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Overexpression of the p75 neurotrophin receptor in the sensori-motor cortex of rats exposed to ethanol during early postnatal life
Neuroscience Letters 342 (2003) 89–92 www.elsevier.com/locate/neulet
Overexpression of the p75 neurotrophin receptor in the sensori-motor cortex of rats exposed to ethanol during early postnatal life Amelia Toescaa, Stefano Giannettia, Alberto Granatob,* b
a Institute of Anatomy, Catholic University, L.go F. Vito 1, 00168, Rome, Italy Department of Psychology, Catholic University, L.go A. Gemelli 1, 20123, Milan, Italy
Received 2 September 2002; received in revised form 7 February 2003; accepted 17 February 2003
Abstract Foetal alcohol syndrome is a known cause of mental retardation. It has been suggested that the anatomical and functional alterations observed in the cerebral cortex could be mediated by an interference of ethanol with developmental processes modulated by neurotrophins and/or their receptors. We have studied by immunohistochemistry the expression of the p75 neurotrophin receptor (p75 NTR) in the sensorimotor cortex of P10 and P20 rats exposed to the inhalation of ethanol during the first week of postnatal life. At both the studied ages, the number of p75 NTR immunoreactive neurons was higher in ethanol treated animals compared to controls. The increase of immunoreactive elements was relatively more marked in the motor than in the somatosensory cortex. The involvement of p75 NTR in ethanol-induced apoptosis and neural plasticity is discussed. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Foetal alcohol syndrome; Development; Apoptosis; Immunohistochemistry; Pyramidal neurons; Nerve growth factor receptor
The exposure to ethanol during brain development, whose effects are globally referred to as foetal alcohol syndrome (FAS), represents one of the chief causes of mental retardation in humans [1]. Several experimental studies have been carried out in order to understand how ethanol affects the development of brain structures concerned with higher cognitive functions, namely the cerebral cortex. It has been demonstrated that the cerebral cortex and its connections undergo deep alterations following early ethanol exposure (e.g. Refs. [11,14]). Moreover, experimental models of FAS allowed clarification of the basic mechanisms involved in the establishment of alcoholinduced brain damage. Among others, the ethanol interference with developmental processes regulated by neurotrophins and neurotrophin receptors has been repeatedly considered to be responsible for the observed anomalies [3, 9]. The present study was designed to investigate the cortical expression of the low-affinity neurotrophin receptor (p75 NTR, reviewed in Ref. [4]) in rats exposed to the
* Corresponding author. Tel.: þ 39-02-72342284; fax: þ 39-0272342280. E-mail address: [email protected] (A. Granato).
inhalation of ethanol during the first postnatal week, when the brain growth spurt occurs [7]. Newborn Wistar rats were exposed to ethanol vapours for 3 h a day from the second postnatal day (P2, P0 is the birthday) through P6 (group Et). The method of ethanol administration was slightly modified from Ruwe et al. [20]. Briefly, an air pump (air flow 3 l/min) was connected to a vaporization chamber kept at a constant temperature of 38 8C, where ethanol (95% v/v) was injected at the rate of 2.5 ml/min. The ethanol atmosphere was then conveyed to a sealed plexiglass cage in which the pups were placed after the separation from the mothers. At the end of each session of ethanol administration, one rat per litter was given a sublethal dose of ketamine (50 mg/kg i.p.) and a blood sample (100 ml) was taken from the left ventricle. The blood alcohol concentration (BAC) was measured using an enzymatic kit (332-A, Sigma, St. Louis, MO). Control animals (group Cont) were represented by pups removed from their mothers for 3 h a day from P2 to P6 omitting the ethanol administration. Four Et and four Cont animals underwent deep anaesthesia (ketamine 90 mg/kg, xylazine 10 mg/kg i.p.) and transcardiac perfusion with 4% paraformaldehyde at P10. An additional four Et and four Cont rats underwent the same procedure at P20. Brains were
