Morphological assessment of ethyl choline mustard aziridinium-induced neurotoxicity in rat brain reaggregate cultures.

Foetal rat brain reaggregate cultures have been employed to investigate the morphological changes associated with the neurotoxic action of ethylcholine mustard aziridinium (ECMA). In a companion study we provided evidence for apparent selective cholinergic neurotoxicity. Exposure of 9-day-old cultures to 12.5 microM ECMA for 3 days produced dilatation of selected axon preterminals and terminals in the outer core tissue layer. Axoplasm in these dilated terminals was electron lucent and contained a flocculent, plasma-like material with remnants of the smooth endoplasmic reticulum. Their synaptic vesicle content was much reduced or, absent. Microglial cells were engaged in phagocytosis of these effete structures and a few necrotic neurons were enveloped by glial processes. Exposure to 50 microM ECMA produced widespread necrosis with some surviving neurons, surrounded by the still-persisting capsular layer. Treatment with 100 microM ECMA generated a greater extent of tissue necrosis, with only a few surviving neurons and glial cells being contained within the necrotic tissue mass. Reaggregates frequently disintegrated following capsule loss. Our results indicate that the initial morphological manifestation of ECMA-induced toxicity is dilatation of axon terminals, that are probably of cholinergic origin and are targeted due to their possession of the high-affinity choline transport system which is unique to these neurons.

Effects of the neurotoxin ethylcholine mustard aziridinium in rat brain reaggregate cultures.

The toxicity was studied of ethylcholine mustard aziridinium (ECMA) in rat brain reaggregate cultures. The objective was to define optimum conditions for selective effects on cholinergic neurones. Single treatments with 12.5-50 mum-ECMA caused approximately 35% loss of choline acetyltransferase (ChAT) activity in 2 hr, increasing to 70-80% in 72-120 hr. The 2-hr, but not the longer-term loss of activity was prevented by the choline transport blocker hemicholinium-3 (40 mum). Significant loss of ChAT activity could not be obtained with less than 12.5 mum-ECMA. Approximately 55% irreversible inhibition of muscarinic receptor binding also occurred in 2 hr. However, after 12.5 mum-ECMA, binding recovered to control values within 48 hr. These persisted throughout the longer-term more extensive loss of ChAT activity, which suggests that receptor recovery localized to cells other than cholinergic neurones. After 25-50 mum-ECMA, muscarinic receptor binding did not recover, which suggests more widespread cytotoxicity destroying cells other than the cholinergic neurones. More pronounced leakage of lactate dehydrogenase and reduced reaggregate concentrations of total and neurofilament protein were consistent with more generalized cytotoxicity after 25 or 50 mum-ECMA. Examination of ECMA treated reaggregates or cerebellar monolayer cultures by light microscopy confirmed this. Concentrations of 12.5 mum-ECMA therefore represent the optimum for achieving selective toxic effects on cholinergic neurones in the cultures. After ECMA, reaggregate concentrations of 5-hydroxyindole acetic acid were markedly increased (100-300%), which suggests increased activity of 5HT neurones. This demonstrates that reaggregate cultures might be used to study trans-synaptic neurochemical sequelae of brain cholinergic neurone loss.

Effect of nerve growth factor and thyrotropin releasing hormone on cholinergic neurones in developing rat brain reaggregate cultures lesioned with ethylcholine mustard aziridinium.

