General considerations in assessing neurotoxicity using neuroanatomical methods.

Neurotoxicology is a major focus of scientists and policy makers. Neurotoxicological investigations provide vital information needed by regulatory scientists to protect public health and can also elucidate fundamental mechanisms governing nervous system function and enhance our understanding of neurodegenerative diseases as well. A definition of neurotoxicity, developed by the Interagency Committee on Neurotoxicology, includes both permanent and reversible adverse effects on the nervous system. There are a number of factors that can greatly affect the outcome of any study designed to assess neurotoxicity, such as the choice of animal species, dose and dosage regimen, and route of administration. In considering neuroanatomical methodologies for assessing neurotoxicity, it is important to evaluate each technique for such factors as: limits of detection (sensitivity and signal to noise ratio), ability to be quantified, sampling problems, what is being measured and what can interfere with this measurement. Other questions relating to the strengths and weaknesses of neuroanatomical techniques that should be addressed include: is the technique difficult to perform? Is it reproducible? Which elements of the nervous system are best evaluated? Does the technique reveal the neuronal circuitry involved in the neurotoxic effect? Is successful application of the technique dependent on timing factors? Clearly, there are many factors that can influence the assessment of neurotoxicity so that it is best to base this assessment on converging data based on complementary techniques.

Effects of adult or neonatal treatment with 6-hydroxydopamine or 5,7-dihydroxytryptamine on locomotor activity, monoamine levels, and response to caffeine.

Rats were treated as neonates or adults with desmethylimipramine (DMI) followed by intraventricular 6-hydroxydopamine (6-HDA) or 5,7 dihydroxytryptamine (5,7-DHT). Locomotor activity of treated rats was measured in photocell cages. Neonatal treatment with 5,7-DHT produced hypoactivity during development while neonatal 6-HDA led to hyperactivity. Treatment of adult rats with 5,7-DHT or 6-HDA, while resulting in equivalent monoamine depletions, was without effect on locomotor activity. The dose response function for caffeine was determined in these rats. Depletion of dopamine by either neonatal or adult treatment with 6-HDA decreased caffeine stimulation of locomotor activity. The adenosine receptor agonist l-phenylisopropyladenosine (L-PIA) decreased locomotor activity in all rats in a dose-dependent fashion.

Modification of electroretinograms in dopamine-depleted retinas.

The functional role of dopamine in frog retina was examined in a combined neurochemical, immunohistochemical and electrophysiological study. Dopamine and serotonin are the primary monoamines present in the retina and they are localized to amacrine cells which have distinct morphologies. Intravitreal injection of 6-hydroxydopamine was found to produce a selective depletion of retinal dopamine content and elimination of tyrosine hydroxylase-like immunoreactivity. Electroretinograms from 6-hydroxydopamine-treated retinas demonstrated enhanced oscillatory potentials and a lengthening of the b-wave implicit time compared to vehicle control retinas; both of these changes in the electroretinogram were reversed by the dopamine agonist apomorphine. These observations support earlier suggestions that dopamine-containing amacrine cells are part of a retinal feedback system that generates oscillatory potentials and plays a role in light adaptation.

Six-hydroxydopamine induced hyperactivity: neither sex differences nor caffeine stimulation are found.

We investigated possible sex differences in the development of locomotor activity in rats treated neonatally with desmethylimipramine (DMI) followed by intraventricular 6-hydroxydopamine (6-HDA). In addition, the locomotor response to the stimulant caffeine was investigated in the male rats after they had reached adulthood. Both male and female 6-HDA-treated rats exhibited increased activity relative to controls. No sex differences were seen in either the development or magnitude of this effect. Male rats were used to determine the dose effects function for caffeine (0.5, 5, 15, 30 mg/kg) on locomotor activity. Control rats exhibited increased locomotor activity whereas 6-HDA-treated rats showed no increases with any dose of caffeine. Large decreases in the dopamine content of the olfactory tubercle (-88%, -82%), nucleus accumbens (-96%, -95%), and striatum (-99%, -99%) were found in both male and female rats. Choline acetyltransferase and glutamic acid decarboxylase activities were unchanged.

