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Early social deprivation alters monoaminergic afferents in the orbital prefrontal cortex of octodon degus
Neuroscience 116 (2003) 617– 620
LETTER TO NEUROSCIENCE EARLY SOCIAL DEPRIVATION ALTERS MONOAMINERGIC AFFERENTS IN THE ORBITAL PREFRONTAL CORTEX OF OCTODON DEGUS G. POEGGEL,a L. NOWICKIa AND K. BRAUNb*
ence on adult behavior may be the result of neurochemical and neuroanatomical alterations of limbic brain circuits, which have emerged during early brain development. There is increasing evidence that, quite comparable to the experience-driven functional maturation of sensory systems (Singer, 1995) emotional experience interferes with the structural and functional maturation of limbic brain circuits. For instance, changes of synaptic densities and composition have been detected in the limbic anterior cingulate cortex (Helmeke et al., 2001b; Helmeke et al., 2001a; Ovtscharoff and Braun, 2001), and altered dopaminergic and serotonergic innervation of the anterior cingulate cortex and other subregions of the medial prefrontal cortical regions have been found after juvenile social deprivation (Braun et al., 2000; Hall, 1998; Winterfeld et al., 1998). Such deprivation induced alterations of monoaminergic innervation of the prefrontal cortex may alter intellectual and socio-emotional competence, since the monoaminergic transmitters dopamine (DA) and serotonin (5-HT) are modulators of emotional cognitive processes (Arnsten, 1997; Driscoll et al., 1983; Goldstein et al., 1996; Murphy et al., 1996; Sokolowski et al., 1994). The prefrontal cortex of primates and rodents consists of medial and orbital parts (Ongur and Price, 2000), which play distinct roles in cognitive and emotional aspects of behavior. For instance, it has been shown that mood alterations are associated with activation of the orbitofrontal cortex (OFC), suggesting that it may be critical to the experience of emotion (Baker et al., 1997), and that positive and negative emotional signals are differentially processed in distinct subregions of the orbitofrontal cortex (Northoff et al., 2000). Animal studies have revealed that orbito-frontal lesions result in enhanced aggressive behavior (de Bruin et al., 1983). Thus, in continuation of our previous work in the medial prefrontal cortex, the present study was focused on the OFC, in which changes of monoaminergic innervation after early social deprivation was investigated.
a
University of Leipzig, Zoological Institute, Talstr. 33, 04103 Leipzig, Germany b
Otto-von-Guericke-University, Institute for Biology, Department of Developmental Neurobiology, c/o Leibniz Institute of Neurobiology, Brenneckestr. 6, 39118 Magdeburg, Germany
Abstract—The influence of early parental deprivation on the development of tyrosine hydroxylase- and 5-hydroxytryptamine-immunoreactive fiber innervation of subregions of the orbital prefrontal cortex (ventrolateral orbital, lateral orbital and agranular insular cortex) was quantitatively investigated in the precocious lagomorph Octodon degus. Forty-five-dayold degus from two groups were compared: 1) degus which were repeatedly separated from their parents during the first three postnatal weeks, and after weaning they were reared in social isolation; and 2) degus which were reared undisturbed in their families. Compared with the normal control animals the ventrolateral orbital prefrontal cortex and agranular insular cortex of the deprived animals displayed significantly increased density of tyrosine hydroxylase-immunoreactive fibers (up to 172% in the ventrolateral orbital prefrontal cortex and up to 143% in the agranular insular cortex). The lateral orbital prefrontal cortex showed increased 5-hydroxytryptamine-positive fiber densities (up to 118%). This altered balance between the serotonergic and dopaminergic cortical innervation in the orbital prefrontal cortex may reflect an anatomical and functional adaptation, which may be triggered by an altered activity of these transmitter systems during the phases of parental separation and social isolation. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: emotional experience, brain development, limbic system, serotonin, dopamine, quantitative immunocytochemistry.
Traumatic emotional experience during early phases of life such as maternal separation and social isolation appears to have a dramatic impact on the shaping of individual’s responsiveness and behavioral strategies at later stages of life. This long-lasting effect of juvenile emotional experi-
EXPERIMENTAL PROCEDURES All experimental protocols were approved by the ethical committee of the government of the state of Saxony-Anhalt according to the German guidelines for the care and use of animals in laboratory research (§8, Abs. 1, 25.05.1998), and the experiments were performed in accordance with the European Communities Council Directive of November 1986 (86/609/EEC). All efforts were made to minimize animal suffering and only the number of animals necessary to produce reliable data were used.
