Evidence that 5 alpha-pregnan-3 alpha-ol-20-one is the metabolite responsible for progesterone anesthesia

Tadpoles (Rana catesbeiana) were anesthetized with [3H]-progesterone (PG) in order to identify the steroid responsible for anesthesia. PG and its metabolites in brain were resolved (HPLC) and quantitated following sacrifice at different behavioral endpoints. The results support the hypothesis that 5 alpha-pregnan-3 alpha-ol-20-one mediates the loss and the return of the righting response. The observed levels for PG were not consistent with such a role. Neither PG nor 5 alpha-pregnan-3 alpha-ol-20-one produced analgesia.     

Disruption of the hyaluronan-based extracellular matrix in spinal cord promotes astrocyte proliferation

Astrocyte proliferation is tightly controlled during development and in the adult nervous system. In the present study, we find that a high-molecular-weight (MW) form of the glycosaminoglycan hyaluronan (HA) is found in rat spinal cord tissue and becomes degraded soon after traumatic spinal cord injury. Newly synthesized HA accumulates in injured spinal cord as gliosis proceeds, such that high-MW HA becomes overabundant in the extracellular matrix surrounding glial scars after 1 month. Injection of hyaluronidase, which degrades HA, into normal spinal cord tissue results in increased numbers of glial fibrillary acidic protein (GFAP)-positive cells that also express the nuclear proliferation marker Ki-67, suggesting that HA degradation promotes astrocyte proliferation. In agreement with this observation, adding high- but not low-MW HA to proliferating astrocytes in vitro inhibits cell growth, while treating confluent, quiescent astrocyte cultures with hyaluronidase induces astrocyte proliferation. Collectively, these data indicate that high-MW HA maintains astrocytes in a state of quiescence, and that degradation of HA following CNS injury relieves growth inhibition, resulting in increased astrocyte proliferation.     

Retinal neurite growth on astrocytes is not modified by extracellular matrix, anti-L1 antibody, or oligodendrocytes.

Two factors that may influence the course of axonal regeneration in the central nervous system (CNS) are extracellular matrix (ECM) and cell surface molecules that may enhance or inhibit neurite outgrowth. Whereas cultured astrocytes have been reported to be a good substratum for neurite outgrowth, there is recent evidence that cultured oligodendrocytes are inhibitory. To test the influences of 1) ECM components, 2) the L1 adhesion molecule, and 3) the inhibitory potential of mature oligodendrocytes in the astrocytic environment, we have utilized a culture system in which neurites from embryonic rat retina grow vigorously on astrocyte monolayers. The major ECM components were assembled in neonatal rat cortical astrocyte-retina co-cultures only when the medium contained serum. In electron microscopic studies of serum containing cultures, retinal neurites were seen to be related to astrocyte surfaces but rarely were found in contact with ECM; in serum-free medium the association between neurites and astrocytes was similar. In addition, the growth of neurites was vigorous whether ECM was present or absent. Presence of antibodies against the cell surface adhesion molecule L1 did not inhibit retinal neurite elongation on glial fibrillary acidic protein-positive astrocytes. When oligodendrocytes from adult rat spinal cord were combined with the astrocytes, retinal neurites grew as well on the mixed glial population as on astrocytes alone. Immunostaining for galactocerebroside showed many oligodendrocyte processes to be aligned in the direction of neurite growth, suggesting association between the two cell types. This association was verified by electron microscopy. Furthermore, retinal explants extended neurites among myelin basic protein-positive oligodendrocytes cultured without astrocytes. Thus, the astrocyte surface is a strong promoter of neurite growth from embryonic rat retina. This growth did not depend upon either ECM or the L1 adhesion molecule. Because neurites grew on astrocytes in the presence of mature oligodendrocytes or among oligodendrocytes alone, we conclude that oligodendrocytes do not inhibit neurite growth under certain conditions.     

