Factors involved in the determination of the neurotransmitter phenotype of developing neurons of the CNS: applications in cell replacement treatment for Parkinson's disease.

The developmental stages involved in the conversion of stem cells to fully functional neurons of specific neurotransmitter phenotype are complex and not fully understood. Over the past decade many studies have been published that demonstrate that in vitro manipulation of the epigenetic environment of the stem cells allows experimental control of final neuronal phenotypic choice. This review presents the evidence for the involvement of a number of endogenous neurobiochemicals, which have been reported to potently influence DAergic (and other neurotransmitter) phenotype expression in vitro. They act at different stages on the pathway to neurotransmitter phenotype determination, and in different ways. Many are better known for their involvement in other aspects of development, and in other biochemical roles. Their proper place, and precise roles, in neurotransmitter phenotype determination in vivo will no doubt be determined in the future. Meanwhile, considerable medical benefits are offered from producing large, long-term, viable cryostores of self-regenerating multipotential neural precursor cells (i.e., brain stem cells), which can be used for cell replacement therapies in the treatment of degenerative brain diseases, such as Parkinson's disease.

Glutamate, GABA and epilepsy.

The nature and value of various animal models of epilepsy for the study and understanding of the human epilepsies are reviewed, with special reference to the ILAE classification of seizures. Kindling as a model of complex-partial seizures with secondary generalisation is treated in detail, dwelling principally on the evidence that the neurotransmitters glutamate and GABA are centrally involved in the kindling process. Kindling in the entorhinal cortex-hippocampus system and its relationship to LTP are analysed in detail. Changes in amino acid content in animal and human brain tissue following onset of the epileptic state are reviewed with special reference to glutamate and GABA. Studies of changes in the extent of basal and stimulus-evoked release of glutamate and GABA both in vivo (microdialysis) and in vitro (brain slices) are evaluated. This includes both kindling and other models of epilepsy, and microdialysis of human patients with epilepsy. Experiments which study the influence of pre-synaptic metabotropic glutamate receptors on glutamate release, and consequently on the extent of electrical kindling, are described. This pre-synaptic control of glutamate release can be studied using synaptosomes. The significance of the ability of focal intracerebrally injected glutamate and NMDA to cause (chemical) kindling and the strong sensitivity of this process to pre-treatment with NMDA receptor antagonists is analysed. Electrical and chemical kindling effects are additive, indicating the existence of mechanisms in common. They are both sensitive to NMDA antagonists and the common mechanism is probably NMDA receptor activation due to the presence of exogenous (chemical) or endogenous (electrically-released) extracellular glutamate. The participation of the NMDA receptor in the generation of the spontaneous hyperactivity which characterises the chronic epileptic state is reviewed. This includes the entry of Ca2+ to stimulate various post-synaptic phosphorylation processes, and possible modulation of NMDA receptor population size and sensitivity. The question of whether neurotransmitter glutamate is involved in initiation and/or spread of seizures is discussed.

Nerve growth factors and the control of neurotransmitter phenotype selection in the mammalian central nervous system.

Determination of neurotransmitter phenotype in the peripheral nervous system (PNS) has been intensively characterized. However, relatively little is known about the underlying molecular and biochemical events involved in determination of transmitter phenotype in the central nervous system (CNS). It has been well established that nerve growth factors regulate cell growth and differentiation. They are increasingly recognized as playing an important role in many decision-making steps during development. Published data suggest that neurotransmitter phenotype is determined largely by exogenous stimuli, such as nerve growth factors--acidic/basic fibroblast growth factor, epidermal growth factor, neurotrophins, etc., working in concert with the genetic programmes. They exert potent effects independently or synergistically with other molecules by acting either on neural precursor cells or differentiated neuronal cells. However, the process of transmitter phenotype determination in the CNS is only beginning to be understood, with more uncharacterized substances, with considerable potency in this respect being reported and in need of isolation and further study. These studies will bring great advances in our existing knowledge of brain development and have potential value for the development of new treatments for neurodegenerative diseases.

Specific lysis of noradrenergic synaptosomes by an antiserum to dopamine-beta-hydroxylase.

An antiserum to dopamine-beta-hydroxylase purified from bovine adrenal medulla, acting in the presence of complement, caused the release of 12% of lactate dehydrogenase, 20% of tyrosine hydroxylase activity, and 40% of noradrenaline (NA) content from synaptosomes prepared from rat brain cerebral cortex. Uptake of [3H]NA was reduced by 54%. Anti-serum alone or complement alone were without action. The antiserum plus complement had no effect on choline uptake and did not release choline acetyltransferase, glutamate decarboxylase, dopamine or 5-hydroxytryptamine. These results suggest selective lysis of noradrenergic terminals had occurred. An enhancement of lysis was not observed when synaptosomes were stimulated with 75 mequiv./lK+ and exposed to a sub-maximal dose of antiserum, plus complement.

