Polyamines and their metabolizing enzymes in human frontal cortex and hippocampus: preliminary measurements in affective disorders.

Affective disorders are associated with maladaptive response to stressful life events. Based on the observation that a transient increase in brain polyamine metabolism is a common response to stressful stimuli, our hypothesis is that a maladaptive polyamine stress response may be involved in the pathophysiology of affective disorders. Our current research efforts, therefore, concentrate on the characterization of this PA response, and on its pharmacological regulation. The present preliminary study is the first to measure the polyamines, putrescine, spermidine, and spermine, and their metabolizing enzymes, ornithine decarboxylase, S-adenosylmethionine decarboxylase, and spermidine/spermine N1 acetyltransferase, in brain autopsy samples from people who suffered from depressive disorders or schizophrenia, or from those who committed suicide. The data of affected individuals did not reveal significant differences when compared to those of suicide cases, or to those of people with no known neurologic or psychiatric abnormalities. The following regional differences were observed: spermidine concentrations and ornithine decarboxylase activity were higher, but S-adenosylmethionine decarboxylase activity was lower in the hippocampus as compared to the frontal cortex. Preliminary studies with rat brain indicate that an increase in polyamine metabolizing enzyme activities occurs within several hours after death and persists for at least 48 hours. These observations, in turn, indicate that earlier autopsies are crucial for detection of changes in polyamine metabolism. We conclude that further studies to test the polyamine hypothesis are warranted.

Age-related reduction in pituitary corticotropin-releasing hormone receptors in two rat strains.

Open field behavior and age-related changes in anterior pituitary corticotropin-releasing hormone (CRH) receptors, as well as plasma ACTH levels, were measured in two inbred rat strains. The strains utilized were Wistar Kyoto (WKY) and Brown-Norway (BN), the former characterized by shorter life-span and hyper-reactivity to stressors as compared to the latter. Behaviorally, WKY rats showed hyper-responsivity to a novel environment as indicated by their delay in entering the open field, increased grooming, reduced rearing, and reduced locomotion. These strain-dependent behavioral differences were not affected by aging. The binding capacity of CRH receptors was similar in both strains and Bmax values were decreased (25-27%) with aging, with no changes in Kd values. In contrast, plasma ACTH levels were 67% higher in WKY than in BN rats but did not change with aging. Thus, despite pituitary CRH receptor down regulation, plasma ACTH levels following decapitation were sustained during aging. This suggests the presence of some compensatory factors in the hypothalamic-pituitary axis regulation which sustain ACTH response during aging. Furthermore, the findings indicate that higher plasma ACTH levels and hyper-reactivity to a novel environment are inversely correlated with longevity in the rat.

Selective influence of rat genotype on age-related regional changes in forebrain muscarinic binding.

Muscarinic binding was analyzed in the hippocampus and frontal cerebral cortex of 3 (young) and 24-month-old (aged) inbred Wistar-Kyoto (WKY), Brown-Norway (BN) and Lewis rats, by the specific binding of the antagonist quinuclidinylbenzilate (QNB). While no age-related changes were detected in the hippocampus of all strains, complex changes were found in the cortex. Maximal binding capacity (Bmax) of [3H]QNB was decreased in the cortex of aged WKY rats, remained unchanged in the BN strain and was elevated in Lewis rats. We conclude that regional differences exist in age-related changes in forebrain muscarinic binding which are probably due to differences in the types and connections of cholinoceptive neurons in these regions. The results suggest that it is the rate of these changes which is affected by the rat genotype.

Strain, stress, neurodegeneration and longevity.

Laboratory studies indicate that the life-span of inbred rodent strains is inversely related to the intensity of their behavioral and neuroendocrine responses to stressful stimuli. In the brain, a shorter life-span is associated with accelerated age-dependent degenerative changes in specific stress-responsive neuronal systems. The evidence suggests a possible genetic linkage between the intensity of the stress response, the rate of age-dependent neurodegeneration and the individual's life expectancy. It is proposed that inherent hyper-reactivity to stressors is genetically linked to a shorter life-span and to accelerated age-dependent neurodegeneration. Several experimental approaches to test 'this stress-longevity-neurodegeneration linkage hypothesis' are outlined.