Foetal rat whole brain reaggregate cultures were prepared in a serum-supplemented (S+) or serum-free medium (S-). Ethylcholine mustard aziridinium (ECMA) was added to the cultures at 9 days in vitro (DIV) at concentrations of 12.5, 25 or 50 microM. Choline acetyltransferase (ChAT) activity was measured at +2, +48 and +96 hr following treatment. In certain experiments the neurotrophic factors, thyrotropin releasing hormone (TRH: 50 micrograms/ml, daily from 9 DIV) or nerve growth factor (NGF: 7S subunit, 5 ng/ml, 0 and +48 hr following ECMA) were added during ECMA treatment. In both types of reaggregate cultured in S+ and S- media there was a 40-80% loss of ChAT activity following ECMA exposure (final concentration = 12.5 microM), presumed to reflect cholinergic cell loss. In both S+ and S- brain reaggregates NGF produced increased ChAT activity with more marked effects in S+ (45-55% increase, +48-96 hr) than in S- medium (20-25% increase, 2-96 hr). No effect on cholinergic muscarinic receptors (specific 3H-QNB binding) was evident after treatment with NGF. TRH had no effect on ChAT activity in the S+ cultures but produced small increases in the S- culture condition (approx 20%, +2-48 hr). Despite a residual "ECMA-resistant" pool of ChAT in the cultures, neither neurotrophic agent was found to cause a reversal of the lesion. In conclusion, the cholinotoxin ECMA appears to produce a cholinergic deficit in both developing S+ and S- reaggregates. This was not reversible by NGF or TRH at the concentrations and under the conditions tested. NGF had marked effects on ChAT activity without affecting muscarinic receptors in untreated developing brain reaggregates cultured in an S+ medium.

The neurotoxicity of ethylcholine mustard aziridinium (ECMA) in rat brain reaggregate cultures.

The cholinergic neurotoxin ECMA causes a biphasic loss of choline acetyltransferase activity in foetal rat whole brain reaggregate cultures. Initial direct inhibition is followed by longer-term loss of cholinergic neurones. Final muscarinic receptor binding, neurofilament protein and Na+, K+-ATPase concentrations suggest that the lesion is specific for cholinergic neurones at 12.5 microM ECMA, but is more generalised at 50 microM ECMA.

Neurochemical development of the striatum in a precocial (guinea pig) and an altricial (rat) species.

In this study, the developmental patterns of key neurotransmitter marker enzymes, namely choline acetyltransferase (ChAT), glutamate decarboxylase (GAD), and tyrosine hydroxylase (TH) in the corpus striatum were compared between the guinea pig and rat. In an attempt to assess cell acquisition and differentiation, striatal DNA and protein content were also determined during development in the two species. The most outstanding feature apparent from these investigations was a sequential maturation of the three neurotransmitter-synthesizing enzymes: TH and GAD first, followed by ChAT in striata of both species. The developmental increase in DNA content suggests that cell acquisition in the rat striatum is approximately 50% at birth, whereas in the guinea pig, the total population is present before birth, and thereafter cell numbers decline. The accretion of protein, reflecting the increase in region size, continued into adulthood in both species. With emphasis on cholinergic neurons, the neurochemical findings are assessed in relation to the morphological descriptions of cellular changes during pre- and postnatal development of the striatum.

High-affinity uptake of choline, a marker for cholinergic nerve terminals, is not specific in developing rat brain.

Developmental changes in the synthesis of acetylcholine (ACh) were investigated in rat hippocampus and frontal cortex. Particular reference was made to the conversion, into ACh, of the choline accumulated by high-affinity uptake as defined using 1 microM hemicholinium-3 (HC-3). Using solutions containing 11.1 mM glucose, conversions were respectively 31 and 55%, in fine slices from 4-8-day-olds. Free choline accounted very largely for the remainder of the choline accumulated. In samples from adults, ACh accounted for 80% of the uptake. The inefficient conversions (into ACh) in immature brain were not the result of a requirement for ketone bodies as the source of acetyl-coenzyme A (acetyl-CoA). Greater rates of release of newly synthesised ACh, than in mature samples, were not responsible, neither were greater cholinesterase activities. The stimulation of high-affinity choline uptake, caused by prior depolarisation of the tissues using K+, also increased during development from 78 to 238% with hippocampus and from 49 to 170% with frontal cortex. Furthermore, prior depolarisation increased the efficiency with which choline, accumulated by high-affinity uptake, was converted into ACh. At all stages of development 80% of the additional choline accumulated, after depolarisation, was converted into ACh. It is concluded that the specificity of HC-3-sensitive uptake is incomplete in immature brain, i.e. high-affinity choline uptake is not exclusively into cholinergic neurones. The cholinergic neuronal compartment becomes more prominent during development so that the specificity is complete in mature brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Supersensitivity of d-amphetamine-induced hyperthermia in rats following continuous treatment with neuroleptics.