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00258-1
90
A. Toesca et al. / Neuroscience Letters 342 (2003) 89–92
removed, cryoprotected in 30% buffered sucrose and cut on a freezing microtome into 30 mm thick coronal sections. Sections of the sensori-motor cortex were processed for immunohistochemistry by the standard avidin-biotin peroxidase complex procedure (Vectastain Elite kit, Vector, Burlingame, CA). Free-floating sections were incubated for 48 h at 4 8C with the primary monoclonal antibody to p75 NTR (MAB365, Chemicon, Temecula, CA) diluted 1:100. Control sections were incubated in a medium lacking the primary antibody. The immunoreaction was revealed using the 3,30 diaminobenzidine (Sigma) as chromogen. Every fifth section was counterstained with thionin for the evaluation of the cytoarchitectonics and rostro-caudal extent of the sensori-motor cortex. Sections were examined under a Nikon E 600 light microscope. Immunoreactive neurons in the sensori-motor cortex have been charted with the aid of the software Neurolucida (Microbrightfield, Colchester, VT). Labelled neuron profiles were counted on every fifth section and cell counts were corrected using Abercrombie’s method [2]. Prior to testing for differences between Cont and Et groups, the section counts were first averaged per individual case. Differences between groups were evaluated by means of ANOVA. The BAC measured in newborn rats at the end of each session of ethanol administration ranged from 226 to 278 mg/100 ml (mean ^ SD 251.2 ^ 21.4 mg/100 ml). No significant differences between Et and Cont cases were observed in the curve of weight gain during the first three postnatal weeks. At the two studied ages, both in Et and Cont animals, most immunoreactive elements observed in the sensori-motor cortex were represented by pyramidal neurons, mainly located in the infragranular layers, with a strong preference for layer 5. As a general feature, the location of the immunoreaction product was mainly perinuclear; immunoreactive apical or basal dendrites were frequently observed in pyramidal neurons of Et animals (inset of Fig. 1B) and were rare in Cont animals. Supragranular neurons, especially in the deep part of layer 3, were more numerous in Et animals than in Cont cases. Immunopositive elements with the size and/or morphological appearance of glial cells were not observed. Both at P10 and P20, the main difference between Cont and Et cases was represented by the tangential location of immunolabelling. Most labelled neurons in Cont animals were located in the primary somatosensory cortex (S1), whereas the medially located primary motor cortex (M1) featured only sparse labelled cells (Figs. 1A and 2A). On the other hand, layers 5 and 6 of M1 in Et animals were characterized by the presence of a great number of p75 NTR immunoreactive profiles (Figs. 1B and 2A). These findings were confirmed by the counts of immunoreactive neurons shown in Fig. 2B. In M1, both at P10 and P20, the mean number of p75 NTR positive neurons per section was higher in Et than in Cont cases and the difference was highly significant. As for S1, a highly significant difference between Cont and Et animals was found only at P10.