Effects of neonatal and adult 6-hydroxydopamine treatment on random-interval behavior.

Rats were given intraventricular injections of 6-hydroxydopamine (6-HDA) or saline-ascorbate vehicle as neonates (3-days old) and as adults (49 and 51 days old). At 73 days of age, they were trained on a random interval 90-sec schedule of water reinforcement. The rats treated with 6-HDA as adults stabilized at response rates approximately twice those of vehicle-treated rats, while rats treated with 6-HDA as neonates showed response rates which were not significantly different from vehicle-treated rats; Both L-Dopa and apomorphine decreased response rates at all doses tested. There were no differences among the groups with respect to the effect of these drugs. Adult-treated rats showed greater response rate decreases following peripheral decarboxylase inhibition with Ro 4-4602. Catecholamine analyses revealed the rats treated with 6-HDA as neonates had greater depletions in the striatum and the remainder of telencephalon than adult-treated rats but an increase in brainstem norepinephrine. These findings suggest that age of treatment is an important determinant of the biochemical and behavioral effects of treatment with 6-HDA.

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.

Age-dependent effects of 6-hydroxydopamine on locomotor activity in the rat.

This experiment examined the effects on locomotor activity of intraventricular 6-hydroxydopamine (6-OHDA) administered to developing and adult rats. 6-OHDA was administered subsequent to pargyline treatment at 3 and 6 days of age; or 6-OHDA was administered subsequent to desmethylimipramine (DMI) treatment (6-OHDA/DMI) at 3 and 6 days of age, 11 and 14 days of age, 20 and 23 days of age, or 46 and 48 days of age. Locomotor activity of vehicle-treated rats assessed in stabilimeter cages peaked between 14 and 16 days of age and subsequently declined to levels characteristic of the adult. Treatment with pargyline and 6-OHDA at 3 days of age, or 6-OHDA/DMI at 3 and 6 or 11 and 14 days of age, did not alter the early rise in locomotor activity but prevented the decline in activity normally seen during the third and fourth weeks of life. When tested as adults, locomotor activity was greater in rats that had been treated with 6-OHDA/DMI at 3 and 6 and at 11 and 14 days of age than in those that had been treated at 20 and 23 days of age. Treatment with 6-OHDA/DMI at 46 and 48 days of age was without significant effect on locomotor activity. 6-OHDA (with pargyline pretreatment) produced large decreases in NE content in telencephalon and diencephalon and in dopamine (DA) content in striatum. 6-OHDA-DMI also produced large decreases in DA content in striatum and, in some of the treatment groups, only small decreases in norepinephrine (NE) content in telencephalon, diencephalon, and brain stem. These data suggest that the maturation of neuronal systems utilizing dopamine as a neurotransmitter is essential for the suppression of locomotor activity normally seen during development. The data further suggest that dopamine depletion per se does not lead to increased locomotor activity, but rather it is the destruction of dopamine-containing fibers prior to the normal period of locomotor suppression that increases locomotor activity.

The spontaneous firing patterns of forebrain neurons. III. Prevention of induced asymmetries in caudate neuronal firing rates by unilateral thalamic lesions.

Unilateral lesions interrupting striatal outputs and inputs (MFB lesions) produce a marked slowing of neuronal firing in the caudate nucleus contralateral to the side of the lesions without affecting neuronal firing in the ipsilateral caudate nucleus. Although the MFB lesion also interrupts the nigrostriatal pathway and depletes the ipsilateral striatum of dipamine and its associated enzymes, the slowing of unit firing rates is apparently due to interruption of striatal outputs rather than inputs. Unilateral thalamic lesions palced ipsilateral to MFB lesions in iether the ventral anterior-ventrolateral nuclei (VA-VL) or in the center median-parafascicular nuclei (CM-PF) prevent the MFB lesion-induced asymmetry in caudate neuronal firing rates. These thalamic lesions do not, however, restore the striatal dopamine content depleted by the MFB lesion. Unilateral CM-PF lesions in otherwise intact cats do not alter caudate unit firing rates nor do they affect striatal dopamine. VA-VL lesions in otherwise intact cats produce a bilateral slowing in the spontaneous firing of neurons in the caudate nuclei, again, whithout altering caudate dopamine concentrations. These results provide further evidence that caudate dopamine concentration per se does not appear to be a potent variable in controlling the spontaneous firing rates of striatal neurons.

