European Journal of Pharmacology (v.405, #1-3)

This review in honor of David de Wied summarizes the work done in my laboratory that first indicated that adrenocorticotropic hormone (ACTH) has a direct effect on the neuromuscular system. Cold stress or ACTH and its related peptides α-melanocyte-stimulating hormone (α-MSH ) and β-lipotropin improve the electromechanical characteristics of adrenalectomized and hypophysectomized rats. ACTH-(1–39) accelerates the return of motor and sensory function and improves the morphological characteristics of the motor endplate after peripheral nerve crush. The non-corticotropic fragments ACTH-(4–10), α-MSH, the ACTH-(4–9) analogue Organon 2766 (Org 2766) or the ACTH-(4–10) analogue Biomeasure 22015 (BIM 22015) improve electrophysiological and morphological parameters of the regenerating neuromuscular system. ACTH-(4–10) immunoreactivity, present in ventral horn motor neurons in low levels, is decreased ipsilaterally following ipsilateral nerve crush but increases both ipsilaterally and contralaterally if injured animals are treated with ACTH-(4–10) indicating a neuroprotective action. Similarly, Org 2766 appears to have a protective action in the brain following nigrostriatal lesions. In developmental studies, perinatal exposure to ACTH peptides improves the structure of the neuromuscular junction, accelerates the maturation of electromechanical properties and enhances nerve–muscle integration and nerve regeneration. Perinatal exposure to these peptides decreases adult male sexual behavior, a change correlated with increased serotinergic input within the medial preoptic area. Similar changes occur in female rats and appear to be long-lasting. In tissue culture studies, both Org 2766 and BIM 22015 promote neurite outgrowth in the absence of nerve growth factor, indicating a neurotrophic role for these peptides.
Keywords: Regeneration; Development; Melanocortin;

Melanocortins and the brain: from effects via receptors to drug targets by Roger A.H Adan; Willem Hendrik Gispen (13-24).
The lack of specific receptors (and antagonists) has hampered the research on the neural mechanism of action of adrenocorticotropic hormone (ACTH)- and melanocyte-stimulating hormone (MSH)-like peptides. Yet the original observations in the 1970s already pointed to cAMP as a possible mediator of ACTH/MSH effects in neurons. The cloning of melanocortin receptors since 1992, the identification of at least two subtypes (melanocortin MC3 and MC4 receptors) that are present in neural tissue and the development of selective and potent agonists as well as antagonists have markedly furthered the position of melanocortins as important neuropeptides. In this paper we discuss the role of especially the receptor subtype melanocortin MC4 in various behaviors including grooming behavior and feeding behavior and consider new insights in the interaction between the opioid and the melanocortin system at the level of the spinal cord (i.e. pain perception). Finally, based on new data obtained in molecular pharmacological studies on brain melanocortin receptors, we suggest a general concept for selective receptor–ligand interaction: ligand residues outside the peptide core-sequence may direct the conformation of the residues in the ligand core-sequence that interact directly with the receptor-binding pocket and thereby determine selectivity.
Keywords: ACTH (adrenocorticotropic hormone); MSH (melanocyte-stimulating hormone); Melanocortin receptor; Brain melanocortin system; Peptide–receptor interaction; Avoidance; Grooming; Food intake; Pain;

Role of melanocortins in the central control of feeding by Anna Valeria Vergoni; Alfio Bertolini (25-32).
The injection of a melanocortin peptide or of melanocortin peptide analogues into the cerebrospinal fluid or into the ventromedial hypothalamus in nanomolar or subnanomolar doses induces a long-lasting inhibition of food intake. The effect keeps significant for up to 9 h and has been observed in all animal species so far tested, the most susceptible being the rabbit. The anorectic effect of these peptides is a primary one, not secondary to the shift towards other components of the complex melanocortin-induced behavioral syndrome, in particular grooming. The site of action is in the brain, and the effect is not adrenal-mediated because it is fully exhibited also by adrenalectomized animals. It is a very strong effect, because the degree of feeding inhibition is not reduced in conditions of hunger, either induced by 24 h starvation, or by insulin-induced hypoglycemia, or by stimulation of γ-aminobutyric acid (GABA), noradrenergic or opioid systems. The microstructural analysis of feeding behavior suggests that melanocortins act as satiety-inducing agents, because they do not significantly modify the latencies to start eating, but shorten the latencies to stop eating. The mechanism of action involves the activation of melanocortin MC4 receptors, because selective melanocortin MC4 receptor antagonists inhibit the anorectic effect of melanocortins, while inducing per se a strong stimulation of food intake and a significant increase in body weight. Melanocortins seem to play an important role in stress-induced anorexia, because such condition, in rats, is significantly attenuated by the blockage of melanocortin MC4 receptors; such a role is not secondary to an increased release of corticotropin-releasing factor (CRF), because, on the other hand, the CRF-induced anorexia is not affected at all by the blockage of melanocortin MC4 receptors. The physiological meaning of the feeding inhibitory effect of melanocortins, and, by consequence, the physiological role of melanocortins in the complex machinery responsible for body weight homeostasis, is testified by the hyperphagia/obesity syndromes caused by mutations in the pro-opiomelanocortin (POMC) gene, or in the melanocortin MC4 receptor gene, or in the agouti locus. Finally, recent evidences suggest that melanocortins could be involved in mediating the effects of leptin, and in controlling the expression of neuropeptide Y (NPY).
Keywords: Melanocortin; Feeding; Central nervous system; Behavior; Body weight homeostasis; Anorexia; Obesity;

