Delayed administration of a potent cyclin dependent kinase and glycogen synthase kinase 3 beta inhibitor produces long-term neuroprotection in a hypoxia-ischemia model of brain injury.

We compared the neuroprotective efficacy of a potent and CNS-penetrant cyclin dependent kinase (CDK) and glycogen synthase kinase 3 beta (GSK3beta) inhibitor (Compound 1) in juvenile (postnatal day 21; P21) and adult C57Bl/6 mice (postnatal day 60; P60) using a model of hypoxic-ischemic brain injury (HI). Neuronal cell counts and density measures from brain sections stained with Cresyl Violet revealed that exposure of P21 mice to 60 min of HI resulted in extensive damage to the ipsilateral cornu ammonis 1 (CA1) region of the hippocampus (40% cell loss) and striatum (30% cell loss) 7 days later. Exposure of P60 mice to 40 min of HI produced a similar pattern of cell loss. Intraperitoneal administration of Compound 1 (3 mg/kg) 1, 5 and 9 h after 60 min of HI did not reduce brain injury in P21 mice relative to vehicle controls. By contrast, in P60 mice, this treatment significantly decreased cell loss in the ipsilateral hippocampus (10% cell loss) and striatum (15% loss) relative to vehicle controls. Terminal uridine deoxynucleotidyl transferase (TUNNEL) positive cell counts and infarct volume were also substantially reduced in P60 mice treated with Compound 1. A motor coordination test performed twice weekly until 5 weeks post-HI confirmed that Compound 1 produced long lasting functional recovery. Our results indicate that Compound 1 produced long lasting neuroprotective effects in adult but not juvenile mice suggesting that inhibition of the CDKs and GSK3beta plays a distinct neuroprotective role in the juvenile and adult brain.

Distribution of immunophilin FKBP-12 protein and mRNA within the mammalian cochlea and cochlear nucleus.

Immunophilin FK binding protein-12 (FKBP-12), the soluble receptor for the immunosuppressant drug FK506, is involved in a number of neuronal activities including increased nerve regeneration in the peripheral nervous system and enhanced recovery in animal models of neurodegenerative diseases. In addition, FKBP-12 is tightly bound to the calcium release channel ryanodine receptor and physiologically interacts with the inositol 1,4,5-trisphosphate receptor. In nearly all cell types, release of intracellular Ca(2+) and subsequent second messenger signaling involves activation of these ion channels. We determined the distribution of FKBP-12 within the mammalian cochlea and dorsal cochlear nucleus (DCN) in order to gain insight into Ca(2+) regulation within the cochlea and to possibly identify potential cellular targets for neuroimmunophilin ligands that may prove useful in protection and recovery following ototoxic insult. FKBP-12 protein and mRNA were found to be abundant throughout rat and guinea pig cochlea and DCN.

Glial cell line-derived neurotrophic factor has a dose dependent influence on noise-induced hearing loss in the guinea pig cochlea.

We examined the effectiveness of glial cell line-derived neurotrophic factor (GDNF) to attenuate cochlear damage from intense noise stress. Subjects were exposed to 115 dB SPL one octave band noise centered at 4 kHz for 5 h. They received artificial perilymph with or without GDNF into the left scala tympani at 0.5 microliter/h from 4 days before noise exposure through 8 days following noise exposure. Different concentrations of GDNF (1 ng/ml, 10 ng/ml, 100 ng/ml, and 1 microgram/ml) were applied chronically directly into the guinea pig cochlea via a microcannula and osmotic pump. Noise-induced hearing loss was assessed with pure tone auditory brainstem responses (at 2, 4, 8 and 20 kHz), measured prior to surgery, 1 day before noise exposure, and 7 days following noise exposure. Subjects were killed on day 8 following exposure for histological preparation and quantitative assessment of hair cell (HC) damage. A dose-dependent protective effect of GDNF on both sensory cell preservation and hearing function was found in the treated ears. At 1 ng/ml, GDNF showed no significant protection; at 10 ng/ml, GDNF showed significant HC protection; and at 100ng/ml, it was greater and bilateral. At 1 microgram/ml, GDNF appeared to have a toxic effect under noise stress in some cochleae. These findings indicate that GDNF at certain concentrations can effectively protect the inner ear from noise-induced hearing loss.

Spiral ganglion neurons are protected from degeneration by GDNF gene therapy.