Specific lysis of GABAergic synaptosomes by an antiserum to glutamate decarboxylase.

An antiserum to pure glutamate decarboxylase (GAD) when incubated with rat cortical synaptosomes in the presence of complement caused release of 33-53% of lactate dehydrogenase (LDH) and 22-41% of total GAD. In addition most of the gamma-aminobutyrate (GABA) present was released. Anti-GAD antiserum alone, or complement alone, were without action. The antiserum plus complement had no effect on noradrenaline or choline uptake, and did not release choline acetylase (ChAT). Anti-ChAT serum plus complement released 30-37% of ChAT and 10-13% of LDH. It prevented choline uptake. This serum did not produce GAD release or prevent GABA, choline or noradrenaline uptake. When cortical synaptosomes were exposed to both antisera plus complement, their actions were strictly additive. The data indicate specific lysis of GABAergic and cholinergic synaptosomal sub-populations.

Specific immunolysis of serotonergic nerve terminals using an antiserum against tryptophan hydroxylase.

An antiserum to tryptophan hydroxylase purified from whole rat brain when incubated with rat striatal synaptosomes in the presence of complement caused release of 18% of LDH, 20% loss of potassium and 60% loss of tryptophan hydroxylase. Uptake of 5-HT was reduced by 60%. Anti-tryptophan hydroxylase alone, or complement alone were without action. The antiserum plus complement had no effect on DA uptake and did not release TH or GAD. These results suggest selective lysis of serotonergic nerve terminals had occurred. The antiserum plus complement reduced choline uptake by 45%. However, this did not seem due to lysis of cholinergic terminals, as ChAT was not released.

Evidence for an inhibitory presynaptic component of neuroleptic drug action.

1 The action of five neuroleptic drugs (haloperidol, cis-flupenthixol, chlorpromazine, fluphenazine and thioridazine) was studied on the synthesis and release of dopamine from rat striatal synaptosomes. 2. In vitro application of the drugs induced an inhibition of synthesis of [14C]-dopamine from L-[U-14C]-tyrosine and a decrease in the tissue content of [14-C]-dopamine, with IC50 values for the latter effect ranging from 3.6 x 10(-7) to 5.9 x 10(-5) M. The rank of their potency was similar to the order of their clinical effectiveness: haloperidol greater than fluphenazine greater than cis-flupenthixol greater than chlorpromazine greater than thioridazine. Trans flupenthixol was without effect up to a concentration of 10(-4) M. 3 The tissue level and release of GABA were not affected by concentration of the neuroleptics up to 10(-4) M. 4 When the neuroleptics were administered in vivo, changes were also detected in the synthesis and release of [14C-]-dopamine from subsequently prepared synaptosomes. A marked inhibition of the K+-induced increase in [14C]-dopamine synthesis was seen following a dose of 2 mg/kg cis-flupenthixol and haloperidol. At this concentration, haloperidol also increased the control release of [14C]-dopamine and reduced the K+-induced increase in release of [14C]-dopamine. 5 Cis-flupenthixol at a dose of 20 mg/kg reduced the K+-induced release of [14C]-dopamine by 48% and to a lesser extent, that of gamma-aminobutyric acid (GABA, 25%). 6 An inhibitory mode of action is proposed for neuroleptics mediated through a presynaptic mechanism.

Inhibitory actions of opioid compounds on calcium fluxes and neurotransmitter release from mammalian cerebral cortical slices.

The effects of opioid agonists on veratrine-stimulated Ca2+ influx and amino acid neurotransmitter release in rat cerebrocortical brain slices were studied. Inhibitory effects were seen on both of these parameters with all of the opioid agonists used. None of the drugs used affected basal 45Ca2+ uptake, basal K+ content or basal amino acid release from the slices. At high concentrations (100 microM) fentanyl, tifluadom, U50,488H, butorphanol and bremazocine greatly inhibited the depolarization of the slices by veratrine as determined by the reduced release of K+. The opioid receptor subtypes at which the drugs were acting were characterized by the antagonistic effects of naloxone and WIN44441-3. The opioid-induced inhibition of stimulated Ca2+ uptake and amino acid release were not antagonized by WIN44441-2, the inactive enantiomer of WIN44441-3. It is concluded that opioid agonists acting through mu- and kappa-receptors and probably through delta- and sigma-receptors, have an inhibitory effect on Ca2+ uptake into cerebrocortical brain slices and the subsequent release of aspartate, glutamate and gamma-aminobutyric acid (GABA).

On the specificity of verapamil as a calcium channel-blocker.