Polyamines modulate the binding of GABAA-benzodiazepine receptor ligands in membranes from the rat forebrain.

The effects of spermine, spermidine and putrescine on the binding of the GABAA-benzodiazepine receptor complex were examined in the hippocampus and frontal cortex membranes of the rat. The results demonstrated modulatory effects of polyamines on the binding of diazepam and flunitrazepam but not on that of GABA, muscimol and Ro 15-1788. When membranes were prepared without detergent, the polyamines enhanced the binding of diazepam. However, while the binding capacity increased after homogenization in the presence of the non-ionic detergent Triton X-100, the polyamines did not enhance the binding but inhibited the binding of diazepam and flunitrazepam at greater concentrations. Considered together with other studies, the present findings indicate that polyamines can modulate the binding characteristics of several different neurotransmitter receptor-ionophore complexes.

Cytochemical localization of ornithine decarboxylase with rhodamine or biotin-labeled alpha-difluoromethylornithine. An example for the use of labeled irreversible enzyme inhibitors as cytochemical markers.

The present work describes a new method for cytochemical localization of enzymes using ornithine decarboxylase (ODC) as an example. The method is based on the preservation of the characteristic-specific and irreversible binding of the inhibitor alpha-difluoromethylornithine (alpha-DFMO) following its conjugation to "label" molecules. The inhibitor has been conjugated to the fluorescent molecule rhodamine-B-isothiocyanate, and its localization in tissue sections was detected directly by fluorescence cytochemistry. Alternatively, alpha-DFMO has been conjugated to biotin and its cytochemical localization determined indirectly following its binding with avidin conjugated to horseradish peroxidase (HRP) and visualization of the HRP reaction product. Both labeled inhibitor molecules were successfully localized cytochemically within specific cells of the developing rat cerebellum and rat liver following thioacetamide injection where ODC activity is greatly enhanced. This novel technique should be of general application 1) in other tissues, 2) for other enzymes, and 3) in electron microscopic studies for ultrastructural localization of the enzyme.

Polyamines in neurotrauma. Ubiquitous molecules in search of a function.

In spite of their abundance, the function of PAs in the adult nervous system remains enigmatic. It is postulated that after trauma, the induction of polyamine metabolism (i.e. the polyamine response), which is inherently transient, is an integral part of a protective biochemical program that is essential for neuronal survival. Several functions ascribed to PAs may assume importance in cellular defense. Thus, regulation of the ionic environment, modulation of signal pathways, control of cellular Ca2+ homeostasis, inhibition of lipid peroxidation, and interaction with nucleic acids are all putative sites for PA action. During maturation, the CNS, unlike the peripheral nervous system, undergoes changes which result in the expression of an incomplete polyamine response after trauma. This may be due to an altered pattern of gene expression, and/or restrictive compartmentalization of the PAs and their metabolizing enzymes. Induction of this partial polyamine response after injury results in a sustained accumulation of putrescine, which by itself may be harmful, without the concomitant increase in spermidine and spermine. Administration of exogenous PAs after trauma exerts a neuroprotective effect. Exogenous PAs are postulated to gain access into cells via an induced uptake system after trauma, and function similarly to newly synthesized PAs. Besides the injured neurons themselves, tissues which are connected or associated with these neurons may be potential targets where PAs could act to stimulate neurotrophic factor production. Based on the neuroprotective effects of PAs in laboratory animals and on their proposed role in mechanisms of neuronal survival, the development of PA-based compounds as therapeutic neuroprotective agents should be pursued.

The polyamine stress response: tissue-, endocrine-, and developmental-dependent regulation.

