and galanin, were investigated to assess specificity. Following treatment with any of the three opioid agonists (mu, DAMGO; delta, DPDPE; kappa, U50488H), the number of CGRP- and SP-IR cultured DRG neurons increased significantly, and in a concentration-dependent manner, with the effects of kappa agonist being less pronounced. NPY and galanin were not affected.Double-immunofluorescence staining showed that the three opioid receptors were co-localized with both CGRP- and SP-like IR.Protein kinase C (PKC)-like IR was found to be significantly increased following a repetitive treatment with DAMGO. Double-immunofluorescence staining showed the co-localization of PKCalpha with CGRP- and SP-IR in cultured DRG neurons. Moreover, a combined treatment with DAMGO and a PKC inhibitor (chelerythrine chloride or Gö 6976) was able to block the effects of the opioid on increased CGRP-like IR. These data suggest that the three opioid receptors may be involved in the induction of CGRP and SP observed following chronic exposure to opiates, and that PKC probably plays a role in the signaling pathway leading to the up-regulation of these neuropeptides. These findings further validate the DRG cell culture as a suitable model to study intracellular pathways that govern changes seen following repeated opioid treatments possibly leading to opioid tolerance.

Autoradiographic distribution of adrenomedullin receptors in the rat brain.

The autoradiographic distribution of putative brain adrenomedullin receptors was investigated using [125I]human adrenomedullin(13-52) as a new radioligand. Specific [125I]human adrenomedullin(13-52) binding sites were very discretely distributed in the rat brain with enrichment seen in the choroid plexus and linings of the third, fourth and lateral ventricles, basolateral amygdaloid nuclei, neural lobe of the pituitary gland, the trigeminal nerves and in the granular cell layer of the cerebellum. To our knowledge, this is the first report on the discrete localization of adrenomedullin receptors in the mammalian brain.

Knockdown of spinal metabotropic glutamate receptor 1 (mGluR(1)) alleviates pain and restores opioid efficacy after nerve injury in rats.

1. Nerve injury often produces long-lasting spontaneous pain, hyperalgesia and allodynia that are refractory to treatment, being only partially relieved by clinical analgesics, and often insensitive to morphine. With the aim of assessing its therapeutic potential, we examined the effect of antisense oligonucleotide knockdown of spinal metabotropic glutamate receptor 1 (mGluR(1)) in neuropathic rats. 2. We chronically infused rats intrathecally with either vehicle, or 50 microg day(-1) antisense or missense oligonucleotides beginning either 3 days prior to or 5 days after nerve injury. Cold, heat and mechanical sensitivity was assessed prior to any treatment and again every few days after nerve injury. 3. Here we show that knockdown of mGluR(1) significantly reduces cold hyperalgesia, heat hyperalgesia and mechanical allodynia in the ipsilateral (injured) hindpaw of neuropathic rats. 4. Moreover, we show that morphine analgesia is reduced in neuropathic rats, but not in sham-operated rats, and that knockdown of mGluR(1) restores the analgesic efficacy of morphine. 5. We also show that neuropathic rats are more sensitive to the excitatory effects of intrathecally injected N-methyl-D-aspartate (NMDA), and have elevated protein kinase C (PKC) activity in the spinal cord dorsal horn, two effects that are reversed by knockdown of mGluR(1). 6. These results suggest that activity at mGluR(1) contributes to neuropathic pain through interactions with spinal NMDA receptors and PKC, and that knockdown of mGluR(1) may be a useful therapy for neuropathic pain in humans, both to alleviate pain directly, and as an adjunct to opioid analgesic treatment.

Acetylcholine induces neuritic outgrowth in rat primary olfactory bulb cultures.