The stimulated uptake of 45Ca2+ into incubated cerebrocortical synaptosomes caused by veratrine (75 microM) was blocked by low concentrations of verapamil (0.5-30 microM) which did not prevent or reduce depolarization as judged by efflux of potassium (K+). However, verapamil did not prevent amino acid neutrotransmitter release at these low concentrations and this is discussed in terms of mobilization of internal calcium (Ca2+) stores. At higher concentrations (30-200 microM) verapamil appeared to act additionally at sodium (Na+) channels since both depolarization-induced K+ efflux and neurotransmitter release were reduced or prevented. When K+, at a high concentration (56 mM), was used as the depolarizing agent, both 45Ca2+ influx and neurotransmitter release were prevented by verapamil across a wide concentration range (0.5-200 microM). The data are discussed in terms of the specificity of action of verapamil on Ca2+ channels.

Enhancement of depolarization-induced synaptosomal calcium uptake and neurotransmitter release by Bay K8644.

Ca2+ uptake into submaximally stimulated synaptosomes was augmented by low concentrations of BAY K8644 (0.1-100 nM). Use of either veratrine or potassium as the depolarizing agent produced similar effects. Stimulated release of aspartate, glutamate and GABA was enhanced over a similar concentration range of BAY K8644. The extent of synaptosomal depolarization induced by veratrine was not enhanced by BAY K8644. The results are discussed in relation to BAY K8644 activation of voltage-dependent Ca2+ channels in neural tissue. This is the first report of BAY K8644 acting on synaptosomes at concentrations that correlate to dihydropyridine binding studies.

Morphine suppression of neurotransmitter release evoked by sensory stimulation in vivo.

The effects of morphine and naloxone on the release of acetylcholine and amino acid neurotransmitters from sensorimotor cortex were studied employing an in vivo superfusion cannula. Morphine (20 mg/kg) reduced the spontaneous release of acetylcholine but had no detectable effect on the spontaneous release of amino acids. It also suppressed the release of acetylcholine and amino acid neurotransmitters evoked by sensory stimulation of the contralateral sensorimotor cortex via the brachial plexus. Naloxone (5 mg/kg) prevented all of these inhibitory actions of morphine. Naloxone also caused a significant increase in spontaneous acetylcholine release.

Morphine inhibition of calcium fluxes, neurotransmitter release and protein and lipid phosphorylation in brain slices and synaptosomes.

Morphine (1-100 microM) was found to inhibit several concomitant events in brain slices and synaptosomes which are augmented by depolarizing agents. Thus, 45Ca2+ uptake, amino acid neurotransmitter release, increases in 3',5' cyclic AMP levels and 32Pi incorporation to proteins and lipids induced by veratrine (25 microM) and by potassium (56 mM), were each inhibited in a dose related manner. These inhibitory actions of morphine were all prevented by naloxone (1 microM). Evidence is presented that morphine binding to a receptor on the synaptic membrane affects intracellular mechanisms involved in neurotransmitter release possibly via a second messenger system. An enhancing action of GTP on the inhibitory influences of morphine suggests that its actions are mediated at least in part, via a coupling of the receptor to adenyl cyclase in the outer membrane. This is supported by its inhibitory action on the capacity of depolarizing agents to increase cyclic AMP levels.

Actions of a tremorgenic mycotoxin on amino acid transmitter release in vivo.

The tremorgenic mycotoxin verruculogen was administered directly into the brain of freely moving rats by the use of cannula systems that superfused either the cortical surface or the lateral ventricular space. The tremor produced by these CNS routes was compared with that produced by i.p. administration of the toxin or the dried mycelium of the fungus that synthesizes the verruculogen. The nature and degree of tremor produced by the central vs peripheral routes suggest that the site of action of verruculogen is not immediately adjacent to the cannula sites in the brain. Measures of the amino acids in the superfusates collected during the verruculogen-induced tremor showed an increase in the excitatory neurotransmitters, glutamate and aspartate in superfusates from the lateral ventricle but not in superfusates from the cortical surface. The differential effect on transmitter release suggests that a subcortical action of verruculogen is responsible for its tremorgenic activity.

Prostaglandins D2 and E2 are not regulators of amino acid release from rat cortical synaptosomes.

Rat cerebral cortex synaptosomes synthesise prostaglandins, and analysis by gas chromatography-mass spectrometry revealed that of the prostaglandins quantified PGD2 (10.9 ng/mg protein) was produced in highest concentration. Treatment with high potassium or veratrine caused release of putative amino acid transmitters, but not of prostaglandins, and prostaglandins D2 and E2 were unable to stimulate release of amino acids. The release of putative amino acid transmitters, evoked by high potassium levels, was not inhibited by these prostaglandins. Prostacyclin receptors could not be identified on synaptosomes by radioligand binding techniques.

Brain-derived neurotrophic factor in human platelets.

