Striatal Cultures Research Articles

Tyrosine hydroxylase immunoreactive neurons in organotypic slice cultures of the rat striatum and neocortex

Organotypic slice cultures of striatum and neocortex were prepared from newborn to seven day old rats and cultured for three to 60 days. When processed for tyrosine hydroxylase (TH) immunocytochemistry medium-sized, aspiny TH immunoreactive (TH-i) neurons with a similar morphology were revealed in the striatum and the neocortex. The neurons had a very similar morphology in both tissues and were present both when the two tissues were grown separately as single cultures and when grown together either en bloc as part of the same tissue slices or as co-cultures. In order to examine whether innervation by dopaminergic fibers would affect the expression of TH-i neurons in the striatal slice cultures, co-cultures of ventral mesencephalon (VM) and striatum were prepared, but the ingrowth of TH-i fibers from the VM did not alter the expression of TH immunoreactivity by a subpopulation of striatal neurons.

Factors regulating the expression of acetylcholinesterase-containing neurons in striatal cultures: effects of chemical depolarization

The influence of chemical depolarization on the survival and differentiation of acetylcholinesterase (AChE)-containing neurons was examined in primary rat striatal cultures, maintained in different types of media (serum-free and serum-supplemented) and substrate (poly-ornithine and astrocyte monolayer). Chronic application of 5 μM veratridine resulted in a vsignificant loss of neurites by AChE-positive cellsa, while a higher concentration (20 μM) reduced the number of stained cell bodies. The effects appeared to be selective with regard to AChE-positive cells, as indicated by morphological observations of the cells in the treated cultures and receptor binding measurements. Similarly, elevation of extracellular KCl levels (20–60 μM) produced a dose-dependent neurite loss by AChE-containing cells. Blockers of voltage-sensitive Ca 2+ channels - verapamil (1 μM) and nifedipine (1 μM) - did not affect the veratridine-induced neurite loss, while tetrodotoxin (0.1 μM) had a partial effect. When cultures treated with 5 μM veratridine were allowed to recuperate for several days, the number of AChE-positive cells possessing neurites returned close to control values, thus indicating the reversibility of the effect of chemical depolarization. The possibility that chronic neuronal depolarization in the striatum might play a role in regulation of the neuronal processes outgrowth by AChE-containing cells is discussed.

The correlation between excitatory amino acid-induced current responses and excitotoxicity in striatal cultures

Neuronal excitotoxic injury is initiated by activation of specific excitatory amino acid receptors and is mediated by ion flow through associated ion channels. In a companion paper we defined the age dependence of glutamate toxicity in striatal cultures. In this study we examined the correlation between age-dependent changes in the electrophysiologic responses to glutamate and N-methyl-D-aspartate (NMDA) and the development of excitotoxicity. Currents were recorded in voltage-clamped neurons in response to the pressure application of glutamate or NMDA. We found that 1-week-old cultures were resistant to excitotoxic effects of 1 or 3 mM glutamate (Freese et al., companion paper). Neurons from cultures of this age often did not exhibit current responses when exposed to NMDA (10 microM or 100 microM). However, most neurons were responsive to 10 microM glutamate. After 12 days in culture the current responses induced by NMDA were more prevalent and shifted toward higher amplitudes. These changes in NMDA current responses coincided temporally with major increases in excitotoxic sensitivity. However, we then found that excitotoxic susceptibility doubled between 12 and 18 days in culture without any parallel increase in glutamate- or NMDA-induced currents. Additionally, neurons which survived 1 mM glutamate exposure for 3 h were found to exhibit glutamate and NMDA responses. These data show that the expression of excitatory amino acid-induced currents is a prerequisite for excitotoxicity, but that additional factors must also contribute to the full maturation of in vitro neuronal vulnerability.

Characterization and mechanism of glutamate neurotoxicity in primary striatal cultures