*Corresponding author: Tel.: ⫹49-391-6263617; fax: ⫹49-3916263618. E-mail address: [email protected] (K. Braun). Abbreviations: ACd, dorsal anterior cingulate cortex; AI, agranular insular cortex; DA, dopamine; 5-HT, 5-hydroxytryptamine (serotonin); LO, lateral orbital cortex; mPFC, medial prefrontal cortex; OFC, orbitofrontal cortex; PFC, prefrontal cortex; TH, tyrosine hydroxylase; VLO, ventrolateral orbital cortex.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00751-0
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G. Poeggel et al. / Neuroscience 116 (2003) 617– 620
Fig. 1. Left: Schematic illustration of the evaluated levels and the measured areas of the OFC of O. degus. Right: Immunocytochemical staining of 5HT- (top) and TH-(bottom) fibers in the Ai. Note the different thickness of the two fiber types.
Octodon degus, is a precocious South American species, which was formerly classified as caviomorph rodent, but is now considered to belong to the lagomorpha (rabbits) (Helmeke et al., 2001a; Ovtscharoff and Braun, 2001). The degus used in this study were bred in our colony as previously described (Braun et al., 2000). Group “parental deprivation”: Four pups were repeatedly separated from their families (three times/day for 1 h from postnatal day 1 (P1) until weaning at P21 (repeated parental deprivation). After weaning these animals were reared isolated in single cages until P45 (social isolation) with acoustic and olfactory but no visual contact to conspecifics. Group “social control”: Five pups were reared under normal undisturbed familial conditions. The histological procedures, including, fixation, sectioning, and immunocytochemical protocols were performed as previously described (Braun et al., 2000). For the detection of serotonergic and dopaminergic fibers, antibodies against 5-hydroxytryptamine (5HT) and tyrosine hydroxylase (TH), respectively, were used. TH is the first and rate limiting enzyme in the synthesis of all catecholamines, and is thus contained in noradrenergic as well as in dopaminergic neurons. Studies in a variety of other species indicate that the majority of TH-labeled fibers are most likely dopaminergic (Akil and Lewis, 1993; Rosenberg and Lewis, 1994; Metzger et al., 1996). Double labeling experiments in rats using anti-TH antibodies and antibodies directed to dopamine--hydroxylase (DBH, a selective marker for noradrenergic neurons and fibers), revealed only ⬍1% of double labeled fibers (Akil and Lewis, 1993). The density of TH-immunoreactive and 5HT-immunoreactive fibers in the OFC (Fig. 1) was quantified in the ventrolateral orbital (VLO), lateral orbital (LO) and agranular insular (Ai) cortex according to the nomenclature of (Uylings and van Eden, 1990). For each animal four sections at a distance of 200 m were selected, and
representative rostro-caudal levels of the pregenual OFC were scanned by a video-equipped system into the computer using a 20⫻ objective. For each section three measuring fields were randomly placed within the respective cortex areas and the relative immunoreactive fiber area of the cortical layers of the different cortical regions was determined using densitometric image analysis (Global LAB Image, Data Translation, Inc., Marlboro, MA, USA). For measuring the area of immunostained structures, the immunoreactive fibers were visualized via adjustments of minimum and maximum gray levels of the DAB precipitate under visual control. The area of marked immunoreactive structures was calculated and divided by the total area of the measuring field, thus indicated as relative ir-area. The aim was not to obtain absolute values of the stained fibers, but to detect deprivation-induced changes of the dopaminergic and serotonergic innervation. The data showed a normal distribution and a Student’s t-test was used to analyze differences between control and deprived groups, significance level was set at P⬍0.05. Table 1. Relative density of TH-immunoreactive fibers (values are relative ir-area, i.e. area of immunoreactive structures/area of the measuring field) Region
Control (N⫽5)
Isolated (N⫽4)
P
Ai LO VLO
0.029⫾0.002 0.036⫾0.003 0.026⫾0.002
0.041⫾0.005 0.045⫾0.006 0.045⫾0.006
0.041 n.s. 0.012
G. Poeggel et al. / Neuroscience 116 (2003) 617– 620 Table 2. Relative density of 5-HT-immunoreactive fibers (values are relative ir-area, i.e. area of immunoreactive structures/area of the measuring field) Region
Control (N ⫽ 5)
Isolated (N ⫽ 4)
P
Ai LO VLO
0.013⫾0.006 0.013⫾0.0007 0.011⫾0.0005
0.016⫾0.0011 0.015⫾0.0003 0.013⫾0.0009
n.s. 0.032 n.s.