Perceptual benefits from the cochlear prosthesis are related to the quantity and quality of the patient's auditory nerve population. Multiple neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), have been shown to have important roles in the survival of inner ear auditory neurons, including protection of deafferented spiral ganglion cells (SGCs). In this study, GDNF gene therapy was tested for its ability to enhance survival of SGCs after aminoglycoside/diuretic-induced insult that eliminated the inner hair cells. The GDNF transgene was delivered by adenoviral vectors. Similar vectors with a reporter gene (lacZ) insert served as controls. Four or seven days after bilateral deafening, 5 microl of an adenoviral suspension (Ad-GDNF or Ad-lacZ) or an artificial perilymph was injected into the left scala tympani of guinea pigs. Animals were sacrificed 28 days after deafening and their inner ears prepared for SGC counts. Adenoviral-mediated GDNF transgene expression enhanced SGC survival in the left (viral-treated) deafened ears. This observation suggests that GDNF is one of the survival factors in the inner ear and may help maintain the auditory neurons after insult. Application of GDNF and other survival factors via gene therapy has great potential for inducing survival of auditory neurons following hair cell loss.

Hair cell protection from aminoglycoside ototoxicity by adenovirus-mediated overexpression of glial cell line-derived neurotrophic factor.

Aminoglycosides are commonly used antimicrobial drugs that often have ototoxic side effects. The ototoxicity often involves permanent loss of cochlear hair cells (HCs). Neurotrophic factors have been shown to protect a variety of tissues, including HCs, from toxic trauma. To determine if glial cell line-derived neurotrophic factor (GDNF) can protect cochlear HCs from trauma, we inoculated an adenoviral vector encoding the human GDNF gene into guinea pig cochleae via the round window membrane 4 days prior to injection of aminoglycosides. Control groups showed little or no negative influence of the viral inoculation on cochlear structure and function. In contrast, ears that were inoculated with the GDNF vector had better hearing and fewer missing HCs after exposure to the ototoxins, as compared with controls. Our results demonstrate the feasibility of gene therapy for cochlear application and suggest that virus-mediated overexpression of GDNF may be developed as a valuable prevention against trauma-induced HC death.

Glial cell line-derived neurotrophic factor. Potential for otoprotection.

Sensorineural hearing loss results from the degeneration of hair cells and/or auditory neurons in the cochlea of the inner ear. BDNF and NT-3 were shown to support survival of auditory neurons both in vitro and in vivo. Cochlea from P3-P4 rats were cultured as floating explants and hair cells in the organ of Corti were identified by phalloidin-FITC immunostaining. Treatment with cisplatin (35 micrograms/mL) or neomycin (0.6 mM) resulted in 21.2 +/- 6.0% and 7.4 +/- 4.7% surviving hair cells, respectively, after 3 days in culture. GDNF, added together with the ototoxins, increased their number to 46.7% and 37.4%, respectively. In cultures of dissociated cochlea from 4-week-old rat, cisplatin (5 mg/mL) added 24 h after seeding resulted in only 6.1 +/- 1.2% surviving neurons. However, when cisplatin was added together with GDNF (10 ng/mL), 32.8 +/- 1.0% of the neurons survived. The efficacy of GDNF in animal models of ototoxicity was tested next. Guinea pigs were pretreated with GDNF in one ear, delivered either by infusion into the inner ear (scala tympani) with Alzet minipumps (50 ng/mL at a 0.5 microL/h), or injected into the middle ear (120 microL at 1 mg/mL) through the tympanic membrane. The ear that did not receive GDNF always served as control. Ototoxicity was induced systemically either by intraperitoneal cisplatin injections (1 mg/kg/day for 15 days or two injections of 7.5 mg/kg at a 5-day interval or by a combination of kanamycin (200-300 mg/kg, administered subcutaneously) and ethacrinic acid (40 mg/kg, intravenous). It was found that the number of surviving hair cells in GDNF-treated ears was about twice that of control ears in animals exposed to the ototoxins. The transducing GDNF receptor (ret) is expressed in the inner ear.

Rescue and regrowth of sensory nerves following deafferentation by neurotrophic factors.