The rat olfactory bulb is innervated by basal forebrain cholinergic neurons and is endowed with both nicotinic and muscarinic receptors. The development of this centrifugal cholinergic innervation occurs mainly in early postnatal stages. This developmental time-course and the demonstration that acetylcholine can modulate some aspects of neuronal proliferation, differentiation or death, suggests the possible involvement of cholinergic afferents in the morphogenesis and/or plasticity of the olfactory bulb. The purpose of the present work was to assess whether acetylcholine could modulate neuronal morphogenesis in the olfactory bulb. Toward this aim, we developed a primary culture model of rat olfactory bulbs. Three major cell types were identified on the basis of their morphological and immunocytochemical phenotype: neuronal-shaped cells expressing the neuronal markers neuron specific enolase, microtubule associated protein 2, neural cell adhesion molecule and beta-tubulin III; glial-like cells immunoreactive for glial fibrillary acidic protein and flattened cells immunolabelled with antibodies against beta-tubulin III and nestin, most likely neuronal precursors. After three to six days of treatment with 100-microM carbachol, a cholinergic agonist, significant increase in neuritic length was observed in cultured olfactory bulb neurons. The neurite outgrowth effect of carbachol was abolished by co-treatment with 1 microM alpha-bungarotoxin, an alpha 7 subunit nicotinic receptor antagonist, but was not affected by the addition of 10 microM atropine, a general muscarinic antagonist. The effect of carbachol was also mimicked by the nicotinic agonists, nicotine (100 microM) and epibatidine (10 microM). This pharmacological profile suggested the involvement of nicotinic receptors of the alpha 7-like subtype as confirmed using 125I-alpha-bungarotoxin receptor autoradiography.Taken together, these data argue for a role for nicotinic receptors in neuritic outgrowth in the rat olfactory bulb and provide a cellular support to the previously described effects of acetylcholine on olfactory bulb plasticity in vivo.

Ontogenic profile of the expression of the mu opioid receptor gene in the rat telencephalon and diencephalon: an in situ hybridization study.

The developmental profile of mu (mu) opioid receptor gene expression has been characterized in the embryonic, postnatal and adult rat brain by in situ hybridization histochemistry. By ED12, mu opioid receptor mRNA was detectable in the deep neuroepithelium of the cortical plate. In the developing rat central nervous system (ED13-PD40), transcripts were seen over numerous telencephalic and diencephalic structures, such as the olfactory bulb, caudate-putamen, nucleus accumbens, amygdaloid complex, hippocampal formation, hypothalamus and thalamus. In the vast majority of brain regions examined, the developmental profile of the mu opioid receptor gene expression is similar to that of its translated protein as established using receptor autoradiography. Once a hybridization signal is detected in the prenatal period, it gradually increased to reach maximal levels during the second and third postnatal weeks. By the end of the third postnatal week, mu opioid receptor mRNA levels decreased to reach amounts seen in adulthood. Our study demonstrates that mu opioid receptor gene expression is seen very early on in the embryonic rat brain with transient increases observed during the critical period of neurogenesis, neuronal migration and synaptogenesis, suggesting a role of this opioid receptor subtype in brain developmental processes.

Expression of the purported sigma(1) (sigma(1)) receptor in the mammalian brain and its possible relevance in deficits induced by antagonism of the NMDA receptor complex as revealed using an antisense strategy.