Excitatory amino acids may play a role in the pathogenesis of cell death in neurodegenerative diseases, including Huntington's disease (HD). In an attempt to develop a tissue culture model for HD, the toxicity of glutamate was examined in primary striatal cultures derived from newborn rats. Morphological criteria were used to determine the toxic effects of glutamate in 6-, 12-, and 18-day-old cultures which were examined before and after 1-3 h of exposure to glutamate. Although younger cultures demonstrated little susceptibility to glutamate relative to controls, the number of neurons in older cultures was significantly depleted in the presence of glutamate. Glutamate toxicity was dose-dependent, with an ED50 of approximately 300 microns glutamate, and a maximal effect was observed within 3 h of initial exposure. Affected neurons demonstrated somal swelling within 1 h of glutamate exposure and disruption of neuritic processes and somal integrity within 3 h. Cell death was significantly increased by raising the extracellular calcium concentration and could be decreased by the addition of magnesium to the incubation medium. Moreover, the N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid, showed a toxicity profile similar to that of glutamate. The NMDA receptor competitive antagonist, 2-amino-5-phosphonovalerate (APV) significantly reduced toxicity, albeit incompletely. An additional component of glutamate mediated toxicity in striatal cultures could be explained by activation of non-NMDA receptor subtypes. These in vitro studies indicate that glutamate is toxic to a subset of mature striatal neurons in the absence of a glutamatergic afferent input, and that this toxicity is mediated partially by the NMDA receptor, with an additional component due to non-NMDA receptors.

Differential expression of ARPP-16 and ARPP-19, two highly related cAMP- regulated phosphoproteins, one of which is specifically associated with dopamine-innervated brain regions

ARPP-16 and ARPP-19 are 2 cAMP-regulated phosphoproteins of Mr = 16,000 and 19,000, respectively, which are identical except for the presence of 15 additional amino acids on the NH2-terminus of ARPP-19. The phosphorylation of these 2 proteins is regulated by cAMP and vasoactive intestinal peptide in reaggregate striatal cultures (Girault et al., 1988). Using immunoblots and immunocytochemistry, we have compared the regional, subcellular, phylogenetic, and ontogenetic distributions of these 2 proteins. ARPP-19 was found in all vertebrate species studied and, at various levels, in all tissues of adult rat. ARPP-19 was also present at high levels in malignant cell lines. During development ARPP-19 concentrations were highest in the embryo and decreased during the pre- and postnatal periods. In contrast, ARPP-16 was detected only in some specific neurons of the dopamine-innervated regions of the basal ganglia and the cerebral cortex, which are known to possess D1 dopamine receptors, in particular the striatonigral neurons. ARPP-16 is phylogenetically recent, being found only in birds and mammals, and appears late in ontogenesis, increasing during the postnatal period. These 2 proteins provide a unique model for studying the specificity of signal transduction and gene expression in dopaminoceptive neurons.

Muscarinic receptors and second-messenger responses of neurons in primary culture

The coupling of muscarinic receptors to second messenger responses was investigated in primary cultures of neurons from the fetal mouse brain. Neurons were mainained in monolayer culture, in serum-free medium; immunocytochemical studies found these cultures to be nearly exclusively neuronal. In striatal cultures, [ 3H]N-methylscopolamine (NMS) bound specifically and with high affinity ( K d= 70 pM) to a homogenous population of receptors on intact neurons (320 fmol/mg cellular protein). Displacement of the binding of [ 3H]NMS by pirenzepine indicated the presence of heterogeneou sites (81% high affinity site, K h= 51nM, K 1= 1.5 μM); AF-DX 116 showed the oppisite selectivity (15% high affinity sites, K rmh= 56nM, K 1= 1.5μM). The dopamine agonist SKF-3893 (1 μM) enhanced the accumulation of cylic adenosine monosphosphate (AMP) in these cultures 2.5-fold; addition of carbachol reduced cyclic AMP levels by 30% (EC 50, 1.7 μM). In the presence of 1 mM lithium, carbachol stimulated the accumulation of inositol monophosphate 5-fold (EC 50, 61 μM). Both responses were antagonized by pirenzepine (apparent K 1 of 23 nM for the phosphoinositide response and 200 nM for the cyclic AMP response) and AF-DX 116 (apparent K i 540 nM and 160 nM, respectively). In binding studies on braiinstem cultures, AF-DX 116 indicated the presence of two sites approximately equal abundance ( K h = 170nM, K 1= 2.9 υM); data for pirenzepine were adequately fit by one-site model ( K d= 630 nM. The efficacy of oxotremorine (compared to oxotremorine-M) in stimulating the phosphoinositide response was equally low in striatal and cortical cultures (0.07), but significantly greater in brainstem cultures (0.26). These studies suggest that the striatal phosphoinositide and cyclic AMP responses were mediated by M 1 and M 2 receptors, respectively, while the brainstem phosphoinositide response was mediated by M 3 receptors.