The tangential and radial distribution of p75 NTR positive neurons observed in the present study are in general agreement with previous reports dealing with the expression of neurotrophin receptors in the sensori-motor cortex of mature rats [15,18]. Our study is not focused on the expression of p75 NTR during normal cortical maturation, yet a certain degree of downregulation occurring during postnatal life may be suggested by the present data. However, the transient expression of p75 NTR has been described for the thalamic ventrobasal nucleus [5], while in the rodent cerebral cortex its level seems to be kept constant through the development [19]. The main result of the present study is the demonstration of the strong and prolonged increase of p75 NTR immunopositivity in the sensori-motor cortex following exposure to ethanol during early postnatal life. An upregulation of p75 NTR as a consequence of early exposure to neurotoxic agents has already been reported [10] and might represent a general feature of several disorders of the nervous system (for review see Ref. [6]). Conversely, other reports pointed out that ethanol is able to reduce the expression of the low-affinity neurotrophin receptor [8,21]. Such discrepancies can be explained by the different experimental strategies (i.e. cell cultures [21]) or the different considered structure (cerebellar Purkinje cells [8]). A conservative interpretation for the increased expression of p75 NTR must necessarily take into account its postulated dual role during neurogenesis. On one hand, p75 NTR is thought to mediate apoptotic neuronal death [4, 13]. Actually, the exposure to ethanol during early postnatal life is able to induce massive apoptosis [12,17]. In addition, the prevalent location of p75 NTR immunoreactive neurons in layer 5, as described in our study, is consistent with the distribution of caspase 3 immunoreactivity reported in one of the studies dealing with the apoptotic effect of ethanol [17]. If p75 NTR represents a marker of susceptibility to apoptosis, the long-lasting overexpression observed in the present study might indicate that cortical cells are prone to apoptotic death well after ethanol withdrawal. This is in agreement with a recent report by Mooney and Miller [16], demonstrating that prenatal exposure to ethanol is able to reduce the cortical bcl-2/bax ratio during the first two postnatal weeks, thus providing a long-lasting permissive step for neuronal death. However, the complex interplay among p75 NTR, Trks, and apoptosis (reviewed in Ref. [4]) should also be taken into account, especially considering the reported colocalization of p75 NTR and trkA within the same cortical neurons [15]. At the other end of the spectrum, the low-affinity neurotrophin receptor can be regarded as a key molecule for neuronal plasticity, mainly because of its capability of modulating neurite outgrowth [22]. According to this view, it is worth mentioning that the extent and complexity of dendritic arbors is increased in layer 5 neurons of the sensori-motor cortex after early exposure to ethanol [14].
A. Toesca et al. / Neuroscience Letters 342 (2003) 89–92
91
Fig. 1. Microphotographs showing the p75 NTR immunoreactivity in the primary motor cortex of a P20 Cont (A) and a P20 Et case (B). Note the higher number and the wider radial distribution of immunopositive elements in the Et case. The immunolabelling of apical and basal dendrites is shown at higher magnification in the inset at the top-left of (B). Scale bar: 50 mm for (A), 40 mm for (B), and 20 mm for the inset.
Fig. 2. (A) Schematic drawing of the p75 NTR immunoreactivity in two representative sections of the sensori-motor cortex from a P10 Cont and a P10 Et case. Each symbol represents one labelled cell. M1, primary motor cortex; S1, primary somatosensory cortex. (B) Bar graphs showing the mean number of labelled cells per section in P10 and P20 cases. T bars represent standard deviations. **P , 0:01.
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A. Toesca et al. / Neuroscience Letters 342 (2003) 89–92
Our results prompt further investigation to clarify the role of the low-affinity neurotrophin receptor in FAS, possibly by assessing the susceptibility to early ethanol exposure in mice lacking p75 NTR.
[11]
[12]
Acknowledgements This work was partly supported by a grant to A.G. from the Alzheimer Research Program, funded by the Italian Ministry of Public Health.
