The functional role(s) of taurine in the CNS has not yet been determined unambiguously. However, its absence is known to have a negative impact on brain development, and it has also been implicated in osmotic regulation, neuromodulation and neurotransmission (Huxtable, 1989). Many of these studies have utilized a variety of in vitro preparations from which the release or efflux of preloaded radiolabeled taurine has been monitored in response to a variety of stimuli. The focus of this chapter is on work done in this laboratory following HPLC analysis of the excitatory amino acid agonist evoked release of endogenous taurine from cultured cerebellar neurons containing a mixture of approximately 90% excitatory granule cells and 57% inhibitory (GABAergic) interneurons.

Neuronal cultures from rat cerebellum consisting of approximately 90% glutamatergic granule neurons, 5-7% GABAergic inhibitory interneurons, and 3-5% glial cells, were treated for four days with 50 microM kainic acid (KA) to determine the cellular origin of released endogenous neuroactive substances. KA, known to be selectively toxic to GABAergic neurons, caused an estimated 80% decrease in glutamic acid decarboxylase (GAD) immunofluorescence. Furthermore, K(+)-stimulated release of GABA decreased to 20% of control values, and did not return to control levels in cultures "recovered" two days in KA-free media, suggesting the loss of inhibitory interneurons. Similarly, adenosine and taurine showed decreased K(+)-stimulated release, which was unrecoverable when KA was removed from the medium. K(+)-stimulated release of glutamate and aspartate also decreased by 50% and 70%, respectively, after chronic KA treatment. In contrast, however, this release returned to control levels in recovered cultures. All decreases in K(+)-stimulated release were prevented by concurrent treatment with KA and the KA antagonist 6-cyano-6-nitroquinoxaline-2,3-dione (CNQX), indicating that a receptor-mediated mechanism was involved. We conclude that, in these cultures, most of the K(+)-stimulated release of adenosine and taurine originates from the GABAergic interneurons, the basket and stellate cells, which are selectively killed by the KA treatment. The data also strongly suggest that glutamate and aspartate, the levels of which recover after KA treatment, originate mainly from the granule neurons.

The evidence presented, together with the lack of solid evidence for a specific receptor site, strongly suggests that taurine does not act as a traditional neurotransmitter in the CNS. In fact, the properties seen to be governing its efflux from both glial cells and neurons argue strongly in favor of a primary role in volume regulation. However, subsequent to its release into the extracellular space, it is possible that the inherent neuroactive properties (e.g., inhibitory neuromodulation and Ca(2+)-level modulation) may be important at the synapse, at the cell plasma membrane, and intracellularly in further directing the level of neuronal activity. Whether or not the levels of released taurine are great enough to sustain these effects has still to be determined.

Primary astrocyte cultures derived from neonatal rat cerebral cortex were treated for 5 min with 0.5 mM or 5.0 mM AlCl3, and the incubation medium was analyzed by HPLC for the content of released glutamate (Glu), taurine (Tau), serine (Ser) and the nucleoside adenosine (Ade). At 0.5 mM, AlCl3 stimulated Tau release to about 170% of basal levels, but did not affect the release of the other compounds. Treatment with 5.0 mM AlCl3 enhanced the release of Tau, Glu and Ade, to 800%, 1000% and 250%, respectively, but decreased the release of Ser to 70% compared to basal levels. The enhanced release of these neuroactive compounds from astrocytes may contribute to changes in neural transmission known to accompany exposure to aluminum.

Cultured neurons from rat cerebellum were used to examine the effects of glutamate receptor agonists on the release of endogenous amino acids and adenosine. Kainic acid exposure resulted in the release of glutamate, taurine, GABA and alanine in a dose- and calcium-dependent manner. Stimulation with quisqualic acid resulted in the dose- and calcium-dependent release of GABA. N-Methyl aspartic acid did not elicit the release of any neuroactive amino acids. These findings suggest that N-methyl aspartate receptors are not coupled to transmitter release in these cultures, and that kainate and quisqualate receptors may have different neuronal distributions.