Postnatal treatment with ACTH-(4-9) analog ORG 2766 attenuates N-methyl-d-aspartate-induced excitotoxicity in rat nucleus basalis in adulthood by Katalin M Horvath; István M Ábrahám; Tibor Harkany; Peter Meerlo; Bela G.J Bohus; Csaba Nyakas; Paul G.M Luiten (33-42).
It has been reported that the ACTH-(4-9) analog H-Met(O2)-Glu-His-Phe-d-Lys-Phe-OH (ORG 2766) administered in adulthood has trophic effects on neuronal tissue and when given postnatally, it can induce long-lasting changes in brain development. In the present study, we investigated whether early postnatal treatment with ORG 2766 affects adult neuronal vulnerability, i.e. the sensitivity of cholinergic neurons against excitotoxic damage. Wistar rat pups received injections of ORG 2766 or saline on postnatal days 1, 3 and 5 and were then left undisturbed until adulthood. At the age of 6 months, the animals were subjected to unilateral lesion of magnocellular basal nucleus by infusion of high dose of N-methyl-d-aspartate (NMDA). The effects of the excitotoxic insult were studied 28 hours and 12 days after the lesion by measuring both the acute cholinergic and glial responses, and the final outcome of the degeneration process. Twenty eight hours after NMDA infusion, postnatally ACTH-(4-9)-treated animals showed stronger suppression of choline-acetyltransferase immunoreactivity and increased reaction of glial fibrillary acidic protein -immunopositive astrocytes in the lesioned nucleus compared to control animals. However, 12 days post-surgery, the NMDA-induced loss of cholinergic neurons, as well as the decrease of their acetylcholinesterase -positive fibre projections in the cortex, were less in ACTH-(4-9) animals. Our data indicate that the early developmental effects of ACTH-(4-9) influence intrinsic neuroprotective mechanisms and reactivity of neuronal and glial cells, thereby resulting in a facilitated rescuing mechanism following excitotoxic injury.
Keywords: Neonatal; ACTH (adrenocorticotropin); Neuroprotection; Magnocellular basal nucleus; Cholinergic neuron; Glia;

Manipulating neuropeptidergic pathways in humans: a novel approach to neuropharmacology? by Horst Lorenz Fehm; Boris Perras; Rüdiger Smolnik; Werner Kern; Jan Born (43-54).
Given the tremendous number of neuropeptides, which are synthesized in the central nervous system, the brain can be viewed as one of the most prominent endocrine organs. Elucidation of the functions of these peptides is hampered by the facts that after intravenous administration access to brain receptors is prevented or impaired by the blood–brain barrier. Here, we provide evidence that intranasal administration can be a way to circumvent the blood–brain barrier. Selected experiments will be reported indicating that peptides after intranasal administration in humans can specifically alter a great variety of brain functions. For vasopressin, we demonstrated improving effects of long-term intranasal treatment on sleep in elderly people. Insulin showed improving effects of short-term memory functions. For adrenocorticotropin/melanocyte stimulating hormone, ACTH/MSH-(4–10), a twofold action was isolated: The melanocortin fragment diminished selective attention and, with subchronic administration, reduced body fat. These results could provide the basis for developing a new, specific, and “soft” neuropharmacology.
Keywords: Neuropeptide; Intranasal administration; Vasopressin; Insulin; Melanocortin; Cognition;

The neuropeptide concept concerns the diverse and broad physiological functions of neuropeptides in behavioral adaptation. Neuropeptides like vasopressin and corticotropin-releasing hormone can coordinate multiple brain functions due to the anatomical organization of the neurons producing them. The cell bodies are focally positioned in the hypothalamus and send long-reaching efferents to limbic and brainstem areas. Likewise, midbrain dopamine systems coordinate emotional behaviors and movement control by specific connectivity of neurons in the midbrain to limbic and striatal centers, respectively. The fundament of the functions of these signalling molecules is laid out during development when transmitter identity and connectivity are specified. This is a highly controlled process involving multiple transcription factors and growth factors acting together in genetic pathways. Here, the genetic pathways enrolling in developing vasopressin, corticotropin-releasing hormone, and midbrain dopamine neurons are discussed.
Keywords: Neuropeptide; Vasopressin; Corticotropin-releasing hormone; Dopamine; Homeobox gene; Transcription factors; Development;

Neuromodulation of memory in the hippocampus by vasopressin by Béatrice Alescio-Lautier; Véronique Paban; Bernard Soumireu-Mourat (63-72).
The involvement of [Arg8]vasopressin in memory processes was analyzed in the hippocampal structure, since we have reported that this is one of the main central target structures of the vasopressin-enhancing effect on memory. This structure is functionally differentiated along its dorsoventral axis, and the expression of the vasopressinergic system is dependent upon whether the dorsal or ventral part of the hippocampus is involved. For this reason, the effect of vasopressin injected into hippocampus was evaluated on the basis of the site of injection. We have shown, using a Go-No Go visual discrimination task with mice that both parts of the hippocampus are involved in the effect of endogenous or exogenous vasopressin, but with higher sensitivity for the ventral part. Based on the expression of Fos protein following intracerebroventricular injection of vasopressin in unconditioned or conditioned mice, we confirmed the greater involvement of the ventral hippocampus in the enhancing effect of vasopressin on memory processes. The effect of the peptide seems specific, since only a few of the hippocampal cells that expressed Fos protein in the unconditioned mice did so in the conditioned mice (cells in the dentate gyrus and the CA3 hippocampal field). Moreover, we have shown that in the ventral hippocampus, vasopressin generates different behavioral effects whether treatment is performed at the beginning or in the middle of the learning process, suggesting that the mnemonic context is an important factor for understanding the effect of vasopressin on memory in the ventral hippocampus.
Keywords: Vasopressin; Hippocampus; Memory process;