[13]
[14]
References [1] E.L. Abel, R.J. Sokol, Fetal alcohol syndrome is now a leading cause of mental retardation, Lancet 2 (1986) 1222. [2] M. Abercrombie, Estimation of nuclear population from microtome sections, Anat. Rec. 94 (1946) 239–247. [3] F. Angelucci, M. Fiore, C. Cozzari, L. Aloe, Prenatal ethanol effects on NGF level, NPY and ChAT immunoreactivity in mouse entorhinal cortex: a preliminary study, Neurotoxicol. Teratol. 21 (1999) 415–425. [4] G.L. Barrett, The p75 neurotrophin receptor and neuronal apoptosis, Prog. Neurobiol. 61 (2000) 205 –229. [5] D.P. Crockett, S.L. Harris, M.D. Egger, Neurotrophin receptor (p75) in the trigeminal thalamus of the rat: development, response to injury, transient vibrissa-related patterning, and retrograde transport, Anat. Rec. 259 (2000) 446–460. [6] G. Dechant, Y. Barde, The neurotrophin receptor p75NTR: novel functions and implications for diseases of the nervous system, Nat. Neurosci. 5 (2002) 1131–1136. [7] J. Dobbing, J. Sands, The brain growth spurt in various mammalian species, Early Hum. Dev. 3 (1979) 79– 84. [8] D.P. Dohrman, J.R. West, N.J. Pantazis, Ethanol reduces expression of the nerve growth factor receptor, but not nerve growth factor protein levels in the neonatal rat cerebellum, Alcohol. Clin. Exp. Res. 21 (1997) 882 –893. [9] K.E. Dow, R.J. Riopelle, Ethanol neurotoxicity: effects on neurite formation and neurotrophic factor production in vitro, Science 228 (1985) 591–593. [10] M. Fiore, L. Aloe, C. Westenbroek, T. Amendola, A. Antonelli, J. Korf, Bromodeoxyuridine and methylazoxymethanol exposure during
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
brain development affects behavior in rats: consideration for a role of nerve growth factor and brain derived neurotrophic factor, Neurosci. Lett. 309 (2001) 113–116. A. Granato, M. Santarelli, A. Sbriccoli, D. Minciacchi, Multifaceted alterations of the thalamo-cortico-thalamic loop in adult rats prenatally exposed to ethanol, Anat. Embryol. 191 (1995) 11–23. C. Ikonomidou, P. Bittigau, M.J. Ishimaru, D.F. Wozniak, C. Koch, K. Genz, M.T. Price, V. Stefovska, F. Horster, T. Tenkova, K. Dikranian, J.V. Olney, Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome, Science 287 (2000) 1056–1060. V. Mamidipudi, M.W. Wooten, Dual role for p75NTR signaling in survival and cell death: can intracellular mediators provide an explanation?, J. Neurosci. Res. 68 (2002) 373 –384. M.W. Miller, N.L. Chiaia, R.W. Rhoades, Intracellular recording and injection study of corticospinal neurons in the rat somatosensory cortex: effect of prenatal exposure to ethanol, J. Comp. Neurol. 297 (1990) 91– 105. M.W. Miller, A.F. Pitts, Neurotrophin receptors in the somatosensory cortex of the mature rat: co-localization of p75, trk, isoforms and cneu, Brain Res. 852 (2000) 355–366. S.M. Mooney, M.W. Miller, Effects of prenatal exposure to ethanol on the expression of bcl-2, bax and caspase 3 in the developing rat cerebral cortex and thalamus, Brain Res. 911 (2001) 71–81. J.W. Olney, T. Tenkova, K. Dikranian, Y. Qin, J. Labruyere, C. Ikonomidou, Ethanol-induced apoptotic neurodegeneration in the developing C57BL/6 mouse brain, Dev. Brain Res. 133 (2002) 115 –126. A.F. Pitts, M.W. Miller, Expression of nerve growth factor, p75, and trk in the somatosensory and motor cortices of mature rats: evidence for local trophic support circuits, Somatosens. Mot. Res. 12 (1995) 329 –342. F.M. Rossi, R. Sala, L. Maffei, Expression of the nerve growth factor receptors trkA and p75NTR in the visual cortex of the rat: development and regulation by the cholinergic input, J. Neurosci. 22 (2002) 912–919. W.D. Ruwe, L. Bauce, W.W. Flemons, W.L. Veale, Q.J. Pittman, Alcohol dependence and withdrawal in the rat. An effective means of induction and assessment, J. Pharmacol. Methods 15 (1986) 225 –234. G.K. Seabold, J. Luo, M.W. Miller, Effect of ethanol on neurotrophinmediated cell survival and receptor expression in cultures of cortical neurons, Dev. Brain Res. 108 (1998) 139–145. T. Yamashita, K.L. Tucker, Y.A. Barde, Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth, Neuron 24 (1999) 585 –593.