Astroglia-released neurite growth-inducing activity for embryonic hippocampal neurons is associated with laminin bound in a sulfated complex and free fibronectin

Neurons from embryonic (E18) rat hippocampus were chosen to identify and characterize neurite growth-stimulating proteins accumulating in serum-free conditioned media (CM) obtained from primary or secondary cultures of cerebral astrocytes (less than 5% nonglial cells) using a quantitative cell culture bioassay. CM were fractionated by FPLC on an anion exchange column (Mono Q) and by gel filtration (Superose 6). Column fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and enzyme-linked immunosorbent assay (ELISA) using antibodies to laminin (LN) and fibronectin (FN). The neurite-promoting activity (NPA) was tested by incubating aliquots of the eluted fractions with poly-L-lysine precoated glass coverslips prior to addition of neurons suspended in chemically defined medium. We provide evidence that the NPA in astroglial CM could be assigned mainly to a negatively charged, highly sulfated LN complex consisting predominantly of the B-chains of LN and presumably a sulfated proteoglycan that was sensitive for chondroitinase and to a lower degree to heparinase degradation. In addition, a smaller proportion of the NPA was associated with uncomplexed LN and free FN. FN reached approximately 10 times the concentration of LN in astroglial CM. As revealed by immunofluorescence microscopy, both LN and FN are simultaneously expressed by cultured astrocytes; however, only the production of FN, measured by ELISA, increased during the time astrocytes were in culture, whereas the release of LN remained unchanged. We conclude that, besides the most active LN complex, FN bound to a polycationic matrix is able to induce neurite growth in hippocampal neurons in vitro.     

Beta-hydroxy-butyrate alters the extracellular content of S100B in astrocyte cultures

Astrocytes have a variety of roles in maintaining neural tissue physiology, including energetic support, uptake and metabolism of glutamate and secretion of neurotrophic factors. Glutamate toxicity has been implicated in neurodegenerative disorders associated with conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. Glucose is the main energetic substrate for brain cells but, in some circumstances, the ketone bodies are used as a supplementary source and have been suggested to be neuroprotective agents against seizure disorders. Here, we investigate some possible biochemical changes in astrocyte cultures induced by beta-hydroxy-butyrate, the predominant blood ketone body. Its effect upon S100B secretion, astrocyte morphology and glutamate uptake was particularly investigated. S100B, a calcium-binding protein expressed and secreted by astrocytes, has neurotrophic activity and a possible role in epileptogenesis. Cell morphology was investigated by phase-contrast microscopy and immunocytochemistry for actin, GFAP and S100B. Our data show that beta-hydroxy-butyrate induces dramatic changes in astrocyte morphology and, independent of this, causes changes in the extracellular content of S100B. We observed an increment in S100B 1 h after beta-hydroxy-butyrate addition and a decrease 24 h later. No changes were observed in glutamate uptake. These astrocytic modifications may be associated with reduced neuronal excitability observed in the ketogenic condition.     

Neural recognition molecule NB-2 of the contactin/F3 subgroup in rat: Specificity in neurite outgrowth-promoting activity and restricted expression in the brain regions.

NB-2, a neural cell recognition molecule of the contactin/F3 subgroup, promoted neurite outgrowth of the cerebral cortical neurons but not the hippocampal neurons. NB-2 in rat became apparent after birth at protein level, reaching a maximum at postnatal day 14 in the cerebrum and postnatal day 3 in the cerebellum. NB-2 in the cerebellum declined abruptly thereafter. In situ hybridization demonstrated that NB-2 mRNA was highly expressed in regions implicated in the central auditory pathway, including the cochlear nuclei, superior olive, inferior colliculi, medial geniculate nuclei, and auditory cortex. In addition, a high level of NB-2 expression was observed in the accessory olfactory bulb, thalamic nuclei, facial nucleus, and inferior olive. By immunohistochemistry, intense immunoreactivity against NB-2 was also detected in the auditory pathway. Thus, NB-2 is expressed in highly restricted brain regions, including the auditory system, suggesting that it plays specific roles in the development and/or maturation of the regions.     

Nardosinone, the first enhancer of neurite outgrowth-promoting activity of staurosporine and dibutyryl cyclic AMP in PC12D cells