RESULTS Compared with the social control group, the parentally deprived animals displayed significantly increased TH-immunoreactive fiber densities in the VLO (P⫽0.012) and Ai (P⫽0.041), no significant differences were found in the LO (Table 1). The density of 5HT-immunoreactive fibers was significantly increased only in the LO of the deprived animals (P⫽0.032), whereas no changes were measured in the VLO and Ai (Table 2).
DISCUSSION The OFC and the structures with which it is interconnected [medial prefrontal cortex (mPFC), amygdala] constitute a circuit that is involved in emotional regulation and its functional development is dramatically shaped by early social influences (Davidson et al., 2000). So far, only few anatomical studies on the cellular and synaptic level have been published on deprivation induced changes of monoaminergic systems. Our results indicate that repeated parental separation followed by early postnatal social deprivation alters the balance between serotonergic and dopaminergic innervation of the OFC. It appears likely that these morphological changes are the result of a deprivation-induced disturbance during the formative period of monoaminergic innervation, a time window which for the medial PFC was determined to occur during the first two postnatal weeks (Braun et al., 2000). Interestingly, the two monoaminergic fiber systems in the three subregions of the OFC responded differentially to the deprivation. While the VLO and Ai displayed an increase of TH-fiber densities but no changes of 5HT-fibers, the LO did not show significant changes of TH- innervation, but increased 5HT-fiber innervation. Interestingly, the direction and magnitude of the deprivation-induced monoaminergic changes are region specific, e.g. in the medial PFC of socially deprived degus and gerbils, reduced dopaminergic fiber densities were found (Braun et al., 2000; Winterfeld et al., 1998). Our anatomical results concerning the dopaminergic system are in line to findings in biochemical studies, in which changes of monoaminergic function in mPFC after preweaning maternal separation and postweaning social isolation have been analyzed. Following maternal separation and exposure to a novel environment increased dopamine release and dopamine turnover was found in several brain regions (Hall, 1998). However, the increased 5HT innervation of medial and orbital prefrontal subregions found in our studies are somewhat discrepant with bio-
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chemical results, where elevated 5-HT turnover and decreased 5HT-concentrations in hippocampus and PFC of maternally deprived rats have been described (Hall, 1998). The experience-induced regulatory adaptations of brain function, which are manifested during early developmental phases of high brain plasticity, may lead to lasting and, once the brain has reached maturity, to more or less stable alterations of functional brain anatomy. Such pathological alterations of ‘emotional’ brain circuits, which in particular appear to affect the higher cortical regions, may underlie the abnormal and inappropriate emotional responses and intellectual retardation, from which the deprived individuals may suffer later in adulthood. For instance, as a result of early deprivation, significantly decreased metabolism bilaterally in the orbital frontal gyrus have been found in PET studies in Romanian orphans, which may be related to their cognitive and behavioral deficits (Chugani et al., 2001). The Ai, a somatic motor region (Freedman and Cassell, 1991), projects directly to brainstem nuclei, and there is evidence that catecholamines can influence autonomic output through these descending pathways (Funk and Stewart, 1996). Furthermore, the Ai plays a role in association of taste-related cognitive processes with feeding behavior (Yamamoto et al., 1984), in food-reward learning tasks (DeCoteau et al., 1997; Ragozzino and Kesner, 1999; Rolls, 2000), in olfactory learning tasks and social behavior (Petrulis et al., 1998). The VLO, which is reciprocally interconnected with the medial agranular cortex, is a component of the cortical circuitry for spatial processing in rodents (Corwin et al., 1994). Unilateral VLO lesions induce neglect, i.e. inability to orient or respond to novel or meaningful stimuli presented to the contralesional side of the body (King et al., 1989). Furthermore, the VLO has been shown to be activated after stress induction (Duncan et al., 1996). Finally, from the consequences of lesion studies in man and animals, it is proposed that the activation of cells in the OFC by a variety of noxious stimuli reflects its more general role in the development and maintenance of behavior in response to negative reinforcement of both social and physical origins (Snow et al., 1992). For instance, OFC dysfunction or lesions results in altered aggressive behavior in animals and humans (Davidson et al., 2000; de Bruin et al., 1983; Kamback, 1973; Orloff and Masserman, 1975). A systematic experimental analysis of deprivation-induced alterations of brain function and behavior in animal models will aid to identify and characterize the critical environmental factors, which shape cortical and subcortical components of emotional circuits and thereby determine normal or pathological behaviors. Acknowledgements—This research was supported by the German Science Foundation, SFB 426.