Spatial, cellular and temporal basis of vasopressin potentiation of norepinephrine-induced cAMP formation by Roberta Diaz Brinton; Richard H Thompson; Elizabeth A Brownson (73-88).
This study investigated the spatial distribution of vasopressin V1 and β1-adrenoceptors within hippocampal subfields and lamina in an attempt to localize the site(s) of interaction between these two receptor systems. In addition, the cell types, neuronal and glial, in which the vasopressin-induced neuromodulation occurs, were identified. Lastly, the temporal constraints of the potentiation induced by vasopressin were investigated. Results of these analyses demonstrated multiple sites within the hippocampus where the interaction between vasopressin and norephinephrine could occur. Moreover, vasopressin-induced potentiation of adrenergic stimulated cyclase occurred in both hippocampal neurons and glia whereas it did not occur in undifferentiated neurons. Analysis of the temporal constraints of vasopressin-induced potentiation revealed that pre-activation of the vasopressin V1 receptor for 1 min yielded greater potentiation than simultaneous exposure to vasopressin and norepinephrine. These data provide insights into the spatial and temporal characteristics for the interaction between the vasopressin receptor and adrenoceptor systems and provide a cellular and biochemical rationale for the behavioral findings of Kovács and De Wied.
Keywords: Vasopressin; Norepinephrine; Neuromodulation; Associative memory mechanism; Hippocampus;

Endogenous opioids and reward by Jan M Van Ree; Raymond J.M Niesink; Leo Van Wolfswinkel; Nick F Ramsey; Marleen (L.) M.W Kornet; Wouter R Van Furth; Louk J.M.J Vanderschuren; Mirjam A.F.M Gerrits; Caroline L Van den Berg (89-101).
The discovery of endogenous opioids has markedly influenced the research on the biology of addiction and reward brain processes. Evidence has been presented that these brain substances modulate brain stimulation reward, self-administration of different drugs of abuse, sexual behaviour and social behaviour. There appears to be two different domains in which endogenous opioids, present in separate and distinct brain regions, are involved. One is related to the modulation of incentive motivational processes and the other to the performance of certain behaviours. It is concluded that endogenous opioids may play a role in the vulnerability to certain diseases, such as addiction and autism, but also when the disease is present, such as alcoholism.
Keywords: Endogenous opoid; Addiction; Reward; Brain stimulation reward; Self-administration; Sexual behaviour; Social behaviour;

Changes in fluid and electrolyte homeostasis may accompany and are likely to modify the clinical symptoms of alcohol-withdrawal reactions. It was of obvious theoretical and practical interest therefore to investigate the changes in the secretion of hormones, which regulate the fluid and electrolyte homeostasis (atrial natriuretic peptide, aldosterone and plasma renin activity) during alcohol withdrawal in chronic alcoholic patients. In a phase of severe withdrawal, there were increased plasma renin activity and aldosterone levels observed. In a phase of partial recovery, on the other hand, the elevated plasma renin activity and aldosterone levels were back to the normal range. In 60% of the patients, delirium tremens was gradually developing during the observation period. In these patients, an elevated level of atrial natriuretic peptide was observed at the time of hospital admission, i.e. days before the actual onset of delirium tremens. It is concluded that the disturbed volume homeostasis and the consequently altered plasma atrial natriuretic peptide secretion might be associated with, and therefore used as an indicator of the onset of delirium tremens. To study the role of central nervous atrial natriuretic peptide, mice were rendered tolerant to and dependent on alcohol with an alcohol–liquid diet for 14 days. Five hours after withdrawal from alcohol, withdrawal hyperexcitability symptoms were analyzed. Intracerebroventricular (i.c.v.) injection of atrial natriuretic peptide attenuated, whereas that of an antiserum against atrial natriuretic peptide intensified the severity of handling-induced convulsions. N-methyl-d-aspartate induced behavioral seizures in a dose-dependent manner, whose effect was more intensive during the alcohol-withdrawal period than in alcohol-naive animals. I.c.v. injections of atrial natriuretic peptide dose-dependently inhibited, whereas that of antiserum against atrial natriuretic peptide potentiated the seizure-inducing effect of N-methyl-d-aspartate in alcohol-dependent mice. Although tentatively, it is concluded that peripheral secretion of atrial natriuretic peptide may be an indicator, whereas central nervous atrial natriuretic peptide a neuropeptide modulator of alcohol-withdrawal symptomatology.
Keywords: Alcohol withdrawal; Delirium tremens; Volume and fluid regulation; ANP (atrial natriuretic peptide) secretion; Withdrawal hyperexcitability; NMDA (N-methyl-d-aspartate) seizure; Glutamate;

Excitatory non-adrenergic–non-cholinergic neuropeptides: key players in asthma by Aletta D Kraneveld; Deborah E James; Annick de Vries; Frans P Nijkamp (113-129).
Professor David de Wied first introduced the term ‘neuropeptides’ at the end of 1971. Later peptide hormones and their fragments, endogenous opioid (morphine-like) peptides and a large number of other biogenic peptides became classified as neuropeptides. All of these peptides are united by a number of common features including their origin (nervous system and peptide-secreting cells found in various organs such as skin, gut, lungs), biosynthesis, secretion, metabolism, and enormous effectiveness. Neuropeptides are biologically active at extremely low concentrations. The past decade, neuropeptide research has revealed that neuropeptides also participate strongly in immune reactions. The neuro-immune concept has opened up a whole new research area. In the last 20 years, significant advances have been made in investigations of the interaction between immune and nervous systems in chronic inflammatory diseases such as asthma. The goal of this review is to bring together the functional relevance of excitatory non-adrenergic–non-cholinergic (NANC) nerves and the interaction with the immune system in asthma.
Keywords: Asthma; NANC (non-adrenergic–non-cholinergic) nerve; Neuropeptide; Animal model; (Human);

The effects of prolactin on animal behavior include the stimulation of novelty-induced grooming in rats. This effect has been demonstrated in hyperprolactinaemic animals bearing pituitary homografts under the kidney capsule or after intracerebroventricular (i.c.v.) administration of prolactin. Since plasma prolactin levels in hyperprolactinaemic rats are similar to those of animals injected with low doses of rat prolactin, we studied the effects of this hormone injected subcutaneously (s.c.) in a dose range of 5–50 μg/kg. Novelty-induced grooming was enhanced only in rats injected with 5 or 10 μg/kg rat prolactin, whereas no effect was observed after the s.c. injection of the higher dose. The sexual behavior of male rats is also affected by prolactin. Male rats with normal mating activity showed enhanced sexual behavior when injected s.c. with rat prolactin (5, 10 or 50 μg/kg). In animals with poor sexual performance or in impotent rats, prolactin (5 or 10 μg/kg, but not 50 μg/kg) restored the full pattern of sexual behavior. An increased lordosis quotient was also observed in ovariectomized rats treated with prolactin 5 or 10 μg/kg. These results suggest that, besides the duration of hyperprolactinaemia, the effective level of plasma prolactin is important for the expression of the behavioral effects of this hormone.
Keywords: Low dose; Prolactin; Grooming; Sexual behavior; Sexual behavior;