Transient alterations in polyamine (PA) metabolism, termed the polyamine stress response (PSR), constitute a common cellular response to stressful stimuli. In contrast to the adult brain and liver, the PSR in the adrenal gland and thymus is characterized by a reduction in PA metabolism. The brain PSR undergoes an early postnatal period of non-responsiveness. The aim of the present study was twofold: i) to determine whether the PSR in the liver, thymus, and adrenal gland is developmentally regulated as that in the brain and ii) to establish whether neuronal and hormonal signals can activate the PSR independently. Ornithine decarboxylase (ODC) activity and tissue PA concentrations served as markers of the PSR. Changes were measured in male Wistar rats during postnatal development and at 2 weeks after adrenalectomy in adults. Unlike the brain, the direction of the PSR in peripheral organs did not undergo developmental changes. After adrenalectomy, the PSR was not activated in the thymus and liver by acute (2-hr) restraint stress, but a characteristic PSR was induced in the hippocampus. However, dexamethasone injection (3 mg/kg) did induce a characteristic PSR in all organs of adrenalectomized rats. The results justify the following conclusions: i) Unlike peripheral organs, the PSR in the brain is developmentally regulated; ii) The developmental switch to a mature PSR in the brain corresponds in time to the cessation of the "stress hypo-responsive period" in the hypothalamic-pituitary-adrenocortical (HPA) axis; iii) In the periphery, the PSR appears to be dependent principally on stress-induced activation of the HPA axis and on increased circulating glucocorticoid concentrations rather than on neuronal activation; iv) In the brain, however, the PSR can be induced independently by glucocorticoids or by direct activation of the neuronal circuitry; and v) up-regulation of the PSR, as in the brain and liver, is constructive and may be implicated in cell survival, while its down-regulation, as in the adrenal and thymus, may be implicated in cell death.

The stress-induced response of the septo-hippocampal cholinergic system. A vectorial outcome of psychoneuroendocrinological interactions.

Considerable data have emerged which strongly indicate that the septohippocampal cholinergic system is involved in the adaptive response to stress. Neurotransmitter regulatory mechanisms in cholinergic synaptic terminals of this part of the limbic system undergo adaptive changes in response to stress and recover slowly after stress. The initial stress-induced response is characterized by activation of hippocampal cholinergic terminals within minutes, as indicated by a rapid and transient elevation in high affinity choline uptake and increased newly synthesized acetylcholine release. The response of this cholinergic system to stress is influenced by both neuronal and hormonal stimuli. Among the several neuronal systems converging in the septum, terminals of the dopaminergic mesolimbic system have been found to be selectively involved in the early response to stress. Pharmacological interference with dopaminergic neurotransmission, with agonist and antagonist treatments, revealed that changes in the tonic inhibitory influence of septal dopaminergic terminals can modulate the response of hippocampal cholinergic terminals to stress. A similar activation of hippocampal cholinergic terminals as after short-term stress was observed after treatments with a large dose of either adrenocorticotropic hormone or corticosterone. Furthermore, glucocorticoids and not adrenocorticotropic hormone can directly enhance acetylcholine release, but only from excited terminals. This indicates that stress-induced activation of the septo-hippocampal system may occur secondary to, but not directly by, increased levels of pituitary-adrenocortical hormones. Yet, it is possible that under stressful conditions the increased glucocorticoid levels may modulate the activity of the stimulated hippocampal cholinergic terminals. Together the findings support the notion that the stress-induced response of the septo-hippocampal cholinergic system represents an integrated output of converging neuronal and hormonal stimuli which convey signals of stress to this limbic brain region.

Metabolism of agmatine into urea but not into nitric oxide in rat brain.

Agmatine is a guanidino compound abundant in bacteria and plants where it serves as a precursor for polyamine synthesis. It can interfere with several neurotransmission-related functions and can exert neuroprotective effects after brain injury. Agmatine was recently identified in mammalian brain and its synthesis by arginine decarboxylation was characterized. Its metabolism by the brain is, however, unknown. Here we report evidence indicating that agmatine can be selectively metabolized in the rat brain (cerebellum) into urea and thus, may lead to formation of putrescine, the precursor of polyamine synthesis. In addition, while agmatine can inhibit brain nitric oxide synthase, it did not serve as a substrate for nitric oxide formation.

