Philip C. Hoffmann

Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion

To facilitate the study of trophic interactions between mesencephalic dopaminergic neurons and their target cells, clonal hybrid cell lines have been developed from rostral mesencephalic tegmentum (RMT) of the 14-day-old embryonic mouse employing somatic cell fusion techniques. Among the hybrid cell lines obtained, one contains a high level of dopamine (DA), another predominantly 3,4-dihydroxyphenylalanine (DOPA), and a third no detectable catecholamines. The hybrid nature of the cell lines is supported by karyotype analysis and by the expression of adhesion molecules as assessed by aggregation in rotation-mediated cell culture. The DA cell line shows neuronal properties including catecholamine-specific histofluorescence, neurite formation with immunoreactivity to neurofilament proteins, and large voltage-sensitive sodium currents with the generation of action potentials. In contrast to the pheochromocytoma cell line (PC12), the dopamine content of the DA hybrid cell line is depleted by low concentrations of N-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Pharmacological analysis of the functional ontogeny of the nigrostriatal dopaminergic neurons

Functional development of the dopaminergic nigrostriatal projection was studied by determining the age at which the biochemical responses of these neurons to physical or pharmacological manipulation are similar to those of adult neurons. Transection of the pathway acutely elevates striatal dopamine in adult and 8- and 10-day-old rats, but not in the 4- or 6-day-old animal. This axotomy-induced increase in striatal dopamine is believed to be a response of the dopaminergic terminals to cessation of impulse traffic and is secondary to a decrease in dopamine release and a concomitant increase in dopamine synthesis resulting from tyrosine hydroxylase activation. Therefore, this response to axotomy acts as an indicator of (1) the presence of impulse traffic in the pathway, and (2) the ability of tyrosine hydroxylase to be activated in response to a reduction in such impulse traffic. In vivo estimation of tyrosine hydroxylase activity showed that axotomy activates the enzyme at 10 days, but not at 4 days, whereas gamma-hydroxybutyrate is effective at both ages. The fact that the enzyme can be activated by gamma-hydroxybutyrate at 4 days indicates that the lack of effect of axotomy at this age is due to the absence of impulse traffic in the system. This conclusion is supported by the finding that the AMT-induced depletion of striatal dopamine is not related to impulse conduction at 4 days since transection of the pathway has no effect on the rate of dopamine loss whereas such transection blocks the AMT-induced depletion at 10 days of age. Nevertheless, despite the absence of neuronal activity at 4 days of age, these neurons are capable of generating and conducting impulse traffic since both 4- and 10-day-old rats showed increased striatal dihydroxyphenylacetic acid (DOPAC) levels when treated with haloperidol indicating increased dopamine release; such increase in DOPAC being dependent on an intact pathway. Given this data, the most parsimonious explanation of the abrupt development of the response to axotomy after the 6th day of age is that an event occurs which physiologically initiates impulse traffic. This event may be activation of afferent neuronal inputs to the cell bodies of the nigrostriatal projection.

The effects of nerve growth factor on the development of septal cholinergic neurons in reaggregate cell cultures.

Recent studies suggest that nerve growth factor is present within the central nervous system where it may exert selective trophic effects on cholinergic neurons. We have measured the effects of nerve growth factor on septal cholinergic neurons in three-dimensional reaggregating cell cultures, a system which closely simulates the cellular environment in situ. Septal cells obtained from 15-day-old mouse embryos were dissociated into a single cell suspension and then allowed to reaggregate in culture in a rotary incubator shaker. After 17 days in culture, half of the reaggregates from a flask were sonicated for measurement of choline acetyltransferase activity, and the remaining reaggregates were processed for acetylcholinesterase histochemistry. Addition of nerve growth factor to medium containing septal reaggregates resulted in greater than a three-fold increase in choline acetyltransferase activity and in the number of acetylcholinesterase-positive cells, as well as an enhancement in the staining of acetylcholinesterase-positive fibers. All of these effects of nerve growth factor could be neutralized by antibodies to nerve growth factor. In order to evaluate the possible role of endogenous hippocampal-derived nerve growth factor, antiserum to nerve growth factor was added to the culture media containing septal-hippocampal coaggregates. After 21 days in culture, the presence of nerve growth factor antibodies did not qualitatively affect the pattern or density of cholinergic fibers observed. Synapse formation between cholinergic axons and hippocampal target cells was still in evidence as revealed by electron microscopy. However, there was a modest decrease in choline acetyltransferase activity (20%) and cholinergic cell number (30%) when compared with coaggregates grown in culture medium either without nerve growth factor antiserum or with non-immune serum. The magnitude of these effects was markedly less than the effects observed when exogenous nerve growth factor was added to septal cells grown alone in reaggregate culture. These results suggest that nerve growth factor may play a role during central cholinergic development, but that additional trophic mechanisms are likely to be required.