A potential injury to the hippocampus has been postulated by the “glucocorticoid cascade hypothesis” as deriving from the life-long exposure to the stress glucocorticoid hormone. This hypothesis has been extensively resorted to in the search of a physio-pathological basis of the cognitive and behavioural impairments of old age, as well as for assigning to the hormone a not-irrelevant pathogenic role in brain degenerative diseases. Here I discuss the experimental evidences that have credited to stress a killing-licence, and pose, on the contrary, that the modest degrees of hypercortisolemia present in the above conditions could be interpreted as a beneficial occurrence.
Keywords: Stress; Glucocorticoid; Cytokine; Ageing; Alzheimer's disease;

Psychobiology is the discipline that attempts to integrate the impact of environmental and psychological variables on biological systems. This paper focuses on the psychobiology of the hypothalamic–pituitary–adrenal (HPA) axis and illustrates several processes that influence the response of the HPA axis. The interaction of the developing rodent or primate with their primary care giver has permanent long-term effects on the HPA axis. Manipulations that alter maternal behavior during critical periods of development permanently modify the HPA axis. The HPA axis can be programmed to be hypo-responsive or hyper-responsive as a function of time and length of maternal separation. In the adult organism, the HPA response to stress is highly dependent on specific psychological factors such as control, predictability, and feedback. In primates, social variables have been shown to diminish or exacerbate the HPA stress response. During the post-natal period of development, the mother appears to actively inhibit the pups' HPA axis. Different aspects of maternal behavior regulate different components of the HPA system.
Keywords: Corticosterone; ACTH (adrenocorticotropin); CRH (corticotropin-releasing hormone); Coping; Social; Maternal behavior;

This short review summarizes the effect of various stressful stimuli on the expression of neuropeptides which co-localize in corticotrophin releasing hormone (CRH)-synthesizing neurons in the hypothalamic paraventricular nucleus, as well as in oxytocin and vasopressin neurons in the supraoptic nucleus. Stress-induced changes failed to act on CRH neurons in the central amygdaloid nucleus but formalin-evoked pain enhanced galanin mRNA expression in the medial subdivision of this nucleus. Changes in the expression of enkephalin, galanin, dynorphin and cholecystokinin mRNA in response to restraint and formalin-induced pain are documented in hypothalamic and amygdaloid nuclei by in situ hybridization histochemical technique.
Keywords: Stressful stimuli; Neuropeptide; c-fos; Paraventricular nucleus; Supraoptic nucleus; Amygdaloid nucleus;

Psychoneuroimmunology, the study of interactions among behavioral, neural and endocrine, and immune processes, coalesced as an interdisciplinary field of study in the late 1970s. Some of the early research that was critical in establishing neuroanatomical, neurochemical and neuroendocrine pathways and functional relationships between the brain and the immune system is outlined here. These and subsequent studies have led to the general acknowledgment that the nervous and immune systems are components of an integrated system of adaptive processes, and that immunoregulatory processes can no longer be studied as the independent activity of an autonomous immune system. This paradigm shift in the study of immunoregulatory processes and the elaboration of the mechanisms underlying behaviorally induced alterations of immune function promise a better understanding and a new appreciation of the multi-determined etiology of pathophysiological states.
Keywords: Conditioned response; Immunity; Stress; Psychoneuroimmunology;

The results of the study reported in Brain Research in 1995 by Isaacson et al. [Isaacson, R.L., Varner, J.A., Baars, J.-M., de Wied, D., 1995. The effects of pregnenolone sulfate and ethylestrenol on retention of a passive avoidance task. Brain Res. 689, 79–84] have been re-examined with special emphasis placed on the distributions of latencies found in the passive avoidance task using rats. This study used two retention tests, one 24 h after training the other at 48 h after training. In the first experiment in that study a range of doses of two anabolic steroids, pregnenolone sulfate and ethylestrenol, were given s.c. just after the footshock training trial. In experiment 2 a similar range of doses of both steroids was given to the rats 1 h before the first retention test. Placing emphasis on the distributions rather than measures of central tendencies revealed that, in contrast to the vehicle treated animals, the anabolic steroid treated animals exhibited bimodal distributions of response latencies. These differences between control and hormone treated animals were observed in both experiments. The new information was interpreted in terms of non-linear dynamics including some aspects of Chaos theory.
Keywords: Anabolic steroid; Pregnenolone sulfate; Ethylestrenol; Passive avoidance behavior; Memory; Non-linear dynamics; Chaos theory; Attractor;

Stress in the brain by E.Ronald de Kloet (187-198).
Part I (first section) reports about research in the period 1964–1976, when the seminal observations were made on which today's concept of corticosteroid action on the brain is based. These key observations concern the discovery of nuclear corticosterone receptors in the limbic brain that mediate control over neuronal circuits underlying hypothalamic–pituitary–adrenal activity and behavioural adaptation. Part II (second section) covers the period of 1977–1989. It is about some aspects of the neuropeptide concept, the implementation of micro-neurochemistry using the “Palkovits punch”, and the application of in vitro autoradiography. Vasopressin and oxytocin receptors were identified and their implication in behaviour was examined using the song control of the canary bird as a model system. Two distinct nuclear receptor types for corticosteroids were identified: mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) which mediate in a coordinate manner the steroid control of hypothalamus–pituitary–adrenal activity and behaviour. Part III (third section) is from 1990 up to 2000. Focus is on the balance of MR- and GR-mediated actions in control of homeostasis as a determinant of health and disease. MR operates in pro-active mode to prevent homeostatic disturbance, while additional GR activation promotes in reactive fashion recovery after stress. An imbalance in MR and GR underlies behavioural deficits and neuroendocrine disturbances increasing vulnerability for stress-related brain disorders. The complete hippocampal genome is screened for corticosteroid responsive genes, which are potential targets for drugs promoting restorative capacity still present in the diseased brain.
Keywords: Stress; Corticosteroid; Hypothalamus–pituitary–adrenal activity;