A neuroglial cooperativity is required for the potentiation by 2- chloroadenosine of the muscarinic-sensitive phospholipase C in the striatum

In rat striatal slices, 2-chloroadenosine, which had no direct effect on inositol phosphate formation, potentiated in a dose-dependent manner the accumulation of inositol phosphates induced either by carbamylcholine (10(-3) M) or by noradrenaline (10(-4) M). Experiments made on pure populations of striatal neurons or striatal glial cells in primary culture from mouse embryos indicated that 2-chloroadenosine potentiated the noradrenaline-elicited phosphoinositide breakdown in striatal glial cultures but did not modify the responses evoked either by noradrenaline or by carbamylcholine in striatal neuronal cultures. However, 2-chloroadenosine enhanced both the carbamylcholine and the noradrenaline-induced accumulation of inositol phosphates in neuroglial cocultures just as it did in rat striatal slices. The potentiation by 2-chloroadenosine of the carbamylcholine response, which is neuron specific, involved a cooperative effect between neurons and glial cells and, as shown by additional experiments, required a brief contact only between the 2 types of cells. The potentiating effect of 2-chloroadenosine was blocked completely by a nonselective A1, A2 adenosine antagonist isobutylmethylxanthine either on rat striatal slices or on mouse embryonic cocultures (noradrenaline and carbamylcholine responses) or on mouse embryonic glial cultures (noradrenaline response). These data indicate the involvement of an extracellular membrane-bound adenosine receptor, possibly of the A1 subtype since N6-cyclohexyladenosine, an A1 adenosine receptor agonist, was more efficient than 5'-N-ethylcarboxamide-adenosine, a rather selective A2 adenosine receptor agonist. We propose that 2-chloroadenosine acts through an adenosine receptor located on glial cells and induces the synthesis of a substance that improves the coupling between carbamylcholine or noradrenaline and phospholipase C located in glial cells or neurons.

Comparison of nerve growth factor's effects on development of septum, striatum, and nucleus basalis cholinergic neurons in vitro.

In the central nervous system, nerve growth factor (NGF) affects basal forebrain cholinergic neurons during early development and in the adult mammalian brain. These neurons are located in medial septum, diagonal band of Broca, and nucleus basalis of Meynert. While the effects of NGF on the development of septal cholinergic neurons are well documented, only little is known about the influence of NGF on development of cholinergic neurons in the nucleus basalis. In addition to the basal forebrain cholinergic neurons, there are cholinergic interneurons in the corpus striatum, which form an anatomically and functionally distinct population of cholinergic neurons. These striatal interneurons have been reported to respond to NGF during early development; however, it is not known whether the effects of NGF on their development are similar to those on septal cholinergic neurons. We prepared cultures of dissociated cells from fetal rat septum, striatum, and nucleus basalis and investigated the development of cholinergic neurons localized in these three different areas in the presence or absence of NGF. We now report that, first, cholinergic neurons of striatum and nucleus basalis develop a more extensive fiber network and contain more acetylcholinesterase (AChE) per neuron than do cholinergic neurons of septum. The amount of choline acetyltransferase (ChAT) per cholinergic neuron is approximately the same in all three culture types when grown in the absence of NGF. Second, NGF treatment increases and anti-NGF treatment decreases the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF is able to promote survival of cholinergic neurons of all three areas studied. Third, NGF increases the total length of fibers and the number of branching points of cholinergic neurons in septal cultures but not in cultures of striatum and nucleus basalis. Fourth, NGF treatment increases AChE activity in septal but not in nucleus basalis or striatal cultures, suggesting that AChE activity reflects the extent of the fiber network of cholinergic neurons of all areas. Fifth, NGF treatment produces severalfold elevations in ChAT activity in septal cultures and more modest increases in cultures of nucleus basalis and striatum, suggesting that NGF is able to stimulate ChAT activity also in the absence of a stimulatory effect on survival and fiber growth. Our results demonstrate that, during early development, NGF is able to affect survival and differentiation of all three populations of forebrain cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation by cAMP and vasoactive intestinal peptide of phosphorylation of specific proteins in striatal cells in culture.