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Arnsten AF (1997) Catecholamine regulation of the prefrontal cortex. J Psychopharmacol. (Oxf) 11:151–162. Baker SC, Frith CD, Dolan RJ (1997) The interaction between mood and cognitive function studied with PET. Psychol Med 27:565–578. Braun K, Lange E, Metzger M, Poeggel G (2000) Maternal separation followed by early social deprivation affects the development of monoaminergic fiber systems in the medial prefrontal cortex of Octodon degus. Neuroscience 95:309 –318. Chugani HT, Behen ME, Muzik O, Juhasz C, Nagy F, Chugani DC (2001) Local brain functional activity following early deprivation: A study of postinstitutionalized Romanian orphans. Neuroimage 14: 1290 –1301. Corwin JV, Fussinger M, Meyer RC, King VR, Reep RL (1994) Bilateral destruction of the ventrolateral orbital cortex produces allocentric but not egocentric spatial deficits in rats. Behav Brain Res 61:79 – 86. Davidson RJ, Putnam KM, Larson CL (2000) Dysfunction in the neural circuitry of emotion regulation—a possible prelude to violence. Science 289:591–594. de Bruin JPC, van Oyen HG, Van de Poll N (1983) Behavioural changes following lesions of the orbital prefrontal cortex in male rats. Behav Brain Res 10:209 –232. DeCoteau WE, Kesner RP, Williams JM (1997) Short-term memory for food reward magnitude: The role of the prefrontal cortex. Behav Brain Res 88:239 –249. Driscoll P, Dedek J, Martin JR, Zivkovic B (1983) Two-way avoidance and acute shock stress induced alterations of regional noradrenergic, dopaminergic and serotonergic activity in Roman high- and low-avoidance rats. Life Sci 33:1719 –1725. Duncan GE, Knapp DJ, Breese GR (1996) Neuroanatomical characterization of fos induction in rat behavioral models of anxiety. Brain Res 713:79 –91. Freedman LJ, Cassell MD (1991) Thalamic afferents of the rat infralimbic and lateral agranular cortices. Brain Res Bull 26:957–964. Funk D, Stewart J (1996) Role of catecholamines in the frontal cortex in the modulation of basal and stress-induced autonomic output in rats. Brain Res 741:220 –229. Goldstein LE, Rasmusson AM, Bunney BS, Roth RH (1996) Role of the amygdala in the coordination of behavioral, neuroendocrine, and prefrontal cortical monoamine responses to psychological stress in the rat. J Neurosci 16:4787–4798. Hall FS (1998) Social deprivation of neonatal, adolescent, and adult rats has distinct neurochemical and behavioral consequences. Crit Rev Neurobiol 12:129 –162. Helmeke C, Ovtscharoff W Jr, Poeggel G, Braun K (2001a) Juvenile emotional experience alters synaptic inputs on pyramidal neurons in the anterior cingulate cortex. Cereb Cortex 11:717–727. Helmeke C, Poeggel G, Braun K (2001b) Differential emotional experience induces elevated spine densities on basal dendrites of pyramidal neurons in the anterior cingulate cortex of Octodon degus. Neuroscience 104:927–931. Kamback MC (1973) The effects of orbital and dorsolateral frontal cortical ablations on ethanol self-selection and emotional behaviors in monkeys (Macaca nemestrina). Neuropsychologia 11:331–335. King V, Corwin JV, Reep RL (1989) Production and characterization of