Trauma and loss of cochlear inner hair cells causes a series of events that result first in the retraction of the peripheral processes of the auditory nerve, scar formation in the organ of Corti, and over the course of weeks to months (depending on the species) the loss of auditory nerve cell bodies (spiral ganglion cells). Neurotrophic factors play an important role in the mature nervous system as survival factors for maintenance and protection and also can play a role in regrowth. Studies in the cochlea now show that application of exogenous neurotrophic factors can enhance survival of spiral ganglion cells after deafness and induce regrowth of peripheral processes, perhaps by replacing lost endogenous factors. Combinations of factors may be most effective for achieving greatest survival and regrowth. Our studies find that brain-derived neurotrophic factor (BDNF) and glial-line-derived neurotrophic factor (GDNF) are very effective at enhancing spiral ganglion cell survival following deafness from ototoxic drugs or noise. It has also been found that BDNF plus fibroblast growth factor (FGF) is very effective at inducing process regrowth. Electrical stimulation also acts to enhance spiral ganglion cell survival, and the combination of electrical stimulation and neurotrophic factors could prove a most effective intervention.

Guinea pig auditory neurons are protected by glial cell line-derived growth factor from degeneration after noise trauma.

For patients with profound hearing loss, cochlear implants have become the treatment of choice. These devices provide auditory information through direct electrical stimulation of the auditory nerve. Prosthesis function depends on survival and electrical excitability of the cochlear neurons. Degeneration of the auditory nerve occurs after lesions of its peripheral target field (organ of Corti), specifically, including loss of inner hair cells (IHCs). There is now evidence that local treatment of the cochlea with neurotrophins may enhance survival of auditory neurons after aminoglycoside-induced deafness. Glial cell line-derived neurotrophic factor (GDNF) has recently been shown to be an important survival factor in other regions of the nervous system. By in situ hybridization, we now show that IHCs of the neonatal and mature rat cochlea synthesize GDNF and that GDNF-receptor alpha, but not c-Ret, is expressed in the rat spiral ganglion. We also show that GDNF is a potent survival-promoting factor for rat cochlear neurons in vitro. Finally, we examined GDNF efficacy to enhance cochlear-nerve survival after IHC lesions in vivo. We found that chronic intracochlear infusion of GDNF greatly enhances survival of guinea pig cochlear neurons after noise-induced IHC lesions. Our results demonstrate that GDNF is likely to be an endogeneous survival factor in the normal mammalian cochlea and it could have application as a pharmacological treatment to prevent secondary auditory nerve degeneration following organ of Corti damage.

GDNF protects the cochlea against noise damage.

Glial-derived neurotrophic factor (GDNF) was tested for its ability to prevent hearing and sensory cell loss in guinea pigs exposed to acoustic trauma. Hearing was measured prior to any treatment. Animals were exposed to damaging levels of noise either before or after local application of GDNF to one ear. Four weeks later, hearing and sensory cell loss was greater in the control ear than in the ear receiving GDNF before acoustic trauma or 2 h after trauma, but not 4 or 6 h after trauma. The results indicate that GDNF treatment in vivo can prevent cochlear sensory cell damage and hearing loss if present during or shortly after acoustic trauma.

Expression of the adrenoleukodystrophy protein in the human and mouse central nervous system.

The gene mutated in X-linked adrenoleukodystrophy (ALD), a progressive demyelinating disease, codes for a protein (ALDP) involved in very-long-chain fatty acid (VLCFA) transport. The expression of ALDP and of two peroxisomal enzymes involved in beta-oxidation of VLCFA, acyl-CoA oxidase, and catalase was studied in human and mouse brain. The pattern of expression was similar in both species. While acyl-CoA oxidase and catalase are found in all types of CNS cells, including neurons and oligodendrocytes, ALDP expression is restricted mostly to the white matter and endothelial cells. ALDP is highly expressed in astrocytes and microglial cells in vivo and in regenerating oligodendrocytes in vitro. In contrast, in vivo, ALDP is detected in much fewer oligodendrocytes and quantitative Western blot analysis confirmed the lower abundance of ALDP in these cells than in astrocytes. Only oligodendrocytes localized in corpus callosum, internal capsules, and anterior commissure express ALDP at levels comparable to those seen in astrocytes. In ALD, demyelination is first detected in these white matter regions, suggesting that the ALD gene mutation selectively affects those oligodendrocytes strongly expressing ALDP. Because of their failure to express ALDP, microglia and astrocytes may also contribute to demyelination in ALD patients.