Nardosinone was isolated as an enhancer of nerve growth factor (NGF) from Nardostachys chinensis [Neurosci. Lett. 273 (1999) 53]. Nardosinone (0.1-100 microM) enhanced dibutyryl cyclic AMP (dbcAMP, 0.3 mM)- and staurosporine (10 nM)-induced neurite outgrowth from PC12D cells in a concentration-dependent manner. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, partially blocked enhancements of dbcAMP (0.3 mM)- or staurosporine (10 nM)-induced neurite outgrowth by nardosinone. Nardosinone alone had no effect on the phosphorylation of MAP kinase. The dbcAMP-induced increase in phosphorylation of MAP kinase was not affected by nardosinone. Staurosporine almost unaffected the phosphorylation of MAP kinase, and nardosinone potentiated the staurosporine-induced neurite outgrowth without stimulation of the phosphorylation of MAP kinase. Since it is known that MAP kinase signaling is required for neurite outgrowth in PC12D cells, these results suggest that nardosinone enhances staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying both the MAP kinase-dependent and -independent signaling pathways of dbcAMP and staurosporine. It is also suggested that nardosinone enhances a downstream step of MAP kinase in the MAP kinase-dependent signaling pathway. Nardosinone is the first enhancer of the neuritogenic action of dbcAMP and staurosporine and may become a useful pharmacological tool for studying the mechanism of action of not only NGF but also both the neuritogenic substances.     

Neuronal migration in cerebellar microcultures is inhibited by antibodies against a neurite outgrowth domain of laminin.

The functional role of laminin in neuronal migration was investigated by using polyclonal antibodies or their divalent (Fab')2 fragments to a neurite outgrowth promoting domain of the B2 chain of laminin in a cerebellar microculture system widely recognized as a model for neuronal migration. We show here that these antibodies or their (Fab')2 fragments totally inhibit migration of the mouse cerebellar granule cells along the glial and other neuronal cell processes. Antibodies to native laminin or other control antibodies have no inhibitory effect. Immunocytochemical analysis of the cerebellar microcultures indicates that the functional role of these antibodies may relate to the fact that the punctate deposits of laminin and its neurite outgrowth promoting domain accumulate in between the migrating neurons and the glial cells. These data provide the first direct evidence for the functional role of laminin and its neurite outgrowth domain in neuronal migration in the mammals. They further suggest that a neuronal cell surface contact with the extracellular deposits of a neurite outgrowth domain of the B2 chain of laminin may mediate neuronal-glial interactions.     

Immunohistochemical study of the extracellular matrix proteins laminin, fibronectin and type IV collagen in secretory meningiomas.

The extracellular matrix plays a pivotal role in numerous cellular functions during normal and pathological processes. Secretory meningiomas are rare histological meningioma subtypes that have benign behavior, are highly vascularized and are frequently accompanied by massive peritumoral edema. The aim of this study was to assess in secretory meningiomas the immunohistochemical expression of laminin, fibronectin and type IV collagen, proteins found in the extracellular matrix. Extracellular matrix proteins were evaluated in samples from six secretory meningiomas using a semiquantitative scale ranging from not detected (0) to marked (3). Laminin expression was not detected in two cases, but was minimal in one, moderate in one and marked in the remaining cases. Fibronectin expression was absent in two cases, minimal in two, moderate in one and marked with generalized distribution in the remaining case. Type IV collagen expression was minimal in three cases, moderate in two and marked with generalized distribution in the remaining case. Our results are indicative of significant neoangiogenic activity. Meningiomas increase in size through increased production of extracellular matrix; furthermore, the proliferation of cells typically associated with neoplasia requires considerable interaction with the extracellular matrix.     

Domain-specific antibodies against the B2 chain of laminin inhibit neuronal migration in the neonatal rat cerebellum

Although the spatial and temporal patterns of neuronal migration have been analyzed in great detail, little direct evidence is available as to what extracellular matrix molecules are involved. Because there is indirect evidence implicating the extracellular matrix protein laminin in neuronal migration, we investigated the effects of antibodies against a synthetic peptide derived from a neurite outgrowth domain of the B2 chain of laminin on neuronal migration in living cerebellar slices. We show by using infrared video microscopy that divalent Fab₂ fragments of these antibodies inhibit granule neuronal movement in living slices of (P8) rat cerebellum. This inhibition of neuronal movement manifests itself by cessation of both radial and horizontal translocations of nuclei inside the granule neuronal processes. Fab₂ fragments of antibodies against the intact (native) laminin molecule or Fab₂ fragments from the preimmune serum do not affect nuclear translocation. Immunocytochemistry shows binding of the divalent Fab₂ fragments of the B2 chain-specific antibodies to the Purkinje and Bergmann glial cell areas, and as punctate deposits in between the cells of the external granule cell layer. Native laminin antibodies bind to the basement membranes, and binding of the Fab₂ fragments from the preimmune sera cannot be demonstrated. These results indicate that neuronal migration in the postnatal rat cerebellum in vivo involves nuclear translocation that can be inhibited by antibodies against a neurite outgrowth domain of the B2 chain of laminin. Thus, migration of cerebellar granule neurons may depend on the interaction between a neurite outgrowth domain of the B2 chain of laminin and neuronal cytoskeleton involved in nuclear movement. © 1995 Wiley-Liss, Inc.     