neglect in rats with unilateral lesions of ventrolateral orbital cortex. Exp Neurol 105:287–299. Metzger M, Jiang S, Wang JZ, Braun K (1996) Organization of the dopaminergic innervation of forebrain areas relevant to learning: A combined immunohistochemical/retrograde tracing study in the domestic chick. J Comp Neurol 376:1–27. Murphy BL, Arnsten AFT, Goldman-Rakic PS, Roth RH, Arnsten AF (1996) Increased dopamine turnover in the prefrontal cortex impairs spatial working memory performance in rats and monkeys. Proc Natl Acad Sci USA 93:1325–1329. Northoff G, Richter A, Gessner M, Schlagerhauf F, Tell J, Baumgart F, Kaulisch T, Kotter R, Stephan KE, Leschinger A, Hagner T, Bargel B, Witzel T, Hinrichs H, Bogerts B, Scheich H, Heinze HJ (2000) Functional dissociation between medial and lateral prefrontal cortical spatiotemporal activation in negative and positive emotions: A combined fMRI/MEG study. Cereb Cortex 10:93–107. Ongur D, Price JL (2000) The organization of networks within the orbital and medial prefrontal cortex of rats, monkeys and humans. Cereb Cortex 10:206 –219. Orloff ER, Masserman JH (1975) Effect of uncertainty on emotionality and ethanol self-selection in monkeys with cortical ablations. Biol Psychiatry 10:245–251. Ovtscharoff W, Braun K (2001) Maternal separation and social isolation modulate the postnatal development of synaptic composition in the infralimbic cortex of Octodon degus. Neuroscience 104:33–40. Petrulis A, DeSouza I, Schiller M, Johnston RE (1998) Role of frontal cortex in social odor discrimination and scent-marking in female golden hamsters (Mesocricetus auratus). Behav Neurosci 112: 199 –212. Ragozzino ME, Kesner RP (1999) The role of the agranular insular cortex in working memory for food reward value and allocentric space in rats. Behav Brain Res 98:103–112. Rolls ET (2000) The orbitofrontal cortex and reward. Cereb Cortex 10:284 –294. Rosenberg DR, Lewis DA (1994) Changes in the dopaminergic innervation of monkey prefrontal cortex during late postnatal development: A tyrosine hydroxylase immunohistochemical study. Biol Psychiatry 36:272–277. Singer W (1995) Development and plasticity of cortical processing architectures. Science 270:758 –764. Snow PJ, Lumb BM, Cervero F (1992) The representation of prolonged and intense, noxious somatic and visceral stimuli in the ventrolateral orbital cortex of the cat. Pain 48:89 –99. Sokolowski JD, McCullough LD, Salamone JD (1994) Effects of dopamine depletions in the medial prefrontal cortex on active avoidance and escape in the rat. Brain Res 651:293–299. Uylings HBM, van Eden CG (1990) Qualitative and quantitative comparison of the prefrontal cortex in rat and in primates, including humans. Prefrontal Cortex 85:31–62. Winterfeld KT, Teuchert-Noodt G, Dawirs RR (1998) Social environment alters both ontogeny of dopamine innervation of the medial prefrontal cortex and maturation of working memory in gerbils (Meriones unguiculatus). J Neurosci Res 52:201–209. Yamamoto T, Azuma S, Kawamura Y (1984) Functional relations between the cortical gustatory area and the amygdala: Electrophysiological and behavioral studies in rats. Exp Brain Res 56:23–31.