B61, a ligand for the Eck receptor protein-tyrosine kinase, exhibits neurotrophic activity in cultures of rat spinal cord neurons.

Although the Eph subfamily represents the largest group of receptor protein-tyrosine kinases, the biological roles of the Eph-related receptors and their ligands are not well understood. B61 has been identified recently by receptor affinity chromatography as a ligand for the Eph-related receptor Eck (Bartley et al.: Nature 368:558-560, 1994). Here we show that Eck immunoreactivity is localized in areas of the embryonic rat spinal cord that are rich in axons, suggesting that Eck plays a role in this region of the developing nervous system. To examine the biological function of Eck, monolayer cultures of dissociated cells from embryonic rat spinal cord were treated with soluble B61. With an ED50 of approximately 10 ng/ml, B61 treatment improved the survival of the overall neuronal population. Furthermore, in the presence of B61 neurites were longer and more elaborated. B61 similarly affected survival and neurite length in cultures enriched in motor neurons. These neurotrophic effects of B61 were not observed in the presence of anti-Eck antibodies, indicating that these effects are likely to be mediated by the Eck receptor.

Inhibition of p185c-erbB-2 proto-oncogene expression by antisense oligodeoxynucleotides down-regulates p185-associated tyrosine-kinase activity and strongly inhibits mammary tumor-cell proliferation.

The c-erbB-2 proto-oncogene codes for a 185-kd putative growth factor receptor that is highly homologous to but distinct from the epidermal growth factor (EGF) receptor. Amplification and overexpression of c-erbB-2 occurs in a number of human tumors, in some of which it is a negative prognostic factor. This study investigates the possibility of inhibiting tumor-cell proliferation by blocking c-erbB-2 expression in the human mammary carcinoma cell line SK-Br-3 using chemically modified antisense oligodeoxynucleotides. Expression of the p185c-erbB-2 protein product was selectively reduced within 48 hours and resulted in a growth arrest of SK-Br-3 cells. Biochemical studies of tyrosine-kinase and S6-kinase activities after antisense inhibition of c-erbB-2 show that p185c-erbB-2 activates the S6-kinase signalling pathway in a nonlinear, dose-dependent manner. This may be relevant for the design of therapeutic strategies involving the inhibition of c-erbB-2 (proto)- oncogene expression.

Effects of ciliary neurotrophic factor on the survival and response to nerve growth factor of cultured rat sympathetic neurons.

The development and maturation of cells depends not only on their genetic history, but also on sequences and combinations of environmental signals appropriate to their developmental age. Early postnatal rat sympathetic neurons are dependent on nerve growth factor (NGF) for survival in vivo and in vitro, but earlier sympathetic neuroblasts may not require NGF. Ciliary neurotrophic factor (CNTF) provides short-term in vitro trophic support to embryonic and neonatal sympathetic neurons, but its role in vivo is not understood. In this study we examined further the capability of CNTF to support neonatal rat superior cervical ganglion (SCG) sympathetic neurons in vitro and the effect of CNTF on the trophic activities of NGF. SCG neurons cultured with either CNTF or NGF survived for 24 hr in low-density cultures depleted of nonneuronal cells, in contrast to neurons with neither factor. However, with CNTF only a fraction of the NGF-maintained number of neurons survived for 6 days. CNTF given in combination with NGF in these nonneuron-depleted cultures produced a significant decrease in the number of neurons surviving for 6 days, compared to the number supported by NGF alone. If such cultures were supplemented with Schwann cells, very different results were obtained: CNTF alone supported the 6-day survival of 80% as many neurons as did NGF, and the combination of CNTF with NGF produced no decrease in neuronal survival. Antibody to NGF did not block the support provided by CNTF and Schwann cells. Immunostaining for the low-affinity NGF receptor (LNGFR), intense in the NGF-supported neurons, was absent in the CNTF-supported neurons and reduced in the neurons exposed to the combination of NGF and CNTF. These results show that CNTF can act synergistically with a Schwann cell-derived agent to provide trophic support to neonatal sympathetic neurons, and that it can down-regulate the responsiveness of those neurons to NGF.

Survival-promoting and protein kinase C-regulating roles of basic FGF for hippocampal neurons exposed to phorbol ester, glutamate and ischaemia-like conditions.