Sigma (sigma) receptors have generated a great deal of interest on the basis of their possible role in psychosis, neuroprotection and various other behaviors including learning processes. The existence of at least two classes of sigma receptor binding sites (sigma(1) and sigma(2)) is now well established. The recent cloning of the mouse, guinea pig and human sigma(1) receptors has allowed the study of the discrete distribution of the sigma(1) receptor mRNA in rodent and human brain tissues using in situ hybridization. Overall, the sites of expression of specific sigma(1) receptor mRNA signals were in accordance to the anatomical distribution of sigma(1) receptor protein first established by quantitative receptor autoradiography. Specific sigma(1) receptor hybridization signals were found to be widely, but discretely distributed, in mouse and guinea pig brain tissues. The highest levels of transcripts were seen in various cranial nerve nuclei. Lower, but still high hybridization signals were observed in mesencephalic structures such as the red nucleus, periaqueductal gray matter and substantia nigra, as well as in some diencephalic structures including such as the habenula and the arcuate, paraventricular and ventromedial hypothalamic nuclei. Superficial (I-II) and deeper (IV-VI) cortical laminae were moderately labeled in the mouse brain. Moderate levels of sigma(1) receptor mRNA were also found in the pyramidal cell layer and the dentate gyrus of the hippocampal formation. Other structures such as the thalamus and amygdaloid body also expressed the sigma(1) receptor mRNA although to a lesser extent. In murine peripheral tissues, strong hybridization signals were observed in the liver, white pulp of the spleen and the adrenal gland. In the postmortem human brain, moderate levels of sigma(1) receptor mRNA, distributed in a laminar fashion, were detected in the temporal cortex with the deeper laminae (IV-VI) being particularly enriched. In the hippocampal formation, the strongest hybridization signals were observed in the dentate gyrus while all other subfields of the human hippocampal formation expressed lower levels of the sigma(1) receptor mRNA. Antisense oligodeoxynucleotides against the purported sigma(1) receptor were used next to investigate the possible role of this receptor in dizocilpine (MK-801)/NMDA receptor blockade-induced amnesia. Following a continuous intracerebroventricular infusion of a specific sigma(1) receptor antisense into the third ventricle (0.4 nmol/h for 5 days), sigma(1)/[3H](+)pentazocine binding was significantly reduced in mouse brain membrane homogenates while a scrambled antisense control was without effect. Moreover, the sigma(1) receptor antisense treatments (5 nmol/injection, every 12 hx3 or 0.4 nmol/h for 5 days) attenuated (+)MK-801/NMDA receptor blockade-induced cognitive deficits in the treated mice while a scrambled antisense control had no effect. Taken together, these results demonstrate the widespread, but discrete, distribution of the sigma(1) receptor mRNA in the mammalian central nervous system. Moreover, antisense treatments against the purported sigma(1) receptor gene reduced specific sigma(1)/[3H](+)pentazocine binding and modulated cognitive behaviors associated with NMDA receptor blockade providing further evidence for the functional relevance of the cloned gene.

Impact of neonatal kainate treatment on hippocampal insulin-like growth factor receptors.

The insulin-like growth factors-I and -II have neurotrophic properties and act through specific membrane receptors. High levels of binding sites for these growth factors are distributed discretely throughout the brain, being concentrated in the hippocampal formation. Functionally, the insulin-like growth factors, in addition to their growth-promoting actions, are considered to play important roles in normal cell functions, as well as in response to pharmacological or surgical manipulations. In adult rats, we have previously shown that systemic injection of kainate produces an overall decrease, in a time-dependent manner, in insulin-like growth factor-I and -II receptor binding sites in the hippocampus [Kar S. et al. (1997) Neuroscience 80, 1041-1055]. Given the evidence that insulin-like growth factors play a critical role during the early stages of brain development, the present study is a logical extension of this earlier report and established the effect of neonatal kainate injection on the developmental profile of insulin-like growth factor receptors. We have evaluated the time-course alteration of these receptors following systemic injection of kainate to newborn rats. After injection of a sublethal dose of kainate (5 mg/kg, i.p.) to postnatal one-day-old pups, [125I]insulin-like growth factor-I, [125I]insulin-like growth factor-II and [125I]insulin binding sites were studied at different postnatal days (7, 14, 21, 28 and 35) using receptor autoradiography. In the developing hippocampus, insulin-like growth factor-I and insulin binding sites are concentrated primarily in the dentate gyrus and the CA2/CA3 subfields, whereas insulin-like growth factor-II binding is discretely localized to the pyramidal layer and the granular layer of the dentate gyrus. Following kainate injection, we observed a slight increase in insulin-like growth factor-I binding sites in given hippocampal subfields starting at postnatal day 14, being significant at day 21. At later days, a progressive decrease was noted. This transient increase may represent an attempt for neuronal plasticity by up-regulating receptor levels. In contrast, insulin-like growth factor-II and insulin receptor binding sites are found to be decreased in various regions of the hippocampus in kainate-treated pups. Taken together, these results provide further evidence for the existence and differential alterations of insulin-like growth factor-I, insulin-like growth factor-II and insulin receptors in the developing rat hippocampus following kainate-induced lesion, suggesting possible involvement of these growth factors in brain plasticity.

Systemic administration of kainic acid induces selective time dependent decrease in [125I]insulin-like growth factor I, [125I]insulin-like growth factor II and [125I]insulin receptor binding sites in adult rat hippocampal formation.