Cell swelling results in regulatory activation of multiple conductive anion pathways permeable toward a broad spectrum of intracellular organic osmolytes. Here, we explore the involvement of extracellular and intracellular Ca(2+) in volume-dependent [(3)H]taurine efflux from primary cultured astrocytes and compare the Ca(2+) sensitivity of this efflux in slow (high K(+) medium induced) and fast (hyposmotic medium induced) cell swelling. Neither Ca(2+)-free medium nor Ca(2+)-channel blockers prevented the volume-dependent [(3)H]taurine release. In contrast, loading cells with the membrane-permeable Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suppressed [(3)H]taurine efflux by 65-70% and 25-30% under high-K(+) and hyposmotic conditions, respectively. Fura 2 measurements confirmed that BAPTA-AM, but not Ca(2+)-free media, significantly reduced resting intracellular Ca(2+) concentration ([Ca(2+)](i)). The calmodulin antagonists trifluoperazine and fluphenazine reversibly and irreversibly, respectively, inhibited the high-K(+)-induced [(3)H]taurine release, consistent with their known actions on calmodulin. In hyposmotic conditions, the effects were less pronounced. These data suggest that volume-dependent taurine release requires minimal basal [Ca(2+)](i) and involves calmodulin-dependent step(s). Quantitative differences in Ca(2+)/calmodulin sensitivity of high-K(+)-induced and hyposmotic medium-induced taurine efflux are due to both the effects of the inhibitors on high-K(+)-induced cell swelling and their effects on transport systems and/or signaling mechanisms determining taurine efflux.

Hypothalamic astrocytes in culture released taurine, a suspected inhibitory amino acid neurotransmitter/neuromodulator/osmoregulator, in response to isoosmotically increasing extracellular K+ in a dose-dependent fashion. In the absence of added Ca2+, basal release levels rose to approach those obtained after exposure to 60 mM K+ in the presence of 2.5 mM Ca2+, and were only partially lowered by the addition of 10 mM Mg2+. Stimulation with K+ (60 mM) did not further increase taurine efflux above the high basal levels seen in the absence of Ca2+. Under standard conditions complete replacement of Na+ with choline Cl had little effect on basal taurine release, but reduced K+-evoked (60 mM) efflux by 60%. The temperature dependence of the basal levels of taurine released from hypothalamic astrocytes was similar to that seen for cultured cerebellar astrocytes and neurons over the range 5–50°C. Taurine release increased from 5 to 15°C, remained constant between 15 and 33°C, decreased between 33 and 37°C and increased thereafter. The infection point of increased basal taurine release seen around 37°C (most prominent in astrocytes), may be of physiological significance. Results presented also show that the ion (Na+, Ca2+ and K+) sensitivities of taurine efflux for cultured hypothalamic astrocytes are similar to those previously reported for cultured astrocytes from the cerebellum.

Partial depolarization of primary cerebellar neuronal cultures with K+ evoked the release of aspartate, glutamate, adenosine, serine, taurine, γ-aminobutyric acid (GABA), alanine and proline. The dihydropyridine calcium channel agonist, BAY K 8644, significantly augmented the K+-induced release of adenosine, aspartate, glutamate and GABA, but not that of serine, taurine, alanine or proline. However, in all cases the dihydropyridine antagonist nifedipine decreased this BAY K 8644-enhanced, K+-evoked efflux to below control levels. Neither BAY K 8644 nor nifedipine alone affected basal efflux levels. The phenylalkylamine calcium channel antagonist, verapamil, was ineffective in antagonizing K+-evoked amino acid release except at very high concentration (100 μM). These findings suggest that L-type Ca2+ channels are present in both excitatory (glutamatergic granule cells) and inhibitory (GABAergic stellate and basket cells) neurons in these cultures, and that they appear to be involved in regulating the release of not only neuroactive amino acids, but also some neutral amino acids and adenosine.