The actions of corticotropin-releasing factor (CRF) and CRF-related peptides in the brain and periphery are mediated through multiple receptors. Two CRF receptor subtypes that differ markedly in their pharmacological profiles and anatomical distribution have been identified and characterized. Important advances have been made in understanding CRF and its actions through the development of specific CRF receptor antagonists, application of antisense oligonucleotides, and the production of transgenic mice lacking functional CRF1 receptors. This chapter describes recent findings with respect to CRF-related peptides and CRF receptors and their role in stress-induced behaviours.
Keywords: CRF (corticotropin-releasing factor); Urocortin; (Antagonist); CRF receptor; Antisense; Stress; Behaviour;

Electrophysiological studies over the past decades have shown that many compounds in addition to ‘classical’ neurotransmitters affect electrical activity in the brain. These compounds include neuropeptides synthesized in brain as well as compounds which are released from peripheral sources and subsequently enter the brain compartment, such as corticosteroid hormones from the adrenal gland. In the present review, this principle is illustrated by describing the effects of two substances, i.e. vasopressin and corticosterone. Neuropeptides and corticosteroid hormones add at least two essential aspects to information processing in the brain. First, they both act conditional, i.e. they modulate the actions of ‘classical’ neurotransmitters, rather than changing basal neuronal activity by themselves. Second, the time-frame in which modulation of electrical properties takes place differs from that generally seen with ‘classical’ neurotransmitters. Neuropeptides modulate electrical activity over a period of minutes, while effects of corticosteroid hormones usually become apparent after at least an hour but then last for hours. In this way, neuropeptides and steroid hormones expand the repertoire of responses through which the brain reacts to environmental challenges.
Keywords: Corticosterone; Vasopressin; Firing pattern; Ion channel; Neurotransmitter;

Long-lasting stress sensitisation by Rianne Stam; Adrie W Bruijnzeel; Victor M Wiegant (217-224).
Stressful experiences in humans can result in a spectrum of long-term changes in behavioural, autonomic and hormonal responsivity. An extreme form of such alterations is found in patients with post-traumatic stress disorder (PTSD). A number of animal models has been developed in which intense stressful experiences (shocks, social confrontations) result in longterm altered responsivity of behavioural, autonomic and hormonal responses to aversive challenges which mimic many of the changes seen in PTSD. These models of stress-induced sensitisation are beginning to generate a better understanding of the vulnerability factors, time-course and underlying neuronal substrates of the long-term disturbances experienced by humans as a result of stressful life events.
Keywords: Behaviour; Autonomic response; Endocrine response; Anxiety; Affective disorder; Post-traumatic stress disorder (PTSD); (Rat);

Role of corticotropin-releasing factor, vasopressin and the autonomic nervous system in learning and memory by Gerda Croiset; Marjoleen J.M.A Nijsen; Patrick J.G.H Kamphuis (225-234).
Learning and memory are essential requirements for every living organism in order to cope with environmental demands, which enables it to adapt to changes in the conditions of life. Research on the effects of hormones on memory has focused on hormones such as adrenocorticotropic hormone (ACTH), glucocorticoids, vasopressin, oxytocin, epinephrine, corticotropin-releasing factor (CRF) that are released into the blood and brain following arousing or stressful experiences.Most of the information have been derived from studies on conditioned behavior, in particular, avoidance behavior in rats. In these tasks, an aversive situation was used as a stimulus for learning. Aversive stimuli are associated with the release of stress hormones and neuropeptides. Many factors play a role in different aspects of learning and memory processes. Neuropeptides not only affect attention, motivation, concentration and arousal or vigilance, but also anxiety and fear. In this way, they participate in learning and memory processes. Furthermore, neuropeptides such as CRF and vasopressin modulate the release of stress hormones such as epinephrine. In turn, systemic catecholamines enhance memory consolidation. CRF and vasopressin are colocalized in neurons from the nucleus paraventricularis, which project to nuclei in the brainstem involved in autonomic regulation. The objective of this paper is to discuss the role of CRF, vasopressin, and the autonomic nervous system (ANS) in learning and memory processes. Both CRF and vasopressin have effects in the same direction on behavior, learning and memory processes and stress responses (release of catecholamines and ACTH). These neuropeptides may act synergistically or in a concerted action aimed to learn to adapt to environmental demands.
Keywords: Corticotropin-releasing factor (CRF); Vasopressin; Autonomic nervous system; Learning; Memory;