Differences in open-field behavior and in learning tasks between two rat strains differing in their reactivity to stressors.

The study characterizes differences between inbred Wistar-Kyoto (WKYs) and Brown-Norway (BNs) rats in open-field behavior, and in discriminative learning and acquisition of an avoidance learning task. Hyper-reactivity of WKYs to novelty was demonstrated in an open-field test. Discriminative learning and retention thereof was slower in WKYs, but as efficient as in BNs. Acquisition of avoidance learning was also slower in WKYs, but their maximal avoidance score was much higher (approximately 85%) than in BNs. Also, recall of avoidance learning was slower for WKYs. We conclude: (1) hyper-reactivity of WKYs to novelty is expressed by their exceptional immobility and excess defecation in the open-field and is paralleled by their known hyper-reactivity to stressful stimuli, and (2) no strain differences exist in the ability to learn a discriminative task, but both acquisition and recall of an avoidance task are slower in WKYs. This may imply that the degree of reactivity to stressful environmental stimuli may play an important role in the acquisition of learning.

Age-related reductions in brain cholinergic and dopaminergic indices in two rat strains differing in longevity.

We have recently reported that inbred Wistar-Kyoto rats which are highly reactive to stressful stimuli, have a much shorter mean life-span (21.5) compared to the less reactive Brown-Norway rats (31.0 +/- 4.5 months). In the present study we found a reduction in forebrain cholinergic neurotransmission indices in 24-month-old Wistar-Kyotos but not in Brown-Norways as compared to their respective young (3-month-old) counterparts. Also only in Wistar-Kyotos dopamine uptake was reduced in the aged striatum, but in the septum it remained unchanged in both strains. In Brown-Norways, age-related changes were observed only in choline acetyltransferase activity and only in brain regions known to contain mainly cholinergic nerve cell bodies. We conclude that at 24 months of age, reductions in brain cholinergic and dopaminergic neurotransmission are more prominent in the highly stress-reactive and shorter-lived Wistar-Kyoto strain, and may be genetically determined.

Early polyamine treatment enhances survival of sympathetic neurons after postnatal axonal injury or immunosympathectomy.

We have recently demonstrated that following injury of their axon, sympathetic neurons of the rat superior cervical ganglion become dependent on polyamine synthesis for their survival. In addition we have observed that the treatment of newborn rats with biogenic polyamines can prevent the naturally occurring reduction in the number of neurons in the ganglion. In the present study groups of newborn rats were subjected to either postganglionic nerve crush (axotomy) or to treatment with antiserum to nerve growth factor (immunosympathectomy), two treatments which result in a massive loss of neurons in the ganglion. Daily injections of the polyamines putrescine, spermidine and spermine (10 mg/kg each), for 7 days after the operation to the axotomized group, and for 9 days starting with the first antiserum injection to the immunosympathectomized group, attenuated the nerve cell loss. The polyamine treatment also attenuated the reduction in the activity of the neurotransmitter-synthesizing enzyme tyrosine hydroxylase observed after both axotomy and immunosympathectomy in the ganglion. These results further indicate that polyamines are important for the survival of sympathetic neurons and, while their mechanism of action is unknown, an interaction with nerve growth factor regulation cannot be excluded. In the iris, the reduction observed in [3H]norepinephrine uptake after the two noxious treatments was unproportionately small when compared to the large drop in the number of parent neurons in the ganglion. This suggests that compensatory mechanisms exist which act to adjust the number of functional axon terminals per neuron so that the number of terminals innervating the target remains relatively constant.

Polyamine biosynthesis is required for survival of sympathetic neurons after axonal injury.