Neurotrophic effects of hippocampal target cells on developing septal cholinergic neurons in culture

The influence of hippocampal target cells on the development of cholinergic septal neurons was studied in rotation-mediated reaggregating cell cultures. Brain cells from 15-day-old mouse embryos were obtained from: septum, containing cholinergic cells which project to the hippocampus; hippocampus which contains target cells for the septal cholinergic neurons; and cerebellum, containing cells which are not targets for the septal cholinergic cells. The cells were then cultured for 3 weeks in a rotary incubator in the following combinations: septal cells alone; hippocampal cells alone; cerebellar cells alone; septal-hippocampal cells together; and septal-cerebellar cells together. After harvesting, fixation, and embedding, 50 micron sections were cut and processed for visualization of acetylcholinesterase activity. Sections from reaggregates containing either hippocampal or cerebellar cells alone contained only a few acetylcholinesterase-positive cells, but no positive fibers. Sections from septal-hippocampal coaggregates revealed a pattern of well-defined, fine-caliber acetylcholinesterase-positive fibers with extensive arborizations and varicosities suggesting axonal proliferation. In septal-cerebellar coaggregates, acetylcholinesterase-positive fibers appeared to be degenerating and distinct areas were observed which were essentially devoid of acetylcholinesterase fibers. In some experiments, either cerebellar or hippocampal cells were labeled with wheatgerm agglutinin-rhodamine prior to culture in order to identify these cells in the resulting reaggregates. Analysis of sections from these studies showed that acetylcholinesterase fibers were excluded from regions of coaggregates containing cerebellar cells, but were present in regions of coaggregates containing hippocampal cells. Finally, cell counts of acetylcholinesterase-positive cells in the various combinations revealed that these putative cholinergic neurons were significantly more numerous in septal-hippocampal coaggregates (271 +/- 19 per 10(6) septal cells added) than in septal reaggregates (38 +/- 6 per 10(6) septal cells added) or septal-cerebellar coaggregates (85 +/- 29 per 10(6) septal cells added). These results, taken together, suggest that hippocampal target cells influence the development and survival of cholinergic neurons.

PENETRATION OF ATP INTO THE MYOCARDIUM.

Summary When the isolated guinea pig heart is perfused with C14- and P32-labeled ATP of known isotope ratio, ATP isolated from the myocardium shows an isotope ratio 100 times greater than the starting material. Inorganic P32 is liberated into the perfusion fluid along with C14-labeled dephosphorylation and deamination products of ATP. UTP, GTP, CTP and ITP are also dephosphorylated when perfused. The results indicate that the ATP molecule does not enter the myocardium as such, but first is degraded and later resynthesized intracellularly.

The effect of development of thermoregulatory function on the biochemical assessment of the ontogeny of neonatal dopaminergic neuronal activity.

Previous studies from this laboratory on the functional ontogeny of impulse flow in the nigrostriatal pathway of the neonatal rat were interpreted as indicating that there was an abrupt onset of impulse flow between 6 and 8 days of age. The functional development was assessed by determining the age at which the biochemical responses of these neurons to physical or pharmacological manipulation were similar to those of adult neurons. Further studies of these phenomena demonstrate that the age-dependency of these biochemical markers of impulse traffic are directly related to the development of thermoregulatory function in the neonatal rat. Accordingly, provided that 4-day-old or even younger rats are maintained at or near litter temperature, they exhibit qualitatively all of the biochemical responses characteristic of the 10-day-old or adult animal in which impulse flow is known to be present.

Ultrastructural evidence for hippocampal target cell-mediated trophic effects on septal cholinergic neurons in reaggregating cell cultures.

We have previously demonstrated at the light microscopic level that when embryonic day-15 septal neurons are co-cultured for 21 days with their target cells from the hippocampus, increased numbers of septal cholinergic neurons are present as compared with co-cultures employing cells from the non-target cerebellum. In addition, fine varicose axon-like cholinergic fibers are found to be associated with the hippocampal cells but not with cerebellar cells. We now provide ultrastructural evidence for hippocampal target cell-enhanced cholinergic neuronal survival, axonal proliferation, and synapse formation in this culture system. Dissociated cell suspensions from septal, hippocampal, and cerebellar areas were obtained from 15-day mouse embryos; and hippocampal and cerebellar cells were internally labeled with rhodamine-conjugated wheat germ agglutinin. Combinations of septal and hippocampal cells, and septal and cerebellar cells were allowed to reaggregate in rotation mediated culture for either 15 or 21 days. The reaggregates were then fixed, embedded, sectioned, and processed for acetylcholinesterase-positive acetylcholinesterase-positive cells and fibers, and under fluorescence to locate rhodamine-labeled cell populations. Representative reaggregate profiles were then re-embedded for electron microscopic examination. In both types of reaggregates, either labeled hippocampal target or cerebellar non-target cells segregated from the septal cells so that areas containing each of the respective cell populations could be studied. In sections of septal-hippocampal reaggregates from 15-day cultures, 571 out of 665 (85%) cholinergic neurons examined were intact, whereas 15% of the cells showed some ultrastructural features of degeneration. Similarly, at day 21, 297 out of 335 (88%) of the cholinergic neurons were intact. In sections of septal-cerebellar reaggregates from 15-day cultures, 473 out of 572 (83%) cholinergic neurons were intact. By day 21 of culture, however, only 15 out of 110 (14%) cholinergic neurons examined were intact from the septal-cerebellar reaggregates. In areas of septal-hippocampal reaggregates occupied by rhodamine-labeled hippocampal cells, profiles of acetylcholinesterase-labeled axons were identified, and synaptic specializations were observed between cholinergic terminals and dendrites as well as somata of hippocampal target cells. In contrast, areas of septal-cerebellar reaggregates occupied by rhodamine-labeled cerebellar cells were devoid of cholinergic fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurotoxic effects of methamphetamine assessed in three-dimensional reaggregate tissue cultures