We have studied three low molecular weight phosphoproteins, ARPP-16, ARPP-19, and ARPP-21 (cAMP-regulated phosphoproteins of Mr 16,000, 19,000, and 21,000, respectively) in reaggregate cultures from various regions of fetal mouse brain. ARPP-16 and ARPP-21 were detected only in striatal and cortical cultures. In contrast, ARP-19, which is structurally related to ARPP-16, was also present in reaggregate cultures prepared from thalamus and ventral and dorsal mesencephalon, as well as in monolayer cultures of astroglial cells. In striatal aggregates cultured over a 3-week period, the relative levels of ARPP-16, ARPP-21, and synapsin I/protein IIIa (synaptic vesicle-associated phosphoproteins closely related to each other and treated as a single entity in the present study) increased with time, whereas the level of ARPP-19 decreased. Incubation of striatal aggregates with 8-Br-cAMP, forskolin, or vasoactive intestinal peptide increased the phosphorylation of all these proteins. We conclude that the state of phosphorylation of two proteins enriched in specific neurons (ARPP-16 and ARPP-21) and two more widely distributed proteins (ARPP-19 and synapsin I/protein IIIa) is regulated by cAMP and vasoactive intestinal peptide in striatal cells in culture. These phosphoproteins may therefore play a role in mediating some of the actions of vasoactive intestinal peptide in the caudate-putamen.

Developmental expression of proenkephalin mRNA in rat striatum and in striatal cultures

Proenkephalin mRNA shows a biphasic developmental profile in rat striatum, with an initial peak at postnatal day 2, a decline to embryonic levels by day 7, and a second increase to adult levels over the course of the second to 4th week after birth. The same 4-fold increase is seen in cultured striatal neurons, over the same time course but without a biphasic response. Cultured fetal glia also contain proenkephalin mRNA.

Three serotonin responses in cultured mouse hippocampal and striatal neurons

Serotonin (5-HT) produced 3 different types of responses in neurons of mouse hippocampal and striatal cell cultures. These 3 responses have been characterized in terms of their pharmacological specificity, physiological mechanism, and dependence on cytoplasmic components. The most frequently observed response was inhibitory and was the result of a receptor-mediated activation of an inwardly rectifying potassium conductance. Typically, the response peaked within 1-3 sec of agonist application and did not exhibit desensitization. 5-Methoxy-N,N-dimethyltryptamine also produced this response in both striatal and hippocampal cultures and had no effect on the other 5-HT currents observed in this study. The selective 5-HT agonists--8-hydroxy-2-(di-n-propylamino)-tetralin, 1-(m-chlorophenyl) piperazine, and 1-(2-methoxyphenyl) piperazine--did not activate this outward current response. Methysergide did not block the 5-HT-activated outward current and often acted as an agonist. The response was lost in low-series-resistance recordings which facilitate solution exchange between the patch electrode and the cell. The loss of this response was prevented by using high-resistance patch electrodes, which retard this exchange. The 2 other responses described in this study were excitatory. They were seen less often than the inhibitory response. One of the excitatory responses was fast, with a time to peak of approximately 200 msec and a duration of 2-4 sec. The other was slow, with a time to peak of 7-10 sec and a duration of approximately 30-40 sec. Both of these responses were accompanied by a conductance increase. The fast excitatory response reversed at depolarized potentials and desensitized with a rate that varied with voltage. Metoclopramide and d-tubocurarine completely and reversibly blocked this fast excitatory response, while methysergide had no effect. The fast excitatory response was not lost during intracellular dialysis of cells in cultures from either striatum or hippocampus. In cultures from both brain regions, the slow excitatory response was blocked by methysergide. The slow excitatory response was lost even in patch-clamp recordings with high-resistance electrodes. This response was similar to responses to dopamine, norepinephrine, and forskolin, all of which are known to activate adenylate cyclase in the CNS.

Toluene induces changes in the morphology of astroglia and neurons in striatal primary cell cultures

Toluene (4.7–150 μmol per ml) was added for 30 or 60 min to astroglial and neuronal primary cell cultures from rat striatum and changes in cell morphology were analyzed by light microscopy. After 60 min incubation in 40 μmol toluene/ml, the cell bodies of the astrocytes appeared contracted, and their processes and nuclei were clearly visible. At higher doses of toluene the astrocytes seemed to be flattened and major cell damage was visualized by the uptake of vital dyes. The neurons, however, became affected and judged by morphological criteria only at the higher toluene doses. In conclusion, toluene induced morphological changes in primary astrocyte cultures and also in primary neuronal cultures at higher toluene concentrations.