(Accepted 20 September 2002)
LETTER TO NEUROSCIENCE EARLY SOCIAL DEPRIVATION ALTERS MONOAMINERGIC AFFERENTS IN THE ORBITAL PREFRONTAL CORTEX OF OCTODON DEGUS G. POEGGEL,a L. NOWICKIa AND K. BRAUNb*
ence on adult behavior may be the result of neurochemical and neuroanatomical alterations of limbic brain circuits, which have emerged during early brain development. There is increasing evidence that, quite comparable to the experience-driven functional maturation of sensory systems (Singer, 1995) emotional experience interferes with the structural and functional maturation of limbic brain circuits. For instance, changes of synaptic densities and composition have been detected in the limbic anterior cingulate cortex (Helmeke et al., 2001b; Helmeke et al., 2001a; Ovtscharoff and Braun, 2001), and altered dopaminergic and serotonergic innervation of the anterior cingulate cortex and other subregions of the medial prefrontal cortical regions have been found after juvenile social deprivation (Braun et al., 2000; Hall, 1998; Winterfeld et al., 1998). Such deprivation induced alterations of monoaminergic innervation of the prefrontal cortex may alter intellectual and socio-emotional competence, since the monoaminergic transmitters dopamine (DA) and serotonin (5-HT) are modulators of emotional cognitive processes (Arnsten, 1997; Driscoll et al., 1983; Goldstein et al., 1996; Murphy et al., 1996; Sokolowski et al., 1994). The prefrontal cortex of primates and rodents consists of medial and orbital parts (Ongur and Price, 2000), which play distinct roles in cognitive and emotional aspects of behavior. For instance, it has been shown that mood alterations are associated with activation of the orbitofrontal cortex (OFC), suggesting that it may be critical to the experience of emotion (Baker et al., 1997), and that positive and negative emotional signals are differentially processed in distinct subregions of the orbitofrontal cortex (Northoff et al., 2000). Animal studies have revealed that orbito-frontal lesions result in enhanced aggressive behavior (de Bruin et al., 1983). Thus, in continuation of our previous work in the medial prefrontal cortex, the present study was focused on the OFC, in which changes of monoaminergic innervation after early social deprivation was investigated.
a
University of Leipzig, Zoological Institute, Talstr. 33, 04103 Leipzig, Germany b
Otto-von-Guericke-University, Institute for Biology, Department of Developmental Neurobiology, c/o Leibniz Institute of Neurobiology, Brenneckestr. 6, 39118 Magdeburg, Germany
Abstract—The influence of early parental deprivation on the development of tyrosine hydroxylase- and 5-hydroxytryptamine-immunoreactive fiber innervation of subregions of the orbital prefrontal cortex (ventrolateral orbital, lateral orbital and agranular insular cortex) was quantitatively investigated in the precocious lagomorph Octodon degus. Forty-five-dayold degus from two groups were compared: 1) degus which were repeatedly separated from their parents during the first three postnatal weeks, and after weaning they were reared in social isolation; and 2) degus which were reared undisturbed in their families. Compared with the normal control animals the ventrolateral orbital prefrontal cortex and agranular insular cortex of the deprived animals displayed significantly increased density of tyrosine hydroxylase-immunoreactive fibers (up to 172% in the ventrolateral orbital prefrontal cortex and up to 143% in the agranular insular cortex). The lateral orbital prefrontal cortex showed increased 5-hydroxytryptamine-positive fiber densities (up to 118%). This altered balance between the serotonergic and dopaminergic cortical innervation in the orbital prefrontal cortex may reflect an anatomical and functional adaptation, which may be triggered by an altered activity of these transmitter systems during the phases of parental separation and social isolation. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: emotional experience, brain development, limbic system, serotonin, dopamine, quantitative immunocytochemistry.
Traumatic emotional experience during early phases of life such as maternal separation and social isolation appears to have a dramatic impact on the shaping of individual’s responsiveness and behavioral strategies at later stages of life. This long-lasting effect of juvenile emotional experi-
EXPERIMENTAL PROCEDURES All experimental protocols were approved by the ethical committee of the government of the state of Saxony-Anhalt according to the German guidelines for the care and use of animals in laboratory research (§8, Abs. 1, 25.05.1998), and the experiments were performed in accordance with the European Communities Council Directive of November 1986 (86/609/EEC). All efforts were made to minimize animal suffering and only the number of animals necessary to produce reliable data were used.
*Corresponding author: Tel.: ⫹49-391-6263617; fax: ⫹49-3916263618. E-mail address: [email protected] (K. Braun). Abbreviations: ACd, dorsal anterior cingulate cortex; AI, agranular insular cortex; DA, dopamine; 5-HT, 5-hydroxytryptamine (serotonin); LO, lateral orbital cortex; mPFC, medial prefrontal cortex; OFC, orbitofrontal cortex; PFC, prefrontal cortex; TH, tyrosine hydroxylase; VLO, ventrolateral orbital cortex.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00751-0
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Fig. 1. Left: Schematic illustration of the evaluated levels and the measured areas of the OFC of O. degus. Right: Immunocytochemical staining of 5HT- (top) and TH-(bottom) fibers in the Ai. Note the different thickness of the two fiber types.