Overexpression of the p75 neurotrophin receptor in the sensori-motor cortex of rats exposed to ethanol during early postnatal life Amelia Toescaa, Stefano Giannettia, Alberto Granatob,* b
a Institute of Anatomy, Catholic University, L.go F. Vito 1, 00168, Rome, Italy Department of Psychology, Catholic University, L.go A. Gemelli 1, 20123, Milan, Italy
Received 2 September 2002; received in revised form 7 February 2003; accepted 17 February 2003
Abstract Foetal alcohol syndrome is a known cause of mental retardation. It has been suggested that the anatomical and functional alterations observed in the cerebral cortex could be mediated by an interference of ethanol with developmental processes modulated by neurotrophins and/or their receptors. We have studied by immunohistochemistry the expression of the p75 neurotrophin receptor (p75 NTR) in the sensorimotor cortex of P10 and P20 rats exposed to the inhalation of ethanol during the first week of postnatal life. At both the studied ages, the number of p75 NTR immunoreactive neurons was higher in ethanol treated animals compared to controls. The increase of immunoreactive elements was relatively more marked in the motor than in the somatosensory cortex. The involvement of p75 NTR in ethanol-induced apoptosis and neural plasticity is discussed. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Foetal alcohol syndrome; Development; Apoptosis; Immunohistochemistry; Pyramidal neurons; Nerve growth factor receptor
The exposure to ethanol during brain development, whose effects are globally referred to as foetal alcohol syndrome (FAS), represents one of the chief causes of mental retardation in humans [1]. Several experimental studies have been carried out in order to understand how ethanol affects the development of brain structures concerned with higher cognitive functions, namely the cerebral cortex. It has been demonstrated that the cerebral cortex and its connections undergo deep alterations following early ethanol exposure (e.g. Refs. [11,14]). Moreover, experimental models of FAS allowed clarification of the basic mechanisms involved in the establishment of alcoholinduced brain damage. Among others, the ethanol interference with developmental processes regulated by neurotrophins and neurotrophin receptors has been repeatedly considered to be responsible for the observed anomalies [3, 9]. The present study was designed to investigate the cortical expression of the low-affinity neurotrophin receptor (p75 NTR, reviewed in Ref. [4]) in rats exposed to the
* Corresponding author. Tel.: þ 39-02-72342284; fax: þ 39-0272342280. E-mail address: [email protected] (A. Granato).
inhalation of ethanol during the first postnatal week, when the brain growth spurt occurs [7]. Newborn Wistar rats were exposed to ethanol vapours for 3 h a day from the second postnatal day (P2, P0 is the birthday) through P6 (group Et). The method of ethanol administration was slightly modified from Ruwe et al. [20]. Briefly, an air pump (air flow 3 l/min) was connected to a vaporization chamber kept at a constant temperature of 38 8C, where ethanol (95% v/v) was injected at the rate of 2.5 ml/min. The ethanol atmosphere was then conveyed to a sealed plexiglass cage in which the pups were placed after the separation from the mothers. At the end of each session of ethanol administration, one rat per litter was given a sublethal dose of ketamine (50 mg/kg i.p.) and a blood sample (100 ml) was taken from the left ventricle. The blood alcohol concentration (BAC) was measured using an enzymatic kit (332-A, Sigma, St. Louis, MO). Control animals (group Cont) were represented by pups removed from their mothers for 3 h a day from P2 to P6 omitting the ethanol administration. Four Et and four Cont animals underwent deep anaesthesia (ketamine 90 mg/kg, xylazine 10 mg/kg i.p.) and transcardiac perfusion with 4% paraformaldehyde at P10. An additional four Et and four Cont rats underwent the same procedure at P20. Brains were