Three-dimensional, rotation-mediated, reaggregate tissue cultures formed from dissociated fetal rostral mesencephalic tegmental (RMT) and corpus striatal (CS) or frontal cortical (FCx) cells were used to study methamphetamine neurotoxicity. Analysis of dopamine (DA), serotonin (5-HT) and gamma-aminobutyric acid (GABA) levels using HPLC techniques revealed decreases in RMT-CS and RMT-FCx reaggregate DA and 5-HT levels after treatment between 14 and 21 days in culture with methamphetamine in concentrations ranging from 10(-6)M to 10(-3)M. Dopamine cell numbers in RMT-CS and RMT-FCx reaggregates were estimated after visualization by histofluorescent techniques. Methamphetamine treatment caused decreases in DA cell numbers which paralleled the decreases in endogenous DA levels. Estimates of the accumulation of exogenous DA by RMT-CS reaggregates treated with methamphetamine showed that the amount of accumulation per cell remained fairly constant despite marked reductions in total DA cell numbers. This suggests that the reductions in endogenous DA levels following methamphetamine were secondary to loss of entire DA neurons rather than of a portion of the terminal axonal fields in the surviving neurons. Reaggregate tissue cultures are a useful tool in the study of potential neurotoxic effects of new or untested psychotherapeutic agents.

Acute and persistent effects of methamphetamine on developing monoaminergic neurons in reaggregate tissue culture

Three-dimensional, rotation-mediated reaggregate tissue cultures composed of rostral mesencephalic cells and corpus striatal cells were used to examine the short-term and persistent effects of methamphetamine on developing monoamine-containing neurons. Reaggregates were exposed to drug for one week. Reductions in reaggregate endogenous dopamine and serotonin levels occurred following treatment with methamphetamine during days 15-22 of culture over the concentration range 10(-7) to 10(-4) M. The highest methamphetamine concentration reduced dopamine and serotonin levels to 29 and 33%, respectively, of control values. Monoamine levels were reduced from control values after 3 days of exposure to 10(-4) M methamphetamine. No further reduction resulted from 4 additional days of drug treatment. In order to determine whether monoaminergic neurons would recover from the drug-induced deficit, reaggregates were exposed to 10(-4) M methamphetamine for 7 days and then grown in drug-free media for an additional 20 days. During the 20 day recovery period, monoamine levels in the control group increased with time in culture. After an initial rapid increase (recovery days 0-9), the level of monoamines in the recovery group remained at a constant proportion to the level in the control group suggesting that the monoaminergic neurons return to a rate of development similar to that seen in untreated cultures. However, this rate was not sufficient to overcome the reduction in monoamine levels produced by 7 days of methamphetamine treatment. The results indicate that the effects of methamphetamine on developing monoaminergic neurons are marked and persistent.

Quantitation of neurochemically identified neurons in reaggregate tissue cultures

The study of cellular interactions during the development of the nervous system is facilitated by the use of the reaggregate tissue culture system. In this system, embryonic neurons are dissociated from one another and allowed to reaggregate in rotatory culture, where they resume their normal differentiation. Reaggregates provide a model for studying neuronal development and cell interactions under conditions which allow for the control of a large number of variables, such as the numbers of cells which are allowed to interact, the presence or absence of appropriate target cells, and the chemical environment in which the neurons develop. For quantitative purposes, it is necessary to estimate the numbers of cells of a given neurochemical or morphologic type. Here we describe a computer-assisted counting system which allows one to determine the numbers of neurochemically identified neurons within reaggregate cultures from the counting of such neurons in a randomly selected sample of thirty 10-micron sections from the reaggregates in a given flask. These sections represent less than 0.5% of the total number of sections derived from the reaggregates in a single flask. In addition, the system allows one to quantitatively compare differences in the numbers of such neurons between experimental flasks utilizing appropriate statistical methods. A comparison of the number of central dopaminergic neurons estimated by this method with the direct counting of such neurons in serial sections of single reaggregates resulted in excellent agreement as to the number of dopaminergic neurons within a given experimental flask.(ABSTRACT TRUNCATED AT 250 WORDS)