Octodon degus, is a precocious South American species, which was formerly classified as caviomorph rodent, but is now considered to belong to the lagomorpha (rabbits) (Helmeke et al., 2001a; Ovtscharoff and Braun, 2001). The degus used in this study were bred in our colony as previously described (Braun et al., 2000). Group “parental deprivation”: Four pups were repeatedly separated from their families (three times/day for 1 h from postnatal day 1 (P1) until weaning at P21 (repeated parental deprivation). After weaning these animals were reared isolated in single cages until P45 (social isolation) with acoustic and olfactory but no visual contact to conspecifics. Group “social control”: Five pups were reared under normal undisturbed familial conditions. The histological procedures, including, fixation, sectioning, and immunocytochemical protocols were performed as previously described (Braun et al., 2000). For the detection of serotonergic and dopaminergic fibers, antibodies against 5-hydroxytryptamine (5HT) and tyrosine hydroxylase (TH), respectively, were used. TH is the first and rate limiting enzyme in the synthesis of all catecholamines, and is thus contained in noradrenergic as well as in dopaminergic neurons. Studies in a variety of other species indicate that the majority of TH-labeled fibers are most likely dopaminergic (Akil and Lewis, 1993; Rosenberg and Lewis, 1994; Metzger et al., 1996). Double labeling experiments in rats using anti-TH antibodies and antibodies directed to dopamine--hydroxylase (DBH, a selective marker for noradrenergic neurons and fibers), revealed only ⬍1% of double labeled fibers (Akil and Lewis, 1993). The density of TH-immunoreactive and 5HT-immunoreactive fibers in the OFC (Fig. 1) was quantified in the ventrolateral orbital (VLO), lateral orbital (LO) and agranular insular (Ai) cortex according to the nomenclature of (Uylings and van Eden, 1990). For each animal four sections at a distance of 200 m were selected, and
representative rostro-caudal levels of the pregenual OFC were scanned by a video-equipped system into the computer using a 20⫻ objective. For each section three measuring fields were randomly placed within the respective cortex areas and the relative immunoreactive fiber area of the cortical layers of the different cortical regions was determined using densitometric image analysis (Global LAB Image, Data Translation, Inc., Marlboro, MA, USA). For measuring the area of immunostained structures, the immunoreactive fibers were visualized via adjustments of minimum and maximum gray levels of the DAB precipitate under visual control. The area of marked immunoreactive structures was calculated and divided by the total area of the measuring field, thus indicated as relative ir-area. The aim was not to obtain absolute values of the stained fibers, but to detect deprivation-induced changes of the dopaminergic and serotonergic innervation. The data showed a normal distribution and a Student’s t-test was used to analyze differences between control and deprived groups, significance level was set at P⬍0.05. Table 1. Relative density of TH-immunoreactive fibers (values are relative ir-area, i.e. area of immunoreactive structures/area of the measuring field) Region
Control (N⫽5)
Isolated (N⫽4)
P
Ai LO VLO
0.029⫾0.002 0.036⫾0.003 0.026⫾0.002
0.041⫾0.005 0.045⫾0.006 0.045⫾0.006
0.041 n.s. 0.012
G. Poeggel et al. / Neuroscience 116 (2003) 617– 620 Table 2. Relative density of 5-HT-immunoreactive fibers (values are relative ir-area, i.e. area of immunoreactive structures/area of the measuring field) Region
Control (N ⫽ 5)
Isolated (N ⫽ 4)
P
Ai LO VLO
0.013⫾0.006 0.013⫾0.0007 0.011⫾0.0005
0.016⫾0.0011 0.015⫾0.0003 0.013⫾0.0009
n.s. 0.032 n.s.
RESULTS Compared with the social control group, the parentally deprived animals displayed significantly increased TH-immunoreactive fiber densities in the VLO (P⫽0.012) and Ai (P⫽0.041), no significant differences were found in the LO (Table 1). The density of 5HT-immunoreactive fibers was significantly increased only in the LO of the deprived animals (P⫽0.032), whereas no changes were measured in the VLO and Ai (Table 2).