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00258-1
90
A. Toesca et al. / Neuroscience Letters 342 (2003) 89–92
removed, cryoprotected in 30% buffered sucrose and cut on a freezing microtome into 30 mm thick coronal sections. Sections of the sensori-motor cortex were processed for immunohistochemistry by the standard avidin-biotin peroxidase complex procedure (Vectastain Elite kit, Vector, Burlingame, CA). Free-floating sections were incubated for 48 h at 4 8C with the primary monoclonal antibody to p75 NTR (MAB365, Chemicon, Temecula, CA) diluted 1:100. Control sections were incubated in a medium lacking the primary antibody. The immunoreaction was revealed using the 3,30 diaminobenzidine (Sigma) as chromogen. Every fifth section was counterstained with thionin for the evaluation of the cytoarchitectonics and rostro-caudal extent of the sensori-motor cortex. Sections were examined under a Nikon E 600 light microscope. Immunoreactive neurons in the sensori-motor cortex have been charted with the aid of the software Neurolucida (Microbrightfield, Colchester, VT). Labelled neuron profiles were counted on every fifth section and cell counts were corrected using Abercrombie’s method [2]. Prior to testing for differences between Cont and Et groups, the section counts were first averaged per individual case. Differences between groups were evaluated by means of ANOVA. The BAC measured in newborn rats at the end of each session of ethanol administration ranged from 226 to 278 mg/100 ml (mean ^ SD 251.2 ^ 21.4 mg/100 ml). No significant differences between Et and Cont cases were observed in the curve of weight gain during the first three postnatal weeks. At the two studied ages, both in Et and Cont animals, most immunoreactive elements observed in the sensori-motor cortex were represented by pyramidal neurons, mainly located in the infragranular layers, with a strong preference for layer 5. As a general feature, the location of the immunoreaction product was mainly perinuclear; immunoreactive apical or basal dendrites were frequently observed in pyramidal neurons of Et animals (inset of Fig. 1B) and were rare in Cont animals. Supragranular neurons, especially in the deep part of layer 3, were more numerous in Et animals than in Cont cases. Immunopositive elements with the size and/or morphological appearance of glial cells were not observed. Both at P10 and P20, the main difference between Cont and Et cases was represented by the tangential location of immunolabelling. Most labelled neurons in Cont animals were located in the primary somatosensory cortex (S1), whereas the medially located primary motor cortex (M1) featured only sparse labelled cells (Figs. 1A and 2A). On the other hand, layers 5 and 6 of M1 in Et animals were characterized by the presence of a great number of p75 NTR immunoreactive profiles (Figs. 1B and 2A). These findings were confirmed by the counts of immunoreactive neurons shown in Fig. 2B. In M1, both at P10 and P20, the mean number of p75 NTR positive neurons per section was higher in Et than in Cont cases and the difference was highly significant. As for S1, a highly significant difference between Cont and Et animals was found only at P10.