Administration of kainic acid evokes acute seizure in hippocampal pathways that results in a complex sequence of functional and structural alterations resembling human temporal lobe epilepsy. The structural alterations induced by kainic acid include selective loss of neurones in CA1-CA3 subfields and the hilar region of the dentate gyrus followed by sprouting and permanent reorganization of the synaptic connections of the mossy fibre pathways. Although the neuronal degeneration and process of reactive synaptogenesis have been extensively studied, at present little is known about means to prevent pathological conditions leading to kainate-induced cell death. In the present study, to address the role of insulin-like growth factors I and II, and insulin in neuronal survival as well as synaptic reorganization following kainate-induced seizure, the time course alterations of the corresponding receptors were evaluated. Additionally, using histological preparations, the temporal profile of neuronal degeneration and hypertrophy of resident astroglial cells were also studied. [125I]Insulin-like growth factor I binding was found to be decreased transiently in almost all regions of the hippocampal formation at 12 h following treatment with kainic acid. The dentate hilar region however, exhibited protracted decreases in [125I]insulin-like growth factor I receptor sites throughout (i.e. 30 days) the study. [125I]Insulin-like growth factor II receptor binding sites in the hippocampal formation were found to be differentially altered following systemic administration of kainic acid. A significant decrease in [125I]insulin-like growth factor II receptor sites was observed in CA1 subfield and the pyramidal cell layer of the Ammon's horn at all time points studied whereas the hilar region and the stratum radiatum did not exhibit alteration at any time. A kainate-induced decrease in [125I]insulin receptor binding was noted at all time points in the molecular layer of the dentate gyrus whereas binding in CA1-CA3 subfields and discrete layers of the Ammon's horn was found to be affected only after 12 h of treatment. These results, when analysed with reference to the observed histological changes and established neurotrophic/protective roles of insulin-like growth factors and insulin, suggest possible involvement of these growth factors in the cascade of neurotrophic events that is associated with the reorganization of the hippocampal formation observed following kainate-induced seizures.

Autoradiographical and immunohistochemical analysis of receptor localization in the central nervous system.

Quantitative receptor autoradiographic methods have been widely used over the past two decades. Some of the advantages and limitations of these techniques are reviewed here. Comparison with immunohistochemical and in situ hybridization methods is also highlighted, as well as the use of these approaches to study receptor gene over-expression in cell lines. Together, data obtained using these various methodologies can provide unique information on the potential physiological roles of a given receptor protein and/or binding sites in various tissues.

Localization of sulfated glycoprotein-2/clusterin mRNA in the rat brain by in situ hybridization.

Sulfated glycoprotein-2 (SGP-2) gene expression seems to be constitutively expressed in a variety of tissues and organs, although levels of expression vary widely from one tissue to the other. SGP-2, also known as clusterin, has been reported to be expressed in the central nervous system (CNS). Some possible roles for brain SGP-2 have been postulated. In order to provide a substrate for a better understanding of the functions of this glycoprotein in the CNS, we investigated the detailed anatomical and cellular distribution of SGP-2 mRNA in the adult rat brain as well as the variation in its cellular expression after excitotoxin lesion. Transcripts for SGP-2 were found to be distributed throughout the rat CNS, although regional differences in their prevalence were readily observed. The ependymal lining of the ventricles showed the highest level of expression followed by various gray matter areas, some of which contained very intensively labeled cells. These cells were mostly found among several hypothalamic and brainstem nuclei, the habenular complex, as well as in the ventral horn of the spinal cord, which displayed striking hybridization signals over motoneurons. Occasional cells expressing high levels of SGP-2 transcripts were found in fiber tracts. Highly SGP-2 mRNA-positive resting glial cells were mainly located near the glial limitans and blood vessels. Two areas of relatively low constitutive SGP-2 mRNA expression are shown to produce strong hybridization signals 10 days after the local administration of the excitotoxin kainic acid. This overexpression of SGP-2 transcripts appears to involve GFAP-positive cells. Taken together, these results indicate that in the intact adult rat CNS, various cell populations, including neurons, constitutively express SGP-2 transcripts, whereas in the injured brain, reactive astrocytes become the major producers.

Quantitative autoradiographic localization of [125I]insulin-like growth factor I, [125I]insulin-like growth factor II, and [125I]insulin receptor binding sites in developing and adult rat brain.