Following short term treatments with the neuroleptic drugs haloperidol, chlorpromazine and oxypertine, there was an increase in the sensitivity of rats to the hyperthermic effects of d-amphetamine. There was no significant difference in the body temperature of control and neuroleptic-treated rats prior to challenge with d-amphetamine. No change in the maximal hyperthermic response was observed. It is possible that these responses may be related to the neuroleptic malignant syndrome occasionally observed in human patients following short term neuroleptic treatments.

Decreased neurotransmitter receptor binding in striatum and cortex from adult hypothyroid rats.

The consequences of propylthiouracil-induced thyroid deficiency on pre- and postsynaptic biochemical markers in the corpus striatum and the visual cortex were investigated in adult rats. Hypothyroidism in adult rats significantly impaired (by 30%) striatal [3H]spiroperidol binding capacity. In the visual cortex, small but significant reductions in [3H]WB.4101 and in [3H]muscimol binding were evident, whereas glutamate decarboxylase activity was increased by 18% in the thyroid-deficient rats. Compatible with previous morphological evidence (Ruiz-Marcos et al.), the present observations may imply alterations in dendritic morphology of neurones in the striatum and cortex during long-term hypothyroidism in adult rats. However, the molecular changes observed suggest a differential sensitivity of transmitter-specific neurones in the central nervous system to thyroid hormone.

The effects of neonatal thyroid deficiency on acetylcholine synthesis and glucose oxidation in rat corpus striatum.

The effects of propylthiouracil (PTU)-induced thyroid deficiency on [14C]acetylcholine synthesis and 14CO2 production from [U-14C]glucose in vitro, by fine prisms of the corpus striatum were investigated in developing rats. Consistent with deficits in choline uptake and choline acetyltransferase activity (Kalaria et al.17), PTU-treatment from two days after birth significantly impaired (27-39%) [14C]acetylcholine synthesis in striatal tissue taken from 3- or 6-week-old animals. In the thyroid-deficient (Tx) animals, 14CO2 production from [14C]glucose was unchanged in incubations in the presence of 5 mM K+ but was significantly reduced (33%) in medium with 31 mM K+ concentration. The addition of 10 mM DL-3-hydroxybutyrate in incubations with 5 mM K+ persistently inhibited 14CO2 production by striatal samples from the Tx rats. The fraction acetylated of [3H]choline accumulated by striatal prisms was unaffected by the PTU-induced thyroid deficiency. These findings suggest the development of fewer cholinergic nerve terminals in striatum during neonatal thyroid deficiency. Cholinergic nerve terminals that develop seem unaffected in their capacity for K+-stimulation and in their ability to acetylate transported [3H]choline.

Effects of thyroid deficiency on the development of cholinergic, GABA, dopaminergic and glutamate neuron markers and DNA concentrations in the rat corpus striatum.

The effects of propylthiouracil (PTU)-induced thyroid deficiency on the postnatal development of synaptic markers for cholinergic, GABA, dopaminergic and glutamate neurons in the rat corpus striatum were investigated. Similar effects were also assessed on ß-alanine uptake by fine prisms and on DNA concentrations in striatal samples from 3- and 6-week-old rats. Thyroid deficiency (Tx) in rats markedly impaired the development of choline acetyltransferase activity and [(3)H]spiroperidol and [(3)H]-glutamate binding capacities. In contrast, small but significant increases were evident in γ-aminobutyric acid uptake and glutamate decarboxylase activity. ß-Alanine uptake, a possible glial marker, was increased by up to 50% in samples from the Tx rats compared to controls. Consistent with deficits in striatal weight and greater DNA concentrations in the striatum of the Tx rats those neuronal markers which showed impairments on a mg tissue basis manifest even greater impairments expressed per whole striatum. Present findings suggest differential effects on neuronal markers, with the greatest impairments in the presynaptic markers for cholinergic interneurons in striatum during neonatal thyroid deficiency. The differential sensitivity on neuronal markers of the relatively late onset of thyroid deficiency seems likely to reflect the timing of the morphological differentiation of cholinergic and the other neurons in striatum.