The tangential and radial distribution of p75 NTR positive neurons observed in the present study are in general agreement with previous reports dealing with the expression of neurotrophin receptors in the sensori-motor cortex of mature rats [15,18]. Our study is not focused on the expression of p75 NTR during normal cortical maturation, yet a certain degree of downregulation occurring during postnatal life may be suggested by the present data. However, the transient expression of p75 NTR has been described for the thalamic ventrobasal nucleus [5], while in the rodent cerebral cortex its level seems to be kept constant through the development [19]. The main result of the present study is the demonstration of the strong and prolonged increase of p75 NTR immunopositivity in the sensori-motor cortex following exposure to ethanol during early postnatal life. An upregulation of p75 NTR as a consequence of early exposure to neurotoxic agents has already been reported [10] and might represent a general feature of several disorders of the nervous system (for review see Ref. [6]). Conversely, other reports pointed out that ethanol is able to reduce the expression of the low-affinity neurotrophin receptor [8,21]. Such discrepancies can be explained by the different experimental strategies (i.e. cell cultures [21]) or the different considered structure (cerebellar Purkinje cells [8]). A conservative interpretation for the increased expression of p75 NTR must necessarily take into account its postulated dual role during neurogenesis. On one hand, p75 NTR is thought to mediate apoptotic neuronal death [4, 13]. Actually, the exposure to ethanol during early postnatal life is able to induce massive apoptosis [12,17]. In addition, the prevalent location of p75 NTR immunoreactive neurons in layer 5, as described in our study, is consistent with the distribution of caspase 3 immunoreactivity reported in one of the studies dealing with the apoptotic effect of ethanol [17]. If p75 NTR represents a marker of susceptibility to apoptosis, the long-lasting overexpression observed in the present study might indicate that cortical cells are prone to apoptotic death well after ethanol withdrawal. This is in agreement with a recent report by Mooney and Miller [16], demonstrating that prenatal exposure to ethanol is able to reduce the cortical bcl-2/bax ratio during the first two postnatal weeks, thus providing a long-lasting permissive step for neuronal death. However, the complex interplay among p75 NTR, Trks, and apoptosis (reviewed in Ref. [4]) should also be taken into account, especially considering the reported colocalization of p75 NTR and trkA within the same cortical neurons [15]. At the other end of the spectrum, the low-affinity neurotrophin receptor can be regarded as a key molecule for neuronal plasticity, mainly because of its capability of modulating neurite outgrowth [22]. According to this view, it is worth mentioning that the extent and complexity of dendritic arbors is increased in layer 5 neurons of the sensori-motor cortex after early exposure to ethanol [14].
A. Toesca et al. / Neuroscience Letters 342 (2003) 89–92
91
Fig. 1. Microphotographs showing the p75 NTR immunoreactivity in the primary motor cortex of a P20 Cont (A) and a P20 Et case (B). Note the higher number and the wider radial distribution of immunopositive elements in the Et case. The immunolabelling of apical and basal dendrites is shown at higher magnification in the inset at the top-left of (B). Scale bar: 50 mm for (A), 40 mm for (B), and 20 mm for the inset.
Fig. 2. (A) Schematic drawing of the p75 NTR immunoreactivity in two representative sections of the sensori-motor cortex from a P10 Cont and a P10 Et case. Each symbol represents one labelled cell. M1, primary motor cortex; S1, primary somatosensory cortex. (B) Bar graphs showing the mean number of labelled cells per section in P10 and P20 cases. T bars represent standard deviations. **P , 0:01.
92
A. Toesca et al. / Neuroscience Letters 342 (2003) 89–92
Our results prompt further investigation to clarify the role of the low-affinity neurotrophin receptor in FAS, possibly by assessing the susceptibility to early ethanol exposure in mice lacking p75 NTR.
[11]
[12]
Acknowledgements This work was partly supported by a grant to A.G. from the Alzheimer Research Program, funded by the Italian Ministry of Public Health.