DISCUSSION The OFC and the structures with which it is interconnected [medial prefrontal cortex (mPFC), amygdala] constitute a circuit that is involved in emotional regulation and its functional development is dramatically shaped by early social influences (Davidson et al., 2000). So far, only few anatomical studies on the cellular and synaptic level have been published on deprivation induced changes of monoaminergic systems. Our results indicate that repeated parental separation followed by early postnatal social deprivation alters the balance between serotonergic and dopaminergic innervation of the OFC. It appears likely that these morphological changes are the result of a deprivation-induced disturbance during the formative period of monoaminergic innervation, a time window which for the medial PFC was determined to occur during the first two postnatal weeks (Braun et al., 2000). Interestingly, the two monoaminergic fiber systems in the three subregions of the OFC responded differentially to the deprivation. While the VLO and Ai displayed an increase of TH-fiber densities but no changes of 5HT-fibers, the LO did not show significant changes of TH- innervation, but increased 5HT-fiber innervation. Interestingly, the direction and magnitude of the deprivation-induced monoaminergic changes are region specific, e.g. in the medial PFC of socially deprived degus and gerbils, reduced dopaminergic fiber densities were found (Braun et al., 2000; Winterfeld et al., 1998). Our anatomical results concerning the dopaminergic system are in line to findings in biochemical studies, in which changes of monoaminergic function in mPFC after preweaning maternal separation and postweaning social isolation have been analyzed. Following maternal separation and exposure to a novel environment increased dopamine release and dopamine turnover was found in several brain regions (Hall, 1998). However, the increased 5HT innervation of medial and orbital prefrontal subregions found in our studies are somewhat discrepant with bio-
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chemical results, where elevated 5-HT turnover and decreased 5HT-concentrations in hippocampus and PFC of maternally deprived rats have been described (Hall, 1998). The experience-induced regulatory adaptations of brain function, which are manifested during early developmental phases of high brain plasticity, may lead to lasting and, once the brain has reached maturity, to more or less stable alterations of functional brain anatomy. Such pathological alterations of ‘emotional’ brain circuits, which in particular appear to affect the higher cortical regions, may underlie the abnormal and inappropriate emotional responses and intellectual retardation, from which the deprived individuals may suffer later in adulthood. For instance, as a result of early deprivation, significantly decreased metabolism bilaterally in the orbital frontal gyrus have been found in PET studies in Romanian orphans, which may be related to their cognitive and behavioral deficits (Chugani et al., 2001). The Ai, a somatic motor region (Freedman and Cassell, 1991), projects directly to brainstem nuclei, and there is evidence that catecholamines can influence autonomic output through these descending pathways (Funk and Stewart, 1996). Furthermore, the Ai plays a role in association of taste-related cognitive processes with feeding behavior (Yamamoto et al., 1984), in food-reward learning tasks (DeCoteau et al., 1997; Ragozzino and Kesner, 1999; Rolls, 2000), in olfactory learning tasks and social behavior (Petrulis et al., 1998). The VLO, which is reciprocally interconnected with the medial agranular cortex, is a component of the cortical circuitry for spatial processing in rodents (Corwin et al., 1994). Unilateral VLO lesions induce neglect, i.e. inability to orient or respond to novel or meaningful stimuli presented to the contralesional side of the body (King et al., 1989). Furthermore, the VLO has been shown to be activated after stress induction (Duncan et al., 1996). Finally, from the consequences of lesion studies in man and animals, it is proposed that the activation of cells in the OFC by a variety of noxious stimuli reflects its more general role in the development and maintenance of behavior in response to negative reinforcement of both social and physical origins (Snow et al., 1992). For instance, OFC dysfunction or lesions results in altered aggressive behavior in animals and humans (Davidson et al., 2000; de Bruin et al., 1983; Kamback, 1973; Orloff and Masserman, 1975). A systematic experimental analysis of deprivation-induced alterations of brain function and behavior in animal models will aid to identify and characterize the critical environmental factors, which shape cortical and subcortical components of emotional circuits and thereby determine normal or pathological behaviors. Acknowledgements—This research was supported by the German Science Foundation, SFB 426.
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(Accepted 20 September 2002)