Insulin-like growth factors I and II (IGF I and IGF II) and insulin itself, which are structurally related polypeptides, play an important role in regulating brain growth and development as well as in the maintenance of its normal functions during adulthood. In order to provide a substrate for the better understanding of the roles of these growth factors, we have investigated the anatomical distribution as well as the variation in the density of [125I]IGF I, [125I]IGF II, and [125I]insulin receptor binding sites in developing and adult rat brain by in vitro quantitative autoradiography. The distributional profile of [125I]IGF I, [125I]IGF II, and [125I]insulin receptor binding sites showed a widespread but selective regional localization throughout the brain at all stages of development. The neuroanatomic regions which exhibited relatively high density of binding sites with each of these radioligands include the olfactory bulb, cortex, hippocampus, choroid plexus, and cerebellum. However, in any given region, receptor binding sites for IGF I, IGF II, or insulin are concentrated in anatomically distinct areas. In the cerebellum, for example, [125I]IGF II receptor binding sites are concentrated in the granular cell layer, [125I]insulin binding sites are localized primarily in the molecular layer, whereas [125I]IGF I receptor binding sites are noted in relatively high amounts in granular as well as molecular cell layers. The apparent density of sites recognized by each radioligand also undergoes remarkable variation in most brain nuclei, being relatively high either during late embryonic (i.e., IGF I and IGF II) or early postnatal (i.e., insulin) stages and then declining gradually to adult levels around the third week of postnatal development. These results, taken together, suggest that each receptor-ligand system is regulated differently during development and thus may have different roles in the process of cellular growth, differentiation, and maintenance of the nervous system. Furthermore, the localization of [125I]IGF I, [125I]IGF II, and [125I]insulin receptor binding sites over a wide variety of physiologically distinct brain regions suggests possible involvement of these growth factors in a variety of functions associated with specific neuronal pathways.

Possible functions of a new genetic marker in central nervous system: the sulfated glycoprotein-2 (SGP-2).

This brief review discusses the recent characterization in the brain of a gene coding for a protein that may be involved in programmed cell death and/or brain plasticity. We will term it sulfated glycoprotein-2 (SGP-2), the name corresponding to the first cDNA characterized. Recent studies have demonstrated the overexpression of this sulfated glycoprotein in various CNS disorders, such as certain gliomas, Alzheimer's disease and epilepsy, as well as after experimental brain injury in animals where different cell types were undergoing tissue remodelling or cell death. In peripheral tissues, SGP-2 gene expression has been found to be strikingly increased following experimental manipulations in which cells of injured tissues were undergoing programmed cell death or apoptosis. The results reported thus far are intriguing and suggest the possible involvement of SGP-2 in apoptotic mechanisms as well as its interaction with components of the immune system possibly associated with cell death in neurodegenerative disorders.

Quantitative autoradiographic localisation of [125I]endothelin-1 binding sites in spinal cord and dorsal root ganglia of the rat.

The autoradiographic distribution of [125I] endothelin (ET)-1 binding sites was studied in the spinal cord and dorsal root ganglia of developing and adult rat. In the spinal cord, high density of [125I]ET-1 binding sites were diffusely distributed throughout the grey matter whereas in the ganglia discrete silver grains were localised primarily on the satellite cells. A variation in the density of binding sites was evident, particularly in the spinal cord, during development. These data, in conjunction with other reports, suggest a possible neuromodulatory role for ET-1 in spinal cord and dorsal root ganglia of the rat.

Human gliomas and epileptic foci express high levels of a mRNA related to rat testicular sulfated glycoprotein 2, a purported marker of cell death.