[13]
[14]
References [1] E.L. Abel, R.J. Sokol, Fetal alcohol syndrome is now a leading cause of mental retardation, Lancet 2 (1986) 1222. [2] M. Abercrombie, Estimation of nuclear population from microtome sections, Anat. Rec. 94 (1946) 239–247. [3] F. Angelucci, M. Fiore, C. Cozzari, L. Aloe, Prenatal ethanol effects on NGF level, NPY and ChAT immunoreactivity in mouse entorhinal cortex: a preliminary study, Neurotoxicol. Teratol. 21 (1999) 415–425. [4] G.L. Barrett, The p75 neurotrophin receptor and neuronal apoptosis, Prog. Neurobiol. 61 (2000) 205 –229. [5] D.P. Crockett, S.L. Harris, M.D. Egger, Neurotrophin receptor (p75) in the trigeminal thalamus of the rat: development, response to injury, transient vibrissa-related patterning, and retrograde transport, Anat. Rec. 259 (2000) 446–460. [6] G. Dechant, Y. Barde, The neurotrophin receptor p75NTR: novel functions and implications for diseases of the nervous system, Nat. Neurosci. 5 (2002) 1131–1136. [7] J. Dobbing, J. Sands, The brain growth spurt in various mammalian species, Early Hum. Dev. 3 (1979) 79– 84. [8] D.P. Dohrman, J.R. West, N.J. Pantazis, Ethanol reduces expression of the nerve growth factor receptor, but not nerve growth factor protein levels in the neonatal rat cerebellum, Alcohol. Clin. Exp. Res. 21 (1997) 882 –893. [9] K.E. Dow, R.J. Riopelle, Ethanol neurotoxicity: effects on neurite formation and neurotrophic factor production in vitro, Science 228 (1985) 591–593. [10] M. Fiore, L. Aloe, C. Westenbroek, T. Amendola, A. Antonelli, J. Korf, Bromodeoxyuridine and methylazoxymethanol exposure during
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
brain development affects behavior in rats: consideration for a role of nerve growth factor and brain derived neurotrophic factor, Neurosci. Lett. 309 (2001) 113–116. A. Granato, M. Santarelli, A. Sbriccoli, D. Minciacchi, Multifaceted alterations of the thalamo-cortico-thalamic loop in adult rats prenatally exposed to ethanol, Anat. Embryol. 191 (1995) 11–23. C. Ikonomidou, P. Bittigau, M.J. Ishimaru, D.F. Wozniak, C. Koch, K. Genz, M.T. Price, V. Stefovska, F. Horster, T. Tenkova, K. Dikranian, J.V. Olney, Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome, Science 287 (2000) 1056–1060. V. Mamidipudi, M.W. Wooten, Dual role for p75NTR signaling in survival and cell death: can intracellular mediators provide an explanation?, J. Neurosci. Res. 68 (2002) 373 –384. M.W. Miller, N.L. Chiaia, R.W. Rhoades, Intracellular recording and injection study of corticospinal neurons in the rat somatosensory cortex: effect of prenatal exposure to ethanol, J. Comp. Neurol. 297 (1990) 91– 105. M.W. Miller, A.F. Pitts, Neurotrophin receptors in the somatosensory cortex of the mature rat: co-localization of p75, trk, isoforms and cneu, Brain Res. 852 (2000) 355–366. S.M. Mooney, M.W. Miller, Effects of prenatal exposure to ethanol on the expression of bcl-2, bax and caspase 3 in the developing rat cerebral cortex and thalamus, Brain Res. 911 (2001) 71–81. J.W. Olney, T. Tenkova, K. Dikranian, Y. Qin, J. Labruyere, C. Ikonomidou, Ethanol-induced apoptotic neurodegeneration in the developing C57BL/6 mouse brain, Dev. Brain Res. 133 (2002) 115 –126. A.F. Pitts, M.W. Miller, Expression of nerve growth factor, p75, and trk in the somatosensory and motor cortices of mature rats: evidence for local trophic support circuits, Somatosens. Mot. Res. 12 (1995) 329 –342. F.M. Rossi, R. Sala, L. Maffei, Expression of the nerve growth factor receptors trkA and p75NTR in the visual cortex of the rat: development and regulation by the cholinergic input, J. Neurosci. 22 (2002) 912–919. W.D. Ruwe, L. Bauce, W.W. Flemons, W.L. Veale, Q.J. Pittman, Alcohol dependence and withdrawal in the rat. An effective means of induction and assessment, J. Pharmacol. Methods 15 (1986) 225 –234. G.K. Seabold, J. Luo, M.W. Miller, Effect of ethanol on neurotrophinmediated cell survival and receptor expression in cultures of cortical neurons, Dev. Brain Res. 108 (1998) 139–145. T. Yamashita, K.L. Tucker, Y.A. Barde, Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth, Neuron 24 (1999) 585 –593.