The brain mineralocorticoid receptor: greedy for ligand, mysterious in function by Johannes M.H.M Reul; Angela Gesing; Susanne Droste; Ingemar S.M Stec; Anja Weber; Cornelius Bachmann; Alicia Bilang-Bleuel; Florian Holsboer; Astrid C.E Linthorst (235-249).
Glucocorticoids exert their regulatory effects on the hypothalamic–pituitary–adrenocortical axis via two types of corticosteroid receptors: the glucocorticoid receptor and the mineralocorticoid receptor. Whereas the glucocorticoid receptor has a broad distribution in the brain, highest levels of mineralocorticoid receptor are found in the hippocampus. Based on the differential occupancy profile by endogenous glucocorticoids, glucocorticoid receptors are thought to mediate negative feedback signals of elevated glucocorticoid levels, whereas mineralocorticoid receptors control the inhibitory tone of the hippocampus on hypothalamic–pituitary–adrenocortical axis activity. Dysfunction of mineralocorticoid receptors and glucocorticoid receptors are thought to be implicated in stress-related psychiatric diseases such as major depression. Because of its intriguing features, we focus in this review on the mineralocorticoid receptor and provide data which reveal novel aspects of the pharmacology and physiology of mineralocorticoid receptors. Newly obtained results are presented, which help to solve the paradox of why dexamethasone binds with high affinity to mineralocorticoid receptors in vitro, yet binds poorly in vivo. Until recently, mineralocorticoid receptor protein and mRNA levels could only be routinely studied with in vitro cytosol binding assays, in vitro and in vivo receptor autoradiography, Northern blot analysis, and in situ hybridization. These methods are unfortunately hampered by several flaws, such as the necessity of adrenalectomy, no or poor neuroanatomical resolution, the fact that mRNA does not provide the same information as protein, or combinations of these factors. We present immunohistochemical data on mineralocorticoid receptors in the brain obtained by using commercially available antibodies, which alleviate many of these shortcomings. Furthermore, an in vivo microdialysis method is presented which allows the assessment of free corticosterone levels in the brain, which is critical for the study of the pharmacological basis of mineralocorticoid receptor (and glucocorticoid receptor) function. Finally, a novel aspect of the regulation of mineralocorticoid receptors is described which provides evidence that this receptor system is dynamically regulated. In conjunction with previously reported effects of antidepressants, these results have initiated a new concept on the cause of the hypothalamic–pituitary–adrenocortical axis disturbances often seen in stress-related psychiatric disorders such as major depression.
Keywords: Mineralocorticoid receptor; Glucocorticoid receptor; Hypothalamic–pituitary–adrenocortical axis; Stress; Antidepressants; Major depression;

Perinatal exposure to androgens permanently transforms certain tissues, e.g., the brain, the genitalia, etc. This process involves both masculinization and defeminization. Immune function also is transformed by early steroid exposure; however, it is not yet known whether the response capabilities of the immunocytes themselves are directly modified or whether they are responding to signals from other masculinized tissues, e.g., the brain. Most evidence points to a direct effect since androgen and estrogen receptors are present in developing immunocytes. Both androgens and estrogens have a role in regulating adult immunity including Th1/Th2 balance. Adult susceptibility to autoimmune and other diseases is also related to steroid exposure. How immune cells respond to gonadal steroids in adulthood may depend on the pattern of androgenic and estrogenic stimulation during early development.
Keywords: Androgen; Th1/Th2; Immune function; Prenatal; Estrogen; Autoimmunity;

Stages of avoidance strategy formation in gerbils are correlated with dopaminergic transmission activity by Holger Stark; Andreas Bischof; Thomas Wagner; Henning Scheich (263-275).
This detailed analysis of behavior is aimed at the differentiation of the components of information processing during associative conditioning. In gerbils, the influences of various acquired non-avoidance strategies as pre-experience were studied during the learning of a standard avoidance task in the same shuttle-box. Identical cue stimuli, frequency-modulated tones as conditioned stimuli and electric footshocks as unconditioned stimuli, were used in various behavioral tasks. In addition to common parameters such as avoidance performance and reaction times, behavioral events such as the attention response and the orienting response were quantified. Thereby, components of shuttle-box learning such as signal detection and signal evaluation were found to be affected by pre-experience-dependent dynamics. Using a microdialysis technique during avoidance learning in the shuttle-box, we found that only strategy formation was correlated with high dopamine levels in medial prefrontal cortex. The increase in dopamine in medial prefrontal cortex may be an indicator of the involvement of working memory principles in signal evaluation stages of conditioning.
Keywords: Dopamine; Microdialysis; Prefrontal cortex; Shuttle-box; Working memory; (Gerbil);

Platelets as a peripheral district where to study pathogenetic mechanisms of Alzheimer disease: the case of amyloid precursor protein by Monica Di Luca; Francesca Colciaghi; Lucia Pastorino; Barbara Borroni; Alessandro Padovani; Flaminio Cattabeni (277-283).
Alzheimer disease is a progressive neurodegenerative disease, characterised by a progressive cognitive and memory decline. From a neuropathological point of view, Alzheimer disease is defined by the presence of characteristic lesions, i.e. mature senile plaques, neurofibrillary tangles (NFTs) and amyloid angiopathy. In particular, accumulation of the amyloid β-peptide in the brain parenchyma and vasculature is an invariant event in the pathogenesis of both sporadic and familial Alzheimer cases. Amyloid β-peptide originates from a larger precursor, the amyloid precursor protein (APP) ubiquitously expressed. Among the different peripheral cells expressing APP forms, platelets are particularly interesting since they show concentrations of its isoforms equivalent to those found in brain. Moreover, a number of laboratories independently described alterations in APP metabolism/concentration in platelets of Alzheimer patients when compared to control subjects matched for demographic characteristics. These observations defined the frame of our work aimed to investigate if a correlation between levels of platelet APP forms and Alzheimer disease could be detected. We have reported that patients affected by Alzheimer disease show a differential level of platelet APP forms. This observation has several implications: APP processing abnormalities, believed to be a very early change in Alzheimer disease in neuronal compartment, do occur in extraneuronal tissues, such as platelets, thus, suggesting that Alzheimer disease is a systemic disorder; further, our data strongly indicate that a differential level of platelet APP isoforms can be considered a potential peripheral marker of Alzheimer disease allowing for discrimination between Alzheimer and other types of dementia.
Keywords: Alzheimer disease; Amyloid precursor protein (APP); Platelet; Diagnosis;

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease in which motor neurons in the nervous system die. The cause is unknown, and no effective treatment exists. Mutations in the gene for superoxide dismutase found in a subpopulation have led to an animal model, but research with these mice has not produced complete insight into the disease mechanism. Studies with isolated motor neurons may produce important information. This review discusses approaches to culture motor neurons — single cells, cocultured with other cells, and in intact preparations, such as the spinal or cortical slice. Motor neurons in monoculture are suitable for acute but not for chronic studies, whereas cocultures and slices survive up to months and are used for chronic studies. Results with toxic substances believed to play a role in the disease, such as oxidants and glutamate, and of studies where the energy status of the cells is manipulated, are presented.
Keywords: Amyotrophic lateral sclerosis (ALS); Motor neuron; Apoptosis; Cell culture; Tissue culture; Neurodegenerative disease;