Treatment of adult rats with specific inhibitors of polyamine synthesizing enzymes prevented the early increase in ornithine decarboxylase activity and polyamine content in the superior cervical ganglion after the postganglionic nerve was cut. Moreover, after axotomy, this treatment led to a marked diminution of the chromatolytic response with a marked neuronal cell death. We conclude that after axonal injury an early increase in polyamine biosynthesis, probably within parent sympathetic neurons, is an obligatory step in the progression of the axon reaction.

Difference in choline acetyltransferase but similarities in catecholamine biosynthetic enzymes in brains of two rat strains differing in their response to stress.

Rats of the Wistar-Kyoto (WKY) strain are more reactive to stress than Brown-Norway (BN) rats as adjudged by their behavioral and plasma catecholamine (CA) responses to stress. Contrary to the strain differences existing in the periphery, brain CA and their biosynthetic enzyme activities were found to be similar in both strains before and after immobilization stress. However, as in the periphery, choline acetyltransferase (CAT) activity was higher in brains of WKY rats. The results suggest that: (1) peripheral and central CA systems may be under different genetic control mechanisms, and (2) strain differences in CAT activity may reflect differences in cholinergic systems both in the brain and periphery, which may be important in the behavioral differences observed.

Collateral sprouting in central mesolimbic dopamine neurons: biochemical and immunocytochemical evidence of changes in the activity and distrubution of tyrosine hydroxylase in terminal fields and in cell bodies of A10 neurons.

The olfactory tubercle of adult rats was examined for the development of collateral sprouts from intrinsic dopaminergic axons following unilateral olfactory bulbectomy. In the ipsilateral tubercle tyrosine hydroxylase (TH) activity began to increase by 10-14 days following the lesion, gradually reaching a maximum of 125% of control (P less than 0.005) by 21 days where it remained permanently elevated. The rise of TH activity in the tubercle reflected changes of the dopaminergic innervation, since dopamine-beta-hydroxylase (DBH) activity was unchanged, and lesions of the dorsal noradrenergic bundle reduced DBH but not TH activity in the tubercle. By immunocytochemical staining the elevation of TH reflected an increased number and altered distribution of TH-containing processes within the olfactory tubercle. By 30 days the uptake of [3H]dopamine into synaptosomes of the olfactory tubercle was also increased to 140% of control (P less than 0.05). In the dopaminergic cell bodies of the ipsilateral A10 group (which innervate the tubercle) TH activity was transiently elevated to 121% (P less than 0.05) by 4 days, returning to control levels by 10 days. Histologically no change in activity was detected. The results indicate that mesolimbic dopaminergic neurons of A10 which innervate the olfactory tubercle will sprout in response to removal of a major non-dopaminergic input, that the new innervation is sustained, and that during collateral sprouting there is a transient elevation of TH activity in the uninjured cell bodies which precedes the period of axonal growth. The activity in the uninjured cell bodies which precedes the period of axonal growth. The findings suggest that (a) the increase of TH activity in the A10 cell bodies during collateral sprouting may be a reflection of an increase in the amount of enzyme protein required for transport into the enlarging terminal fields, (b) that as in development sprouts are in place before they reach biochemical maturity, (c) the biochemical mechanisms underlying collateral sprouting of uninjured neurons are not necessarily the same as those associated with regenerative sprouting in response to axonal injury, and (d) the development and the acquisition of biochemical maturation of collateral sprouts in the CNS involves complex two-way signaling between terminal field and cell bodies. The development of collateral sprouts of dopaminergic neurons may be the cellular substrate of the development of behavioral hyperactivity and aggression produced by bilateral olfactory bulbectomy in rat.

Failure to detect collateral sprouting of mesolimbic dopaminergic neurons during early postnatal development.