Recent evidence suggests that protein kinase C (PKC) is involved in the pathophysiology of neurodegenerative diseases. We examined the effect of basic fibroblast growth factor (bFGF) on the survival of cultured rat hippocampal neurons exposed to conditions in which PKC is likely to play a role. bFGF reduced neuron damage caused by the PKC-activating phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), glutamate and ischaemia-like culture conditions. bFGF was able to counteract the excessive activation of PKC caused by these treatments. Moreover, bFGF prevented the loss of PKC occurring after prolonged exposure to TPA or ischaemia-like conditions. These results indicate that both the overactivation and the abnormal degradation of PKC can lead to neuron degeneration, and that the neurotrophic competence of bFGF may reside in its ability to regulate and normalize the PKC phosphorylating system.

CNTF promotes the survival of neonatal rat corticospinal neurons in vitro.

Corticospinal neurons were identified in cell cultures of neonatal rat cortex by immunostaining of cholera toxin B subunit (CTB), retrogradely transported from the cervical part of the spinal cord. The CTB-immunoreactive neurons were larger than the neurons in the overall (unstained) neuronal population and represented a small fraction of it (average of 0.3%) after 6 hours in vitro. The number of both total and CTB-labeled neurons declined progressively with time in culture. The neuronal death was, however, markedly faster in the CTB-labeled neuronal population than in the overall neuronal population. Ciliary neurotrophic factor (CNTF) promoted the survival of CTB-positive corticospinal neurons in a dose-dependent manner; with CNTF, the death rate of the CTB-labeled neurons became identical to that of the overall population.

Convergent regulation by ciliary neurotrophic factor and dopamine of tyrosine hydroxylase expression in cultures of rat substantia nigra.

Ciliary neurotrophic factor and dopamine were found to enhance the expression of tyrosine hydroxylase immunoreactivity in cultured neurons from the substantia nigra of 16-day-old rat fetuses. The number of tyrosine hydroxylase-positive cells decreased progressively to approximately 30% by 96 h. Treatment with 5 microM dopamine maintained the tyrosine hydroxylase-positive neurons at 60% for 48 h, but not for longer. Concurrent treatment with 5 microM dopamine and 20 trophic units/ml ciliary neurotrophic factor had a greater impact on tyrosine hydroxylase-positive cells, resulting in the maintenance of 70% of the initial number for up to 72 h, but not beyond that time. When dopamine or dopamine/ciliary neurotrophic factor treatments were applied for 24 h after a 48-h delay, the number of tyrosine hydroxylase-positive cells was restored to 60 and 80%, respectively, but not restoration was observed with 96-h delayed treatments. These results suggest that dopamine and ciliary neurotrophic factor, alone or in combination, are not able to support the survival of tyrosine hydroxylase-positive neurons, but reduce their apparent numerical loss by enhancing the expression of tyrosine hydroxylase. The effects of dopamine, alone or in combination with ciliary neurotrophic factor, were predominantly mediated by D2 receptors, since they were blocked by selective D2 receptor antagonists and since the D2 receptor agonist quinpirole was able to substitute for dopamine. The effects of dopamine and ciliary neurotrophic factor were similar in astroblast-rich and in astroblast-depleted cultures, suggesting that they were not mediated through glial cells. These results extend our previous observations on locus coeruleus cultures, in which the concurrent treatment with ciliary neurotrophic factor and norepinephrine was shown to enhance tyrosine hydroxylase expression (but not survival) of noradrenergic neurons. They also consolidate the view that ciliary neurotrophic factor and the neuron's own transmitter act in convergence and in an autocrine/paracrine mode as regulators of the corresponding neurotransmitter phenotype.

CNTF protection of oligodendrocytes against natural and tumor necrosis factor-induced death.

A proportion of developing oligodendrocytes undergo natural cell death by apoptosis, and mature oligodendrocytes die, either by apoptosis or necrosis, in response to injurious signals such as cytotoxic cytokines and complement. Ciliary neurotrophic factor (CNTF), a trophic factor found in astrocytes in the central nervous system (CNS), promoted the survival and maturation of cultured oligodendrocytes. This trophic factor also protected oligodendrocytes from death induced by tumor necrosis factors (apoptosis) but not against complement (necrosis). These results suggest that CNTF functions in the survival of oligodendrocytes during development and may lead to therapeutic approaches for degenerative diseases of the CNS that involve oligodendrocyte destruction.