Ability of developing epithelia to attract neurite outgrowth in culture is not correlated with the appearance of laminin

Using a new technique for organ explants that facilitates the visualization of developing epithelia, we tested the abilities of salivary gland and lung rudiments to support de novo axonal outgrowth from the embryonic submandibular ganglion. We confirmed that salivary gland epithelia, but not lung epithelia, are able to support axonal outgrowth. This neurite outgrowth is also supported by salivary gland epithelia that have been lightly fixed with paraformaldehyde. When given a choice of both salivary gland and lung epithelia as a substrate for axonal outgrowth, the submandibular ganglion neurons showed an absolute preference for the salivary gland. Immunohistochemical localization of laminin was performed on whole mounts of developing epithelia after growing neurites were localized with a histochemical stain for esterase. Areas of lung epithelium devoid of any neurite outgrowth contained substantial immunoreactivity for laminin. In addition, Western blot analyses of extracts of embryonic lung and salivary gland indicate that the same amount of laminin or more is present on a per protein basis in the lung as in the salivary gland. An antiserum directed against laminin and a monoclonal antibody that blocks axonal regeneration on basal laminae in vitro (INO) were unable to block the outgrowth of axons over fixed epithelia. This suggests that molecules other than laminin are responsible for the preferential growth of axons over the salivary gland epithelia.     

Evidence that changes in spine neck resistance are not responsible for expression of LTP

From modeling studies it is known that changes in spine neck resistance can influence the shape of the non-linear curve relating synaptic current to synaptic conductance if the resistance of the neck approaches the synaptic input resistance. Such work also indicates that the effects of resistance will be much more pronounced for fast rather than slow synaptic currents. Accordingly, a reduction in neck resistance could produce an increase in the rapid responses generated by the quisqualate/AMPA class of glutamate receptors while only minimally affecting the slower NMDA receptor-mediated responses and thus account for the pattern of changes known to be associated with long-term potentiation (LTP). This hypothesis predicts that large reductions in synaptic conductance should have disproportionate effects on potentiated versus control responses. This was tested by using field potential recordings of synaptic currents in CA1 pyramidal cells in hippocampal slices in response to stimulation of Schaffer/commissural inputs that either received LTP-inducing stimulation or did not. Two manipulations were used to systematically reduce synaptic conductances: reductions of extracellular Ca++ and partial blockade of postsynaptic receptors. Reductions of synaptic field potentials by 40-75% by either method at control synapses were accompanied by equivalent reductions at previously potentiated synapses. These results suggest that LTP expression is not due to a change in the curves relating synaptic current to synaptic conductance as would be predicted by the spine resistance hypothesis.     

Evidence that cholecystokinin-enhanced retention is mediated by changes in opioid activity in the amygdala

Mice, partially trained to avoid footshock in a T-maze, showed enhanced retention relative to vehicle-injected mice when treated peripherally with arecoline, D-amphetamine, cholecystokinin octapeptide (CCK-8), epinephrine or naloxone. Both intra-amygdaloid and intraventricular injections of beta-endorphin resulted in amnesia. D-amphetamine and arecoline blocked the amnestic effect of beta-endorphin administered into the amygdala but it required higher doses for CCK-8, epinephrine and naloxone to block the amnestic effect of beta-endorphin. The effects of CCK-8, epinephrine and naloxone showed a differential ability to block amnesia induced by beta-endorphin intraventricularly with epinephrine and naloxone preventing amnesia but CCK-8 not improving retention. This data suggests that the memory enhancement produced by peripherally administered CCK-8 involves the amygdala and that both CCK-8 and epinephrine interact with opioid amnestic mechanisms within the amygdala to alter memory processing.     

