KOSTRZEWA, R. M., J. W. KLARA, J. ROBERTSON AND L. C. WALKER. Studies on the mechanism of sprouting of noradrenergic terminals in rat and mouse cerebellum after neonatal 6-hydroxydopa. BRAIN RES. BULL. 3(5) 525-531, 1978.-The effect of various pharmacologic agents on the noradrenergic innervation of rat cerebellum was observed. It was found that the neurotoxin 6-hydroxydopa (6-OHDOPA), when given to rats at birth, caused a 46% reduction at 5 weeks of age in tyrosine hydroxylase activity in the locus coeruleus, the nucleus of origin for noradrenergic fibers innervating the cerebellum. At the same time, however, both tyrosine hydroxylase activity and NE content were elevated by 50% in the cerebellum. By treating gravid mice with the 6-OHDOPA, which crosses the placental barrier to affect the brains of developing pups, a dissociation has been shown between the elevated cerebellar NE levels and reduced telencephalic NE content. None of the other assorted pharmacological agents-namely amphetamine, metaraminol, apomorphine, α-methyl-ρ-tyrosine, L-dihydroxyphenylalanine and tyramine-when given at birth, caused a permanent elevation in cerebellar NE content. This series of studies suggests that a reduced number of noradrenergic perikarya are providing a greater innervation of the cerebellum than in control rats. Also, alteration of the telencephalic noradrenergic fibers, which are also derived from the locus coeruleus, does not appear to be a necessary event for the initiation of sprouting of noradrenergic fibers in the cerebellum. Because none of the acute-acting pharmacological agents caused a permanent elevation of NE in the cerebellum, it appears that damage, and not mere stimulation or blockade, is a necessary event for initiation of sprouting.

Litters of female rats were treated at birth and 48 hr later with either saline or 6-hydroxydopa (60 μg/g, IP), were ovariectomized in adulthood and tested on a number of behavioral tasks including age of vaginal opening, sexual receptivity, open-field activity, equilibrium, and habituation to acoustic startle. Results of the open-field test indicated that the treated animals were more active overall, were more likely to enter inner segments, reared more often, and defecated less than the control animals. On a rod- balancing task, the treated animals exhibited impaired equilibrium. Treated animals were more reactive than controls in response to acoustic startle, but there were no differences between the groups in rate of habituation or sensitization to the startle stimulus. Norepinephrine content of treated animals was significantly lower than controls in the cortex, amygdala, hippocampus, and spinal cord, but higher in the cerebellum and brainstem. There was no difference between the groups in cardiac norepinephrine nor in striatal dopamine.

Treatment of newborn rats with 6-hydroxydopa (6-OHDOPA, 60 μg/g IP) increased the levels of norepinephrine in the adult cerebellum and hindbrain. Concurrent treatment with morphine sulfate (20 μg/g IP) potentiated the response to 6-OHDOPA in the cerebellum and pons-medulla. In addition, increased noradrenergic neurite density in 4 week cerebellar cortex (as observed with histofluorescent staining by glyoxylic acid) suggests that neonatal morphine increased the sprouting of noradrenergic neurons in the 6-OHDOPA treated rats.

In order to demonstrate the influence of morphine on the developmental localization of regenerated noradrenergic fibers in rat cerebellum, a glyoxylic histofluorescent method and radiometric assay for norepinephrine (NE) were utilized. An initial reduction of NE in the cerebellum after 6-hydroxydopa [6-OHDOPA; 60 µg/g intraperitoneally (i.p.)] was followed by a return to control levels at 3 days, and an elevation above control levels at 7 days. The initial rates of recovery of NE in the cerebellum of the 6-OHDOPA group of rats and the group receiving morphine (20 µg/g i.p.) in combination with 6-OHDOPA were identical up to 7 days. However, by 14 days NE content was further elevated in the cerebellum of the morphine+6-OHDOPA group. Histofluorescent microscopic observations of the cerebellar cortex correlated with the biochemical findings. A reduction in cerebellar NE content at 3 days was associated with a reduction in the number of visible histofluorescent fibers in the cerebellar cortex. By 7 days the relative number of fibers in the 6-OHDOPA groups was similar to that seen in the control group, but by 9 days the relative number of fluorescent fibers in the cerebellar cortex was increased above control. By 13 days there was a further increase in the relative number of fluorescent fibers in the cerebellar cortex of the morphine+6-OHDOPA group, as compared to the group treated with 6-OHDOPA alone. These findings provide an anatomic correlate for recovery of noradrenergic fibers after 6-OHDOPA, and demonstrate an action of morphine in enhancing regenerative sprouting.