Primary cultures of cerebellar neurons obtained from 7-9-day-old rats and grown 7-9 days in vitro (DIV) were used to study the effects of Na+ and Ca++ on K+-evoked taurine release. These cultures, made up largely of granule neurons (90%) and inhibitory interneurons (5-7%), produced a dose-dependent, depolarization-evoked taurine release that was Ca++-dependent at 40 mM K+, and Ca++-independent at K+ concentrations above 40 mM. The dihydropyridine Ca++ channel agonist BAY K 8644 (1 microM) augmented 30 mM K+-evoked release, while the antagonist nifedipine (5 microM) abolished both the BAY K 8644- and K+-enhanced release. Depolarization with the Na+ channel agonist veratridine (50 microM) stimulated taurine efflux, which was completely blocked by pretreatment with tetrodotoxin (2 microM). However, 50 mM K+-evoked taurine release was not affected by tetrodotoxin pretreatment. Substitution of choline Cl for NaCl partially antagonized 50 mM K+-evoked release, and by itself, the Na+ ionophore monensin (50 microM) stimulated release. These results suggest that both K+-evoked and basal taurine release from primary cerebellar neuronal cultures are sensitive to the levels of both intracellular and extracellular Na+ and Ca++. In contrast to previous findings using cerebellar astrocytes, neuronal L-type Ca++ channels, but not voltage-dependent Na+ channels, also appear to be necessary. The implications of these results on taurine's status as a putative neurotransmitter are discussed.

In 16–18 days in vitro (DIV) primary astrocyte cultures prepared from 7- to 9-day-old rats, 48 h exposure to 12,13-phorbol dibutyrate (PDBU) (1 μM) or dibutyryl cAMP (dbcAMP) (1 mM) reduced cellular taurine content, and both basal and 50 mM K+-evoked taurine efflux, but did not alter cellular glutamate or total protein content. Decreases in cellular taurine content first became apparent between 1 and 6 h and were maximal after 24 h. Treatment also rapidly altered astrocyte morphology to a more processbearing form within 1 h. In contrast, fibroblast growth factor (FGF), epidermal growth factor (EGF), dbcGMP and α-PDBU did not affect cellular morphology, amino acid content or taurine efflux at any time tested. These findings suggest that, while protein kinase C translocation and adenylate cyclase activation may be only indirectly involved in the regulation of astrocyte morphology, long-term decreases in cellular taurine content and efflux may be the more direct result of these second messenger systems.

The K+‐stimulated efflux of endogenous taurine from primary rat cerebellar astrocyte cultures prepared from 7–9‐day‐old rats was studied at 16–18 days in vitro using HPLC analysis. Taurine efflux was dose‐dependent at K+ concentrations between 10 mM and 80 mM, with an EC50 of approximately 50 mM. Maximum stimulation of efflux above basal levels ranged from 56% at 10 mM K+ (204 pmol/min/mg protein) to 470% at 80 mM K+ (960 pmol/min/mg protein). Removal of Ca2+ from the buffer and the addition of either 1 mM EGTA or 10 mM Mg2+ abolished K.+‐stimulated efflux. Taurine efflux peaked and fell in parallel with the K+ concentration, but with an approximate lag of 3–5 min. The time course and amount of preloaded [3H]taurine released did not differ significantly from that seen for endogenous efflux. Basal taurine efflux varied inversely with the extracellular concentration of Ca2+ over the concentration range 0–5.0 mM. The observed Ca2+ dependence is consistent with a role for Ca2+ in the regulation of taurine release. Furthermore, taurine release from astrocytes in response to elevated K+ may reflect a neuromodulatory role for this amino acid in the CNS.

An HPLC method is described that measures amino acids (putative neurotransmitters and/or neuromodulators) released from primary, dissociated cerebellar cells maintained in monolayer culture. Precolumn derivatization with phenylisothiocyanate, followed by reverse phase chromatography with UV detection was used to quantitate the phenylcarbamyl amino acid derivatives in a chemically defined medium. Quantitation was linear, reproducible and sensitive to one picomole. This method is useful for the measurement of putative neurotransmitters GABA, glutamate, aspartate, taurine and adenosine, and can easily be modified to analyze other amino acids in physiological samples.