Development of left ventricular hypertrophy in young spontaneously hypertensive rats after peripheral sympathectomy.

The effects of peripheral sympathectomy with nerve growth factor antiserum (NGFAS) on blood pressure, systemic hemodynamics, myocardial function, myocardial hypertrophy, and renin were studied in male spontaneously hypertensive (SH) rats of the Okamoto strain and normotensive control Kyoto-Wistar (WKY) rats. NGFAS prevented the developing of hypertension in the SH rats but did not alter blood pressure in the WKY rats. The NGFAS-treated SH rats developed the same hemodynamic abnormalities as the sham-treated rats, including increased peripheral vascular resistance and depressed cardiac output; Indices of left ventricular performance, including peak flow velocity, stroke power, stroke work, dP/dtmax, and flow acceleration (dF/dt), were diminished in the SH rats compared to the WKY rats. NGFAS treatment further depressed ventricular function in the SH rats, but had little effect on the WKY rats; Plasma renin activity in both the SH and WKY rats was unaffected by NGFAS treatment. Although NGFAS treatment effectively prevented the development of hypertension in the SH rats, it did not influence the development of left ventricular hypertrophy as reflected by increases in left ventricular mass, RNA, DNA, and hydroxyproline content. The data suggest that the development of myocardial hypertrophy and myocardial dysfunction in the SH rat is in part independent of hypertension and plasma renin activity.

Catecholamines, blood pressure, renin and myocardial function in the spontaneously hypertensive rat.

1. Neither nerve-growth-factor antiserum (NGFAS) administered subcutaneously nor 6-hydroxydopamine administered intraventricularly to immature spontaneously hypertensive rats (SHR) inhibited the development of the hypertensive syndrome. In contrast, NGFSA did not affect blood pressure in normotensive Kyoto/Wistar rats. 2. Peripheral vascular resistance was increased and cardiac index decreased in both NGFAS and 6-hydroxydopamine-treated SHR despite preservation of normal blood pressure. 3 NGFAS treatment did not influence the development of left ventricular hypertrophy in SHR, despite the lowering of blood pressure. In contrast, 6-hydroxydopamine caused an attenuation in the development of left ventricular hypertrophy. 4. Indices of left ventricular contractility were depressed by NGFAS treatment but not by 6-hydroxydopamine. 5. Plasma renin activity was unaffected by NGFAS treatment and increased by 6-hydroxydopamine.

Prevention of hypertension in the SH rat: effects of differential central catecholamine depletion.

Six-hydroxydopamine (6-OHDA) was administered intraventricularly to 6-week-old male spontaneously hypertensive (SH) rats of the Okomoto strain and to normotensive rats of the Kyoto-Wistar strain. In addition, bilateral lateral tegmental lesions were placed in 35-40-day-old SH rats to interrupt ascending noradrenergic pathways. SH rats treated with 6-OHDA did not develop hypertension and had lower heart rates than control rats. Blood pressure and heart rate of Kyoto-Wistar animals were unaffected by the drug treatment. 6-OHDA produced widespread depletion of norepinephrine throughout the CNS of both SH and Kyoto-Wistar rats. Bilateral lateral tegmental lesions interrupted the dorsal noradrenergic bundle and depleted forebrain norepinephrine. These lesions did not prevent the development of hypertension and led to an increased heart rate. It is concluded that 6-OHDA does not produce its effect through a nonspecific lowering of blood pressure, but rather, that it interferes with the expression of the hypertensive syndrome. The lack of effect seen following depletion of forebrain norepinephrine as the result of interruption of the dorsal noradrenergic bundle indicates that the fibers destroyed by this lesion are not essential for the development of genetically determined hypertension.