The amino acid, 6-hydroxydopa (6-OHDOPA), found at the active site of amine oxidases, exists as a keto-enol. Exogenously administered 6-OHDOPA is an excitotoxin like β-N-oxalylamino-L-alanine (BOAA) and β-N-methylamino-L-alanine (BMAA), acting at the non-N-methyl-D-aspartate (non-NMDA) α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor. BMAA and BOAA are causal factors of neurolathyrism in humans. Much exogenously administered 6-OHDOPA is biotransformed by aminoacid decarboxylase (AADC) to the highly potent and catecholamine-(CA) selective neurotoxin, 6-hydroxydopamine (6-OHDA). 6-OHDOPA destroys locus coeruleus noradrenergic perikarya and produces associated denervation of brain by norepinephrine-(NE) containing fibers. Opiopeptides and opioids enhance neurotoxic effects of 6-OHDOPA on noradrenergic nerves, by a naloxone-reversible process. An understanding of mechanisms underlying neurotoxic effects of 6-OHDOPA can be helpful in defining actions of known and newfound amino acids and for investigating their potential neurotoxic properties.

6-hydroxydopa (6-OHDOPA) was synthesized with the expectation that it would be able to cross the blood-brain barrier to be enzymatically decarboxylated to 6-hydroxydopamine (6-OHDA), the newly discovered neurotoxin for noradrenergic and dopaminergic neurons. In part, 6-OHDOPA fulfilled these criteria. When administered experimentally to rodents, 6-OHDOPA destroyed peripheral sympathetic noradrenergic nerves and did exert neurotoxicity to noradrenergic nerves in brain—in large part, from its conversion to 6-OHDA. However, the efficacy of 6-OHDOPA was less than that of 6-OHDA; also, 6-OHDOPA was relatively selective for noradrenergic neurons; near-lethal doses of 6-OHDOPA were required to damage dopaminergic nerves; and ultimately, 6-OHDOPA was found to be an agonist at AMPA receptors, thus accounting for more non-specificity. Nevertheless, 6-OHDOPA was found to be a particularly valuable tool in uncovering processes and mechanisms associated with noradrenergic nerve regeneration and sprouting, particularly when administered to perinatal rodents. Also, 6-OHDOPA was a good tool for selective mapping of noradrenergic nerve tracts in brain, since dopaminergic tracts were unaffected and did not interfere with the histofluorescent methodology used for this purpose in the early 1970s. As an experimental research tool, 6-OHDOPA was valuable in a short time-window, but its utility is largely limited because of newer research technologies that provide better means today for nerve tract mapping, and for experimental approaches engaged toward study of processes and mechanisms attending nerve regeneration. AMPA actions of 6-OHDOPA have not been extensively studied, so this avenue may enliven use of 6-OHDOPA in the future.

Although it is well-recognized that catecholamines are generally unable to penetrate the developed blood-brain barrier (BBB) to gain entry into brain, except at circumventricular sites where the BBB is absent or deficient, onto-genetic development of this barrier seems to have escaped systematic study. To explore BBB development, several approaches were used. In the first study rats were treated once on a specific day of postnatal ontogeny, as early as the day of birth, with the neurotoxin 6-hydroxydopa-mine (6-OHDA; 60 mg/kg), and then terminated in adulthood for regional analysis of endogenous norepinephrine (NE) content of brain. In another study, rats were treated once, on a specific day of postnatal ontogeny, with the BBB-perme-able neurotoxin 6-hydroxydopa (6-OHDOPA; 60 mg/kg) following pretreatment with the BBB-impermeable amino acid decarboxylase inhibitor carbidopa (100 mg/kg IP), then terminated in adulthood for regional analysis of endogenous NE content of brain. In the third study rats were treated once, on a specific day of postnatal ontogeny, with the analog [3H]metaraminol, and terminated 1 hour later for determination of regional distribution of tritium in brain. On the basis of [3H]metaraminol distribution and NE depletions after neurotoxin treatments, it is evident that the BBB in neocortex, striatum, cerebellum and other brain regions forms in stages over a period of at least 2 weeks from birth. Moreover, because the BBB consists of several element (physical-, ion-restrictive-, and enzymatic-barrier), the method employed will derive data mainly applicable to the targeted aspect of the barrier, which may or may not necessarily coincide with elements of the barrier that have a different rate of ontogenetic development. Accordingly, it is evident that some aspects of physical- and ion-restrictive elements of the BBB form within approximately the first week after birth in rat neocortex and striatum, while enzymatic elements of the BBB form more than than 2 week later. Regardless, the BBB forms at earlier times in forebrain vs hindbrain regions.