Cooperative effects of ciliary neurotrophic factor and norepinephrine on tyrosine hydroxylase expression in cultured rat locus coeruleus neurons.

Ciliary neurotrophic factor (CNTF) was found to promote the expression of tyrosine hydroxylase (TH) immunoreactivity by cultured noradrenergic neurons from the locus coeruleus (LC) of E18 rat fetuses, but only in the concomitant presence of norepinephrine (NE), their own neurotransmitter. The number of TH-positive cells in LC cultures was shown to decrease by 65% within 3 days and by 75% after 6 days. Treatment with 10 TU/ml human recombinant CNTF together with 1 microM NE was able to fully maintain the initial number of TH-positive neurons for 3 days. This effect, however, was no longer seen after 6 days of continuous exposure. A 24-hr treatment with CNTF/NE was capable of completely restoring the initial number of TH-positive cells, even if its addition was delayed for 2 days. Moreover, when its addition was delayed for 5 days, CNTF/NE restored approximately 80% of the TH-positive neurons that were initially present. These results suggest that the disappearance of TH-positive neurons in LC cultures is not due to their death, but rather to the reduced expression of TH and that the simultaneous exposure to CNTF and NE upregulates TH. Effects on TH-positive cell number were not evoked by CNTF or NE alone. The CNTF/NE effect was dependent on protein synthesis, but was only partially inhibited by RNA synthesis inhibitors, suggesting that both transcription from preexisting mRNA and synthesis of new RNA were stimulated. The effect of CNTF/NE was mediated by alpha 2-adrenoceptors, since it was blocked by alpha 2-antagonists and since alpha 2-agonists were able to substitute for NE. Our results suggest a novel mechanism of regulation of the phenotype of the noradrenergic LC neuron, involving the collaborative influences of CNTF and norepinephrine, their own neurotransmitter.

Potential regulation by trophic factors of low-affinity NGF receptors in spinal motor neurons.

Developing spinal motor neurons (SMN) express low-affinity nerve growth factor receptors (LNGFR) but not high-affinity transducing NGF receptors. Moreover, SMN are not supported by NGF in vitro. In the normal adult rat most SMN are not LNGFR immunoreactive (LNGFR-IR), but they transiently reexpress LNGFR (though not the high-affinity receptor) after peripheral nerve injury. With a cut lesion of the sciatic nerve (when only a neuroma forms), the number of LNGFR-IR SMN at L4-L6 rapidly increases to a maximum between day 1 and 7 and returns to baseline levels by day 30. After a crush lesion (accompanied by regeneration to the muscle), LNGFR-IR SMN appear in about the same numbers, but they start to disappear 1 week later. We speculate that the similar appearance and differential decline of LNGFR-IR seen after the two types of lesions are regulated by the availability of a common signal such as ciliary neurotrophic factor. The adult SMN model provides a good opportunity to investigate the reexpression of LNGFR after peripheral nerve injury, and more generally, the unknown role and regulation of LNGFR.

Receptor-mediated toxicity of norepinephrine on cultured catecholaminergic neurons of the rat brain stem.

The mechanisms associated with the neurotoxic responses caused by prolonged exposure (48 hr) to norepinephrine (NE) were examined in cultures of brain stem of 18-day-old rat fetuses. Two separate components of NE neurotoxicity were identified and differentiated according to dose dependency, sensitivity to catalase and blockade by adrenoceptor antagonists. The first component of NE toxicity was responsible for the death of the overall cell population, affecting both neurons and astroblasts, and was mediated by NE auto-oxidation products. This toxicity was observed at high doses of NE (LD50: 100 microM), was mimicked by other catecholamines (epinephrine, isoproterenol, dopamine), was fully antagonized by catalase and could not be blocked by adrenoceptor antagonists. The second component of NE toxicity was specifically targeted at noradrenergic neurons and was mediated by alpha 1 adrenoceptors. The specific toxicity for noradrenergic neurons was seen at lower doses of NE (LD50: 20 microM) and epinephrine (LD50: 40 microM). It was mimicked by the alpha 1 agonist phenylephrine and blocked by the alpha antagonists prazosine and nicergoline. These results indicate that protracted exposure to catecholamines may be a possible cause of damage to noradrenergic neurons that can be prevented by alpha 1 adrenoceptor blockade.