Evidence to suggest that extracellular acetate is accumulated by rat hippocampal cholinergic nerve terminals for acetylcholine formation and release

It is well established that extracellular choline is transported into central cholinergic nerve terminals by `high' and `low' affinity processes to form the neurotransmitter acetylcholine (ACh). The intent of the present investigation was to ascertain whether extracellular acetate might also be transported into central cholinergic nerve terminals to form ACh. To test this possibility, rat hippocampal tissue was incubated with varying concentrations of extracellular [1-¹⁴C]acetate (0.1–100 μM) and the uptake of [1-¹⁴C]acetate and the amount of [¹⁴C]ACh formed by the tissue determined. The results indicated that the uptake of extracellular [1-¹⁴C]acetate was temperature-dependent and saturable having an apparent Michaelis constant (K_m) of 22 μM. The formation of [¹⁴C]ACh in the tissue as a function of extracellular [1-¹⁴C]acetate appeared to occur by both `high' and `low' affinity processes with apparent K_m values of 0.5 and 19.6 μM, respectively. In other experiments, three inhibitors (lithium, allicin and sodium) of acetyl CoA synthetase (EC 6.2.1.1 acetate: CoA ligase), the enzyme which converts acetate to acetyl CoA when ATP and CoA are present, inhibited [1-¹⁴C]acetate uptake and the amount of [¹⁴C]ACh formed from that [1-¹⁴C]acetate. Additionally, vesamicol, an inhibitor of ACh transport into synaptic vesicles, blocked the filling of a synaptic vesicle-enriched fraction of hippocampal tissue with newly synthesized [¹⁴C]ACh formed from extracellular [1-¹⁴C]acetate. High K⁺ depolarization of hippocampal tissue loaded with extracellular [1-¹⁴C]acetate not only increased the synthesis but also the release of [¹⁴C]ACh. These results suggest that extracellular acetate is recycled by rat hippocampal cholinergic nerve terminals for the formation and release of ACh. They also suggest that the enzyme acetyl CoA synthetase mediates extracellular acetate uptake into hippocampal cholinergic nerve terminals by metabolizing it to acetyl CoA and thereby creating a diffusion gradient for it to follow. © 1997 Elsevier Science B.V. All rights reserved.     

Sequential effects of astroglial-derived factors on neurite outgrowth: initiation by protease inhibitors and potentiation by extracellular matrix components.

Astroglial-conditioned medium (GCM) induced two distinct, but intimately related, phases of neuritogenesis in NB2a/d1 neuroblastoma cells--a "rapid-outgrowth," unstable phase, and a delayed, relatively stable phase, which are apparently regulated by glial-derived protease inhibitors and laminin, respectively. The initial rapid outgrowth (less than 4 hr) may be mediated by inhibition of a thrombin-like protease, present as a serum component and/or adsorbed to the outer neuronal surface, since (1) a similar effect was obtained by serum removal or by adding the specific thrombin inhibitor, hirudin; (2) exogenous thrombin inhibited the rapid outgrowth of neurites by GCM; and (3) cell-free enzyme assays confirmed the presence of thrombin-inhibitory activity in GCM. Although neurites induced by removal of serum removal or hirudin addition are rapidly resorbed following serum replenishment or hirudin depletion, GCM-induced neurites continued to elongate after GCM removal, indicating that GCM contained additional neurite-promoting factors. Anti-laminin antiserum did not inhibit the initial elaboration of neurites by GCM but prevented their continued elongation. Anti-laminin antiserum had no affect on neurite outgrowth induced by serum deprivation. The more protracted, second phase of neurite outgrowth could also be achieved by the addition of soluble purified laminin to undifferentiated cells. Unlike neurites at 4 hr, neurites at 24 hr were no longer dependent on the protease inhibitors in GCM, since exogenous thrombin no longer caused them to retract. Simultaneous addition of thrombin and anti-laminin antiserum with GCM had identical inhibitory effects on continued neurite elaboration at 24 hr as did anti-laminin antiserum without thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMDAR LTP and LTD induction: 2B or Not 2B...is that the question?

NMDA receptor (NMDAR) dependent forms of synaptic plasticity are thought to play critical roles in many aspects of CNS function and dysfunction, from learning and memory to addiction. NMDARs are heteromeric tetramers principally comprised of two NR1 subunits and two of four varieties of NR2 subunits (NR2A-2D). Recently, it has been proposed that specific NR2 subtypes subserve distinct roles in NMDAR-dependent long-term potentiation (LTP) and long-term depression (LTD). Here, we will review this literature, and describe an existing countervailing hypothesis, the charge-transfer hypothesis, which postulates that the total charge transfer through NMDARs, rather than specific subunits, dictates the polarity of synaptic plasticity. We will propose that a modification of the charge-transfer hypothesis, to include the possible involvement of protein-protein interactions imparted by distinct NR2 subunits, best fits the existing data.     