Monoclonal antibody, 1A9, prepared against bovine white matter, recognizes a proteinaceous, myelin‐specific domain in the CNS that is restricted to the surface of oligodendroglia in primary dissociated cell cultures. The antigen is not detected in the PNS or non‐neural tissues. Antibody binding is abolished by heating, exposure to SDS and delipidation, indicating that a conformationally sensitive epitope is recognized. The antigen is present in tracts of developing white matter in rat cerebellum beginning at 5 days postnatally. In developing cultures of fetal rat brain the period of rapid onset for the phenotypic expression of 1A9 antigen is similar to that of galactocerebroside, corresponding to 2–4 postnatal days of age. The 1A9 antigen is not observed in white matter or cultured oligodendroglia of the hypomyelinating jimpy mutant mouse, but its expression is qualitatively normal in the quaking mutant. The possibility is raised that 1A9 may be the primary target of the jimpy mutation.

A rat brain fraction enriched with microvessels was used as the immunogen to produce mouse hybridoma cell lines secreting monoclonal antibodies. One of these antibodies, selected from 156 supernatants by enzyme-linked immunosorbent and immunofluorescent assays, reacted only with the endothelium of microvessels in the brain. The endothelium-specific antibody labelled the cytoplasm of microvascular endothelial cells, their luminal membranes, and an extracellular layer, the endocapillary coat, which covered the luminal surface of these cells. In the kidney, the antibody specifically stained the brush border of the proximal tubuli, and in the liver, the antibody specifically stained bile canaliculi. This demonstrates the 3 morphological structures with important transport functions, cerebral microvascular endothelium, brush border of kidney proximal tubuli, and liver bile canaliculi, express the same epitope.

The dendritic processes and perinuclear cytoplasm of stellate-shaped perisinusoidal cells in frozen sections of rat liver were specifically labeled with antisera raised independently to glial fibrillary acidic protein (GFAP), the major component of intermediate filaments in astrocytes. A liver protein co-migrating with authentic GFAP and immunoreactive with GFAP antisera was demonstrated with immunoblots of brain and liver extracts enriched in intermediate filament proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This study presents yet another example of immunoreactivity to GFAP, or a highly similar protein localized outside the CNS, in cells of mesenchymal origin exhibiting some morphological features common to astroglia.

We examined the ability of developing cerebellar cell cultures to synthesize a 71,000 MW stress protein (SP71) in response to heat shock and Cd2+ treatment. The induction of SP71 synthesis appeared to be dependent on both the age of the culture and the stressor used. Heat shock induced SP71 synthesis in freshly prepared cells and in cell cultures at each age examined, whereas Cd2+ was effective only in cultures at 7 days of age and older. These findings are discussed with reference to the development of various cell types in these cultures.

A solid-phase, indirect β-galactosidase-linked immunoassay (ELISA) is described for screening large numbers of monoclonal antibodies that recognize cell surface antigens of primary monolayer cerebellar cultures. Target cultures were prepared from perikaryal suspensions of postnatal rodent cerebellum seeded into poly-l-lysine pre-coated, flat-bottom microtiter wells and fixed with glutaraldehyde after growth in vitro. Hybridoma supernatants were then incubated on these cultures. After the addition of β-galactosidase-linked anti-mouse IgG F(ab′)2 fragments, antigen-positive supernatants were detected with the enzyme substrate o-nitrophenyl-β-d-galactopyranoside. Using a monoclonal antibody specific for rat brain Thy-1 glycoprotein, this solid-phase ELISA was found to be useful in quantifying changes in the developmental expression of cerebellar surface antigens in these cultures.

Abstract: The effect of tetanus toxin pretreatment on K+ ‐stimulated [3H]γ‐aminobutyric acid release from neuron‐enriched cerebellar cell cultures at various stages during their development in vitro was assessed. Tetanus toxin had little inhibitory effect on immature (1‐3‐day‐old) cultures, but markedly reduced K+‐evoked [3H]γ‐aminobutyric acid release from 7‐ and 14‐day‐old cultures (∼80% inhibition). It is suggested that cerebellar neurons in culture develop tetanus toxin‐sensitive transmitter release mechanisms similar to their in vivo counterparts.