To determine if greater amounts of hydroxyl radical (·OH) are formed by dopamine (DA) denervation and treatment with L-dihydroxyphenylalanine (L-DOPA), the neostriatum was DA denervated (99% reduction in DA content) by 6-hydroxydopamine treatment (134μg icv, desipramine pretreatment) of neonatal rats. At 10 weeks the peripherally restricted dopa decarboxylase inhibitor carbidopa (12.5mg/kg i.p.) was administered 30min before vehicle, L-DOPA (60mg/kg i.p.), or the known generator of reactive oxygen species, 6-hydroxydopa (6-OHDOPA) (60mg/ kg i.p.); and this was followed 30min later (and 15 min before termination) by the spin trap, salicylic acid (8 μmoles icv). By means of a high performance liquid chromatographic method with electrochemical detection, we found a 4-fold increase in the non-enzymatically formed spin trap product, 2,3-dihydroxybenzoic acid (2,3-DHBA), with neither L-DOPA nor 6-OHDOPA having an effect on 2,3-DHBA content of the neostriatum. Basal content of 2,5-DHBA, the enzymatically formed spin trap product, was 4-fold higher vs. 2,3-DHBA in the neostriatum of untreated rats, while L-DOPA and 6-OHDOPA each reduced formation of 2,5-DHBA. We conclude that DA innervation normally suppresses ·OH formation, and that the antiparkinsonian drug L-DOPA has no effect (2,3-DHBA) or slightly reduces (2,5-DHBA) ·OH formation in the neostriatum, probably by virtue of its bathing the system of newly formed ·OH.

A selective neurotoxin takes many forms: as an antibody to a neurotrophin, as an alkylator, as an excitotoxin, as a blocker of requisite neuronal excitation during ontogenetic development, as a generator of oxidative stress, as an inhibitor of vital intraneuronal processes, and as an agent adversely affecting a host of multiple sites in neurons. Neurotoxins have been invaluable for elucidating cellular mechanisms attending or preventing neuronal necrosis and apoptosis, and for modeling and thereby discerning mechanisms invoked in neurological and psychiatric disorders. Neuroprotectants, endogenous and exogenous, are being explored as potentially useful agents to ward off diseases. Finally, hypothesized as posing a risk to humans as environmental constituents, neurotoxins are now being remodeled as adjuncts for therapeutic intervention in a variety of human medical disorders.

Over the past 60 years, a large number of selective neurotoxins were discovered and developed, making it possible to animal-model a broad range of human neuropsychiatric and neurodevelopmental disorders. In this paper, we highlight those neurotoxins that are most commonly used as neuroteratologic agents, to either produce lifelong destruction of neurons of a particular phenotype, or a group of neurons linked by a specific class of transporter proteins (i.e., dopamine transporter) or body of receptors for a specific neurotransmitter (i.e., NMDA class of glutamate receptors). Actions of a range of neurotoxins are described: 6-hydroxydopamine (6-OHDA), 6-hydroxydopa, DSP-4, MPTP, methamphetamine, IgG-saporin, domoate, NMDA receptor antagonists, and valproate. Their neuroteratologic features are outlined, as well as those of nerve growth factor, epidermal growth factor, and that of stress. The value of each of these neurotoxins in animal modeling of human neurologic, neurodegenerative, and neuropsychiatric disorders is discussed in terms of the respective value as well as limitations of the derived animal model. Neuroteratologic agents have proven to be of immense importance for understanding how associated neural systems in human neural disorders may be better targeted by new therapeutic agents.