There is increasing evidence that stem cell populations can undergo a transition between mesodermal and neural ectodermal cell fates. Bone marrow-derived cells have been shown to be extremely versatile: they can become brain and liver cells and muscle, while other mesodermal derived cells have been shown to migrate into the brain and differentiate into neurons. Moreover, under the appropriate conditions, neural stem cells can differentiate into hematopoietic and muscle cell fates. It is now well established that newly differentiated cell types are continuously generated from immature stem cells throughout development and in adult mammals, including humans. This review addresses the contribution that bone marrow-derived stem cells may play during neurogenesis. We transplanted male bone marrow into female recipients to track and characterize the Y chromosome containing cells in the CNS (central nervous system) of mice.
Keywords: Stem cell; Differentiation; Hematopoietic; Central nervous system;

The dopamine D4 receptor: one decade of research by James N Oak; John Oldenhof; Hubert H.M Van Tol (303-327).
Dopamine is an important neurotransmitter involved in motor control, endocrine function, reward, cognition and emotion. Dopamine receptors belong to the superfamily of G protein-coupled receptors and play a crucial role in mediating the diverse effects of dopamine in the central nervous system (CNS). The dopaminergic system is implicated in disorders such as Parkinson's disease and addiction, and is the major target for antipsychotic medication in the treatment of schizophrenia. Molecular cloning studies a decade ago revealed the existence of five different dopamine receptor subtypes in mammalian species. While the presence of the abundantly expressed dopamine D1 and D2 receptors was predicted from biochemical and pharmacological work, the cloning of the less abundant dopamine D3, D4 and D5 receptors was not anticipated. The identification of these novel dopamine receptor family members posed a challenge with respect to determining their precise physiological roles and identifying their potential as therapeutic targets for dopamine-related disorders. This review is focused on the accomplishments of one decade of research on the dopamine D4 receptor. New insights into the biochemistry of the dopamine D4 receptor include the discovery that this G protein-coupled receptor can directly interact with SH3 domains. At the physiological level, converging evidence from transgenic mouse work and human genetic studies suggests that this receptor has a role in exploratory behavior and as a genetic susceptibility factor for attention deficit hyperactivity disorder.
Keywords: Dopamine D4 receptor; G protein-coupled receptor; Structure; Pharmacology; Signaling; Physiology; Genetics;

Structure and function of the dopamine transporter by Nianhang Chen; Maarten E.A Reith (329-339).
The dopamine transporter mediates uptake of dopamine into neurons and is a major target for various pharmacologically active drugs and environmental toxins. Since its cloning, much information has been obtained regarding its structure and function. Binding domains for dopamine and various blocking drugs including cocaine are likely formed by interactions with multiple amino acid residues, some of which are separate in the primary structure but lie close together in the still unknown tertiary structure. Chimera and site-directed mutagenesis studies suggest the involvement of both overlapping and separate domains in the interaction with substrates and blockers, whereas recent findings with sulfhydryl reagents selectively targeting cysteine residues support a role for conformational changes in the binding of blockers such as cocaine. The dopamine transporter can also operate in reverse, i.e. in an efflux mode, and recent mutagenesis experiments show different structural requirements for inward and outward transport. Strong evidence for dopamine transporter domains selectively influencing binding of dopamine or cocaine analogs has not yet emerged, although the development of a cocaine antagonist at the level of the transporter remains a possibility.
Keywords: Dopamine transporter; Cocaine; Cocaine analog; Phosphorylation; Chimera; Site-directed mutagenesis;

Psychoneuroendocrinology is a branch of neuroscience that developed in the beginning of the last century, which investigates the possibility of a cause–effect link between endocrinopathies and mental disorders — with these studies ending in negative results. Psychoneuroendocrinology was then used as a methodological approach for the investigation of neurotransmitter function, on the basis of the observation that neurotransmitters regulate neurohormone and peripheral hormone secretions. Data were obtained for hypothalamic noradrenergic, serotoninergic, dopaminergic, gabaergic and acetylcholinergic functions, which could not be automatically extended to higher brain centers whose impairments might be etiopathogenetically involved in the development of mental disorders. Future studies should focus on new methodological approaches to brain biochemistry, on investigation of genetic, molecular biology, brain imaging, psychoneuroimmunoendocrinology, neuropeptide and neurosteroid secretion in relation to brain endocrine function in mental diseases.
Keywords: Psychoneuroendocrinology; Psychoneuroimmunoendocrinology; Genetic; Molecular biology;

Antidepressants: past, present and future by Jerzy Vetulani; Irena Nalepa (351-363).
Since the discovery of first antidepressants in mid-1950's, the field has been intensively studied. Several new classes of compounds emerged and several hypotheses on the mechanism of their action were proposed. The novel antidepressants are either selective and reversible monoamine oxidase inhibitors, (e.g., moclobemide), or selective serotonin reuptake inhibitors (e.g., citalopram or paroxetine), or serotonin and noradrenaline reuptake inhibitors (e.g., venlafaxine). Recently neuropeptides (e.g., thyrotropin-releasing hormone,TRH) or antagonists of neuropeptide receptors (e.g., tachykinin NK1 receptor) undergo clinical tests. Several hypotheses proposed the predominant involvement of one or few neurotransmitter receptors in the mechanism of antidepressant action, but it is now assumed that several distinct receptor mechanisms' trigger different but converging intracellular signal cascades that activate transcription factors, which, in turn, promote the expression of genes encoding for proteins, that play a crucial role in restoring of neuronal functions involved in mood regulation.
Keywords: Antidepressant; Mechanism of action; Monoamine; Neuropeptide; Intracellular signaling; Transcription factor;