Dependence of an adenosine-activated potassium current on a GTP-binding protein in mammalian central neurons

Neurons in hippocampal and striatal cell cultures respond to adenosine with an inhibitory potassium current. This response disappears during whole-cell patch-clamp recording in which the cell is filled with minimal saline. We have found that this loss of sensitivity to adenosine can be prevented by including 100 microM GTP in the patch electrode filling solution. GDP is less effective than GTP in supporting the adenosine response, while GMP has little, if any, effect. Treatments known to inhibit GTP-binding proteins (G-proteins) block the adenosine-activated potassium current: The adenosine response is inhibited by including poorly metabolized analogs of guanine nucleotides along with GTP in the recording electrode. Diphosphate and triphosphate analogs appear to achieve this effect through different mechanisms. The adenosine response is also blocked by incubating cultures in islet-activating protein (pertussis toxin), an inhibitor of a class of G-protein. Thus, our data implicate a G-protein in the activation of a potassium current by adenosine. Intracellular ATP can increase the effectiveness of GMP, GDP, or low concentrations of GTP, suggesting that even during internal dialysis, neurons can maintain GTP levels through phosphotransferase reactions. Intracellular ATP also appears to suppress an outward current that is different from the adenosine-activated current. Raising intracellular cAMP levels either with bath-applied forskolin or by including a cAMP analog in the recording electrode did not alter the adenosine response. These results indicate that a G-protein is involved in the coupling between the adenosine receptor and a potassium channel, and that this coupling is not mediated by cAMP.

Nerve growth factor promotes cholinergic development in brain striatal cultures.

We have examined the effect of the trophic protein, nerve growth factor (NGF), on organotypic cultures of fetal rat striatum. Treatment of cultures with NGF for 10-11 days resulted in a 5- to 12-fold increase in the specific activity of the cholinergic enzyme choline acetyltransferase (CAT; EC 2.3.1.6). in a dose-dependent fashion. This effect was not elicited by insulin, ferritin, or cytochrome c, proteins similar in structure or physicochemical properties to NGF. The effect of NGF on CAT activity was specifically blocked by anti-NGF antiserum, whereas treatment with the antiserum alone did not have a significant effect on the enzyme. Immunocytochemical studies of the treated cultures, using a monoclonal antibody directed against CAT, revealed positively stained neurons exhibiting dendritic and axonal processes. NGF did not have an effect on total protein content of the striatal cultures, suggesting a highly specific effect. Moreover, levels of substance P, a peptide localized to other, noncholinergic neurons, were not altered by NGF. Substance P remained unchanged after treatment with NGF for 12 days, whereas CAT activity increased 12-fold in sister cultures. Although the mechanisms of action of NGF on striatal cholinergic interneurons remain to be determined, the marked, specific response of CAT suggests that this well-defined trophic protein may play a critical role in normal brain development.

Primary cultures from defined brain areas; effects of seeding time on the development of β-adrenergic- and dopamine-stimulated cAMP-activity during cultivation

The influence of seeding time on the expression of β-adrenergic- and dopamine-stimulated cAMP activity was studied in primary astroglial cultures from various brain areas. In all cultures isoproterenol (10 −7–10 −5 M) caused an increased cAMP accumulation with time in culture. In brainstem cultures from 17-day-old embryos there was a decreased intracellular cAMP accumulation between 3 and 4 weeks. In all newborn rats and in striatal cultures from 7-day-postnatal rats there was a similar decrease between 2 and 3 weeks. These changes are discussed and interpreted as a differentiation of the cells concerning β-receptors. The most prominent cAMP accumulation caused by isoproterenol was found in striatal cultures from newborn rat. Although isoproterenol and noradrenaline both caused intracellular cAMP accumulation, isoproterenol was the most effective. There was a cAMP accumulation specific to dopamine (10 −6–10 −4 M) in 2-week-old striatal cultures from newborn rat, although there was an interaction with β-adrenergic receptors. In the other cultures studied there was only a very small dopamine stimulation of cAMP. The expression of this heterogeneity among astroglial primary cultures from various brain areas seems to be influenced by the genetic program for cellular maturation and by specific cellular contacts in vivo until seeding and in culture.

Simultaneous determination of leu-enkephalin localization and [3H] gamma-aminobutyric acid uptake in rat striatal cell cultures.

The purpose of this study was to investigate the coexistence of gamma-aminobutyric acid (GABA) and leu-enkephalin in single neurons from the corpus striatum. Monolayer cell cultures, started from newborn rat corpus striatum, were grown in serum-free medium and examined using GABA autoradiography and leu-enkephalin immunocytochemistry in a double-label protocol. Examples of cells were found which were positive for one or the other neurotransmitter or for neither transmitter, but not for both. Furthermore, cells which appear similar by morphological criteria alone differed in transmitter specificity. We conclude that the two transmitters tested are not localized within single cells and that morphology alone is inadequate to identify functional cell classes in this area.