Using biochemical parameters the present study sought to assess the normal developmental pattern of the dopaminergic innervation of the olfactory tubercle (OT) and how it is affected by olfactory bulbectomy. In rats, the adult pattern of cellular organization is achieved in the OT gradually over the first 7 days after birth. On the other hand, tyrosine hydroxylase (TH) and [3H]dopamine ([3H]DA) uptake, while present at low levels, start to increase rapidly only after the first 7 days reaching adult levels by 40 and 20 days after birth, respectively. TH in dopamine (DA) cell bodies of A10 was already high, 40% of adult value, at birth, reached 150% by day 14 and decreased back to adult values by day 21 after birth. In 10-day-old rats, bulbectomy resulted, 30 days later, in an increase to 123% of control in TH activity and 137% in [3H]DA uptake within the OT. Comparable changes were found following bulbectomy in adults. However, bulbectomy in 1-day-old rats did not produce any significant changes 40 days later. The findings suggest that during postnatal growth TH activity is increased in DA cell bodies, preceding the changes in DA terminals of the OT, resembling the changes occurring during collateral sprouting in adults. In addition, changes indicative of collateral sprouting do not occur in response to deafferentation of the OT in 1-day-olds but do in 10-day-olds or older animals, a phenomenon probably related to a critical development period of the OT.

Stain-dependent differences between the septo-hippocampal cholinergic system and hippocampal size.

The activity of choline acetyltransferase is over twice as high in the hippocampus of Wistar Kyoto (WKY) than in Brown Norway (BN) rats, and this is paralleled by a comparable difference in acetylcholinesterase staining intensity within the hippocampal formation. However, the size of the whole hippocampus is smaller in WKY than in BN rats. There are no strain differences in the activities of the neurotransmitter-synthesizing enzymes: tyrosine hydroxylase in the septum and glutamic acid decarboxylase in the hippocampus. The findings indicate the existence of strain-dependent inverse relationship between the septo-hippocampal cholinergic system and the size of the hippocampus.

Modes of adaptation of peripheral neuroendocrine mechanisms of the sympatho-adrenal system to short-term stress as studied in two inbred rat strains.

Alterations in mechanisms involved in catecholamine (CA) and corticosterone (B) regulation following short periods of stress have been investigated in two inbred rat strains: a strain more reactive to stress, Wistar-Kyoto (WK), and a less reactive strain, Brown-Norway (BN). Measurements after decapitation stress alone and after decapitation following different periods of immobilization stress, indicate that: (a) plasma CA levels immediately after severe stress (i.e. decapitation) are directly related to the behavioral reactivity of the two rat strains to stress but inversely related to adrenal gland size, their content of CA and biosynthetic enzymes, and to plasma levels of MHPG; (b) levels of B in adrenal glands and in plasma after stress are similar in both strains, in spite of different gland sizes; (c) CAT activity in presynaptic sympathetic terminals is directly related to plasma CA after short-term stress; and (d) liver COMT activity is directly related to plasma CA levels after stress, but inversely plasma levels of MHPG. The implications of the findings are discussed further in the text and lead to the conclusion that the BN strain represents a mode of a slower response to stressful stimuli than WK.

Increased choline kinase activity in the rat superior cervical ganglion after axonal injury.

The activity of choline kinase (CK) was examined in the rat superior cervical ganglion (SCG) during development and following postganglionic axotomy. The highest specific enzyme activity (nmol phosphorylcholine/mg protein/h) 52 +/- 8, is observed 5 d before birth, then it rapidly decreases by about 50%, reaching at the day of birth levels observed in the ganglion throughout life. During development the total enzyme activity per ganglion is increased steadily until it reaches a 5-fold increase which parallels the increase in protein content. Following axotomy the enzyme activity per ganglion is rapidly increased by about 2-fold between 1 and 5 d postoperative and then gradually decreases reaching control levels at 30 d. The transient increase in enzyme activity parallels the increase in protein content of the axotomized ganglia. The peak increase in enzyme activity coincides with the peak chromatolytic response of the axotomized ganglion. We conclude that choline kinase activity is transiently increased within neurons after axonal injury, and that this event represents an effort of the nerve cell body to enhance its phosphatidylcholine biosynthesis essential for new membrane synthesis during the regeneration of the cut axon.