Clone pTB16 has been isolated by differential screening of a human glioma cDNA library. Northern blot analysis has shown that pTB16 expression is several times (greater than 11-fold) higher in gliomas than in a primitive neuroectodermal tumor. This observation was supported by in situ hybridization and extended to nine other gliomas. Expression was virtually absent in adenocarcinoma cells metastasized to brain. Malignant gliomas showed stronger hybridization than benign gliomas, while blood capillaries did not show hybridization. pTB16 mRNA was also shown to be expressed in established glioma cell lines and at high levels in epileptic foci, indicating that expression of the gene may be limited to certain cell types and that its upregulation is not merely a consequence of cellular proliferation. Nucleotide sequence analysis identified pTB16 as the human counterpart for rat testicular sulfated glycoprotein 2 (SGP-2), whose function in the reproductive system remains unknown. Although SGP-2 transcripts, and hence pTB16, were recently shown to be increased in neurodegenerative diseases such as scrapie in hamsters and Alzheimer disease in humans, our observations with brain tumors and epilepsy are suggestive of a role for pTB16 in neuropathologies in general and support the hypothesis of its involvement in tissue remodeling and cell death.

Arginine-vasopressin in anterior pituitary cells: in situ hybridization of mRNA and ultrastructural localization of immunoreactivity.

The hypothalamic nonapeptide arginine-vasopressin (AVP) exerts several distinct receptor-mediated actions on pituitary cells. Although hypothalamic AVP reaches the anterior pituitary via well-defined pathways, there is now accumulating evidence that AVP may also be produced endogenously in anterior pituitary cells. Using in situ hybridization, we demonstrate here the presence of AVP mRNA in the anterior pituitary of the rat. The observed grain density over pituitary cells was, however, greater than 10-fold lower than the one observed over AVP producing neurons present in the supraoptic and paraventricular nuclei of the hypothalamus. Immunoelectron microscopic analysis using two different AVP-specific antibodies revealed that the distribution of AVP-like immunoreactivity (AVP-LI) in the anterior pituitary is cell-specific. AVP-LI is most abundant in corticotrophs, followed by lactotrophs, gonadotrophs and thyrotrophs. On the other hand, there is complete absence of AVP-LI from somatotrophs. Interestingly, all pituitary cells in which AVP-LI is detected also represent potential target sites for AVP action. A minor fraction of AVP-LI was found to be membrane-associated and may originate, at least in part, from extrapituitary sources. This fraction likely represents receptor-bound peptide. The bulk of AVP-LI, however, was present in the cellular cytoplasm, not associated with any specific ultracellular structure. Specifically in corticotrophs, AVP-LI was excluded from secretory granules. However, our finding of AVP mRNA in anterior pituitary cells indicates that intracellular AVP-LI includes endogenously produced peptide, suggesting a paracrine and/or autocrine action.

Growth factors and lymphokines: modulators of cholinergic neuronal activity.

It is well known that various markers of the cholinergic synapse are altered in Alzheimer's Disease. Much interest is currently focussing on the evaluation of the possible efficacy of certain growth factors, especially nerve growth factor (NGF), to reduce or reverse cholinergic neuronal losses. Here we report that other growth factors (epidermal growth factor and insulin-like growth factor I) and a lymphokine, interleukin-2, are able to block acetylcholine release in the rat hippocampus. This suggests that while certain growth factors like NGF may have positive effects on the cholinergic neuron, others may act as "negative" factors on this neuronal population.

Neurochemical deficits in pathological brain aging: specificity and possible relevance for treatment strategies.

Normal brain aging is accompanied by the losses of certain neuronal populations and the appearance of structures such as neuronal plaques and neurofibrillary tangles. Additionally, various neurotransmitter systems are altered in the elderly, although marked variations are observed between individuals, suggesting important differences between successful and unsuccessful aging. In certain pathological conditions, only certain features of normal aging are exacerbated. For example, the densities of forebrain cholinergic neurons are markedly decreased in cortical and hippocampal (but not striatal) areas in Alzheimer's disease. We discuss here the comparative alterations of cholinergic markers in certain neurological disorders such as Alzheimer's disease, Parkinson's disease, and the combined pathology. Differential alterations of brain cholinergic profile are observed in each disorder, this most likely having functional significance. We also found that muscarinic receptors of the M2 subtype act as negative autoreceptors to decrease brain acetylcholine release, whereas nicotinic agonists induced the opposite effect. This suggests that blockade of negative M2 or stimulation of positive nicotinic autoreceptors could have beneficial effects in Alzheimer's disease. Additionally, modulation of heteroreceptor activation such as the serotonergic or interleukin-2 sites located on or in proximity to cholinergic nerve terminals could offer alternate strategies for the treatment of cholinergic deficits in pathological brain aging.