A sensitive, capture enzyme-linked immunosorbent assay (CELISA) has been applied to the accurate and reproducible measurement of brain-derived neurotrophic factor (BDNF) protein in normal human blood platelets, a mean concentration of 1.03 +/- 0.04 ng (SEM)/mg of platelet protein being observed. The method, which requires only 10 ml blood, is now suitable for the investigation of a variety of clinical disorders.

Choline acetyltransferase in mammalian synaptosomes: Evidence for an integral membrane-bound form.

Choline acetyltransferase activity was detected in extensively washed membranes prepared from rat and guinea pig synaptosomes. When these preparations were treated with the non-ionic detergent Triton X-114 and heated to 37 degrees C to cause phase separation, a significant percentage was found to associate with the detergent-rich phase, indicating that the enzyme might be an integral membrane-bound protein. In guinea pigs receiving septal lesions, a large reduction in both total and in Triton X-114-extractable choline acetyltransferase in hippocampal synaptosomes was observed indicating that the detergent-extracted form is associated with cholinergic nerve terminals. When membrane-bound choline acetyltransferase from lysed, washed synaptosomes was incubated in Triton X-114, 30% of the membrane-associated enzyme could be extracted into the detergent-rich phase. This extraction could be improved by reducing the chloride content of the extraction medium. When the chloride content of synaptosomes, prepared from rat cerebral cortex, was manipulated, by either exposure to ?-aminobutyric acid, muscimol or to a medium containing reduced levels of chloride, the ability of antibodies against choline acetyltransferase to specifically immunolyse (in the presence of complement) the cholinergic synaptosome population was enhanced. These results suggest that the choline acetyltransferase found in the nerve terminal region exists in at least two forms (a soluble and a lipophilic form) which are partially interconvertible. The conversion between the two forms can be influenced by chloride ions.

The neuronal survival effects of rasagiline and deprenyl on fetal human and rat ventral mesencephalic neurones in culture.

The neuronal survival properties of rasagiline (R(+)-N-propargyl-1-aminoindane mesylate or TVP-1012), a novel monoamine oxidase B inhibitor, have been investigated using neuronal cell cultures from fetal rat and human ventral mesencephalon. The ability of rasagiline to reduce the rate of neuronal cell loss in vitro was tested using primary neuronal cell lines and immunohistochemistry to quantify the reduction in cell death. Direct comparison was made with deprenyl, a widely used and long established monoamine oxidase B inhibitor. Rasagiline was shown to act 15-20% more effectively as a neuronal survival agent than deprenyl, increasing both the survival of the total number of neurones and selectively increasing the survival of dopaminergic neurones with no statistically significant increase in survival of GABAergic neurones.

The presence of neuropeptides in GABAergic and cholinergic rat cerebrocortical synaptosome sub-populations.

GABAergic and cholinergic synaptosomes from rat cerebral cortex were isolated by a magnetic immunoaffinity technique, i.e. immunomagnetophoresis. These subpopulations were extracted and subjected to radioimmunoassay for four neuropeptides: Neuropeptide Y (NPY); vasoactive intestinal peptide (VIP); substance P (SP); and somatostatin (SRIF). In each of the sub-populations three of the four peptides were enriched in the sorted fraction compared with the mother fraction with respect to the cytosolic marker lactate dehydrogenase (LDH). In the GABAergic sub-population the order was SP > SRIF > NPY > or = VIP whilst in the cholinergic sub-population they were enriched in the order VIP > or = NPY > SP > SRIF. The presence of NPY has not previously been reported in cortical cholinergic neurons.

Kindling of full limbic seizures by repeated microinjections of excitatory amino acids into the rat amygdala.

Fully developed limbic seizures were kindled by repeated (every second day) microinjections of an L-glutamate plus L-aspartate (Glu/Asp) mixture (1:3 ratio; 1.5 mumol total dose). Glu alone (1.5 mumol) or Asp alone (1.5 mumol), into the rat amygdala. This excitatory amino acid (EAA)-induced kindling was durable, persisting for at least several months, and showed strong positive transfer to electrical kindling. Fully EAA kindled seizures were inhibited by focally applied NMDA-receptor antagonists. EAA kindling and electrical kindling are shown to have many similar properties. This strongly suggests that they may also have neurochemical mechanisms in common. These results further highlight the important role of EAAs in basic mechanisms involved in the generation and expression of epilepsy.

NMDA receptor blockade inhibits glutamate-induced kindling of the rat amygdala.

Full limbic seizures were kindled in rats by repeated, bi-daily microinjections of glutamate (1.5 mumol) into the basolateral amygdala. Co-administration of the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoic acid with the glutamate, prevented the development of both electroencephalographic and motor signs of the kindling response. These results indicate an important functional role for NMDA receptors in the development of excitatory amino acid-induced kindling.