Evidence that 5α-pregnan-3α-ol-20-one is the metabolite responsible for progesterone anesthesia

Tadpoles (Rana catesbeiana) were anesthetized with [³H]-progesterone (PG) in order to identify the steroid responsible for anesthesia. PG and its metabolites in brain were resolved (HPLC) and quantitated following sacrifice at different behavioral endpoints. The results support the hypothesis that 5α-pregnan-3α-ol-20-one mediates the loss and the return of the righting response. The observed levels for PG were not consistent with such a role. Neither PG nor 5α-pregnan-3α-ol-20 produced analgesia.     

Central noradrenergic activity is responsible for nitroglycerin-induced cardiovascular effects in the nucleus tractus solitarii

The studies show that unilateral microinjection of nitroglycerin (NTG) into nucleus tractus solitarii (NTS) produce dose-dependent decreases in mean arterial pressure (MAP) and heart rate, but injection of sodium nitroprusside (SNP) into the area induced slight effects. Hypotensive responses to NTG injected into the NTS showed that the compound was 20 times more potent than after i.v. administration. Responses to NTG injected into the NTS were abolished in an additive fashion by either rauwolscine, an alpha 2-adrenoceptor antagonist, or guanethidine which inhibits release of norepinephrine (NE). Injection of prazosin, an alpha 1-adrenoceptor antagonist, into the NTS reduced the hypotensive responses of NTG, but did not alter the bradycardia induced by the drug. Tachycardic responses following i.v. administration of either NTG or SNP were attenuated by bilateral injection of rauwolscine into the NTS, whereas only hypotensive responses to i.v. NTG were reduced by the pretreatment. NTG produced a dose-dependent increase in concentrations of 3,4-dihydroxyphenylalanine in media bathing medulla-pons, which were quantified using high-performance liquid chromatography with electrochemical detection. NE and 3,4-dihydroxyphenylglycol concentrations in media of incubated medulla-pons slices were simultaneously increased following higher concentrations of NTG. The results suggest that NTG in the NTS induces hypotensive and bradycardiac responses, and an increase in turnover of NE may stimulate alpha 2-adrenoceptors and be responsible for the effects of the drug. The NTS may contribute a component of action to the cardiovascular effects of intravenous NTG. The cardiovascular responses of intravenous SNP appear to involve peripheral action.     

Cultured astrocyte proliferation induced by extracellular guanosine involves endogenous adenosine and is raised by the co-presence of microglia

Extracellular adenosine (Ado) and ATP stimulate astrocyte proliferation through activation of P(1) and P(2) purinoceptors. Extracellular GTP and guanosine (Guo), however, that do not bind strongly to these receptors, are more effective mitogens than ATP and Ado. Exogenous Guo, like GTP and 5'-guanosine-betagamma-imidotriphosphate (GMP-PNP), dose-dependently stimulated proliferation of rat cultured astrocytes; potency order GMP-PNP > GTP > or = Guo. The mitogenic effect of Guo was independent of the extracellular breakdown of GTP to Guo, because GMP-PNP, a GTP analogue resistant to hydrolysis, was the most mitogenic. In addition to a direct effect on astrocytes, Guo exerts its proliferative activity involving Ado. Exogenous Guo, indeed, enhanced the extracellular levels of endogenous Ado assayed by HPLC in the medium of cultured astrocytes. Culture pretreatment with Ado deaminase (ADA), that converts Ado into inosine, reduced but did not abolish Guo-induced astrocyte proliferation whereas erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), that inhibits ADA activity, amplified Guo effect. Moreover, the mitogenic activity of Guo was partly inhibited by 8-cyclopentyl-1,3-dipropylxanthine and alloxazine, antagonists of Ado A(1) and A(2B) receptors, respectively. Also microglia seem to be a target for the action of Guo. Indeed, the mitogenic effect of Guo on astrocytes was: i) increased proportionally to the number of microglial cells present in the astrocyte cultures; ii) amplified when purified cultures of astrocytes were supplemented with conditioned medium deriving from Guo-pretreated microglial cultures. These data indicate that the mitogenic effects exerted by exogenous Guo on rat astrocytes are mediated via complex mechanisms involving extracellular Ado and microglia-derived soluble factors.