The role of dopamine in the mechanism of action of antidepressant drugs by Paolo Stefano D'Aquila; Maria Collu; Gian Luigi Gessa; Gino Serra (365-373).
The present paper reviews evidence on the effect of antidepressant treatments on dopamine transmission. Chronic treatment with antidepressant drugs potentiates the behavioural stimulant responses elicited by the stimulation of dopamine receptors, including reward-related behaviours. Moreover, antidepressants affect dopamine release in several brain areas. The reviewed literature is discussed in terms of the possible mechanisms underlying antidepressant-induced supersensitivity to dopamine-mediated behavioural responses, and of the possible implications for the therapeutic effect of these drugs. It is concluded that the potentiation of dopaminergic neurotransmission induced by chronic antidepressant treatments might contribute to their therapeutic effect.
Keywords: Antidepressant drug; Dopamine; Depression; Mania; Mesolimbic dopamine system;

Stress in schizophrenia: an integrative view by Christine C Gispen-de Wied (375-384).
Stress and the development of a (schizophrenic) psychosis are inextricably related. The process by which stress actually affects psychosis is far less clear. The hypothalamic–pituitary–adrenal system, and in particular the release of corticosteroids, has been attributed an essential role. However, schizophrenia is a disorder in which many functions are distorted. Dysfunctions can be found in behavior, cognition, coping, physiology, pituitary–adrenal and immune functioning. In this short paper, these functions are discussed as to how they contribute to the way stress is appraised and processed. Schizophrenic patients are impaired in their biological response to stress by showing a blunted cortisol response to psychosocial stress. It is hypothesized that this reflects rather cognitive dysfunction, based on biological dysfunctions in those brain structures that are responsible for these processes, i.e. the prefrontal cortex and the limbic system. Considering the blunted cortisol response as a maladaptive stress response, its consequences are commented on with an emphasis on the immune system. Finally, the role of neuroleptics, and in particular the atypical ones, is discussed for their beneficial effect, beyond their fear-and anxiety-reducing properties, in restoring some of the cognitive dysfunctions schizophrenic patients display. By doing so, they may improve perception of the environment, enhance adjustment and thus a proper stress response. Integration of these processes in stress research described, may provide new vistas of the stress concept in schizophrenia.
Keywords: Schizophrenia; Stress; Cognition; Coping; Hypothalamic–pituitary–adrenal system; Immune system;

Cell adhesion molecule proteins play a diverse role in neural development, signal transduction, structural linkages to extracellular and intracellular proteins, synaptic stabilization, neurogenesis, and learning. Three basic mRNA isoforms and potent posttranslational modifications differentially regulate these neurobiological properties of the neural cell adhesion molecule (N-CAM). Abnormal concentrations of N-CAM 105–115 kDa (cN-CAM), N-CAM variable alternative spliced exon (VASE), and N-CAM secreted exon (SEC) are related to schizophrenia and bipolar neuropsychiatric disorders. These N-CAM isoforms provide potential mechanisms for expression of multiple neurobiological alterations between controls and individuals with schizophrenia or bipolar illness. Multiple processes can trigger the dysregulation of N-CAM isoforms. Differences in neuropil volume, neuronal diameter, gray matter thickness, and ventricular size can be related to N-CAM neurobiological properties in neuropsychiatric disorders. Potential test of the N-CAM dysregulation hypothesis of neuropsychiatric disorder is whether ongoing dysregulation of N-CAM would cause cognitive impairments, increased lateral ventricle volume, and decreased hippocampal volume observed in schizophrenia and to a lesser extent in bipolar disorder. An indirect test of this theory conducted in animal experiments lend support to this N-CAM hypothesis. N-CAM dysregulation is consistent with a synaptic abnormality that could underlie the disconnection between brain regions consistent with neuroimaging reports. Synapse stability and plasticity may be part of the molecular neuropathology of these disorders.
Keywords: Schizophrenia; Bipolar disorder; Depression; Memory; Synaptic plasticity; Ventricle enlargement; Neurocognitive impairment;

A variety of stressful events, including emotional stress, cause a marked increase in noradrenaline release in several brain regions, and especially in the hypothalamus, amygdala and locus coeruleus, in the rat brain. These findings suggest that an increased noradrenaline release could be closely related to the provocation of negative emotions such as anxiety and/or fear. In order to confirm this hypothesis, we carried out several studies. Diazepam, a typical benzodiazepine anxiolytic, significantly attenuated not only the immobilization stress-induced increase in noradrenaline release in the three rat brain regions but also the emotional changes of these animals, and these effects were antagonized by flumazenil, a benzodiazepine antagonist. Naloxone and opioid agents, such as morphine, β-endorphin and [Met5]-enkephalin, significantly enhanced and attenuated the stress-induced increase in noradrenaline release in these regions and the stress-induced emotional change, respectively. Two stressful events which predominantly involve emotional factors, i.e., psychological stress and conditioned fear, caused significant increases in noradrenaline release selectively in these three brain regions and these increases were also significantly attenuated by pretreatment with diazepam in a flumazenil reversible manner. Yohimbine, an α2-adrenoceptor antagonist which caused a marked increase in noradrenaline release in the several brain regions, had an anxiolytic action in the two behavioral tests involving anxiety, i.e., the conditioned defensive burying test and the modified forced swim test. β-Carbolines, which possess anxiogenic properties, significantly increased noradrenaline release in the hypothalamus, amygdala and locus coeruleus. Taken together, these findings suggest that the increased release of noradrenaline in the hypothalamus, amygdala and locus coeruleus is, in part, involved in the provocation of anxiety and/or fear in animals exposed to stress, and that the attenuation of this increase by benzodiazepine anxiolytics acting via the benzodiazepine receptor/GABAa receptor/chloride ionophore supramolecular complex may be the basic mechanism of action of these anxiolytic drugs.
Keywords: Anxiety; Stress; Noradrenaline; Hypothalamus; Amygdala; Locus coeruleus; Benzodiazepines; Opioid agent; Anxiolytic drug;

Index (407-408).

Index (409-414).