Timed changes of synaptic zinc, synaptophysin and MAP2 in medial extended amygdala of epileptic animals are suggestive of reactive neuroplasticity

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Timed changes of synaptic zinc, synaptophysin and MAP2 in medial extended amygdala of epileptic animals are suggestive of reactive neuroplasticity Germán l. Pereno, Carlos A. Beltramino⁎ Cátedra de Neurofisiología y Psicofisiología, Facultad de Psicología, Universidad Nacional de Córdoba, 5000, Córdoba, Argentina Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra, Friuli 2434, 5016, Córdoba, Argentina

A R T I C LE I N FO

AB S T R A C T

Article history:

Repeated seizures induce permanent alterations of the brain in experimental models and

Accepted 29 January 2010

patients with intractable temporal lobe epilepsy (TLE), which is a common form of epilepsy

Available online 6 February 2010

in humans. Together with cell loss and gliosis in many brain regions, synaptic reorganization is observed principally in the hippocampus. However, in the amygdala this

Keywords:

synaptic reorganization has been not studied. The changes in Zn density, synaptophysin

Synaptogenesis

and MAP2 as markers of reactive synaptogenesis in medial extended amygdala induced by

Amygdala

kainic acid (KA) as a model of TLE was studied. Adult male rats (n = 6) were perfused at

Sprouting

10 days, 1, 2, 3 and 4 months after KA i.p. injection (9 mg/kg). Controls were injected with

Temporal lobe epilepsy

saline. The brains were processed by the Timm's method to reveal synaptic Zn and analyzed by densitometry. Immunohistochemistry was used to reveal synaptophysin and MAP2 expression. A two-way ANOVA was used for statistics, with a P < 0.05 as a significance limit. Normal dark staining was seen in all medial extended amygdala subdivisions of control animals. At 10 days post KA injection a dramatic loss of staining was observed. A slow but steady recovery of Zn density can be followed in the 4 month period studied. Parallel, from 10 days to 2 months stronger synaptophysin expression could be observed, whereas MAP2 expression increased from 1 month with peak levels at 3–4 months. The results suggest that a process of sprouting exists in surviving neurons of medial extended amygdala after status epilepticus and that these neurons might be an evidence of a reactive synaptogenesis process. © 2010 Published by Elsevier B.V.

⁎ Corresponding author. Cátedra de Neurofisiología y Psicofisiología, Facultad de Psicología, Universidad Nacional de Córdoba, Enfermera Gordillo esquina Enrique Barros, Ciudad Universitaria, 5000, Córdoba, Argentina. Fax: +54 351 433 4119. E-mail address: [email protected] (C.A. Beltramino). Abbreviations: BSTM, medial division of the bed nucleus of the stria terminalis; BSTMa, anterior medial bed nucleus of the stria terminalis; BSTMp, posterior medial bed nucleus of the stria terminalis; KA, kainic acid; MeA, medial amygdaloid nuclei; MeAD, medial amygdaloid nuclei, anterodorsal division; MeAV, medial amygdaloid nuclei, anteroventral division; MePD, medial amygdaloid nuclei, posterodorsal division; MePV, medial amygdaloid nuclei, posteroventral division; MEXA, medial extended amygdala; SE, status epilepticus; TLE, temporal lobe epilepsy 0006-8993/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.brainres.2010.01.087

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1.

Introduction

Temporal lobe epilepsy (TLE), the most common type of epilepsy in adult humans, is characterized clinically by the progressive development of spontaneous recurrent seizures of temporal lobe origin and pathologically by hippocampal neuron loss and mossy fiber sprouting (Tauck and Nadler, 1985; Sutula et al., 1989). Studies performed in the last 2 to 3 decades have shown that “seizures beget seizures,” triggering a cascade of events that transform a naive network into one that generates seizures (Ben-Ari, 2008). The events that follow epilepsy seizures are not restricted to the immediate period, but include long-term alterations and synaptic rearrangements, which have an impact on the brain circuit's mode of operation, as seen in animal models of temporal lobe epilepsy (Kienzler et al., 2009; Leite et al., 2005; Pitkänen et al., 2002; Sloviter et al., 2006; Zhang et al., 2002) and epileptic humans (Andrade-Valença et al., 2008; Bausch, 2005; Proper et al., 2000; Swartz et al., 2006). However, the substrate for seizure generation is distributed over several limbic structures, including the amygdaloid complex (Aroniadou-Anderjaska et al., 2008; Bertram, 2009; Majores et al., 2007; McIntyre and Gilby, 2008; Pitkänen et al., 1998). There have been several studies on the organization of the amygdaloid complex of the temporal lobe (Price et al., 1987; Swanson, 2003). A neuroanatomical theory on this structure has been proposed by de Olmos et al. (2004), termed the extended amygdala. On the basis of experimental anatomical data this construct was divided into the medial (MEXA) and central extended amygdala. The medial division of the extended amygdala is a large complex of nuclei recognizable by their extensive connectional relations with the medial hypothalamus where they can influence hormonal and somatomotor aspects of behavior and emotional states. It participates in the processing of pheromonal signals concerned with the control and modulation of neuroendocrine, appetitive, reproductive and emotional behaviors (Beltramino and Taleisnik, 1985; Commins and Yahr, 1984). A great number of studies with animal models have revealed that the amygdala is sensitive to changes induced by status epilepticus (SE) (Gurbanova et al., 2008; Qashu et al., 2009; Riba-Bosch and Pérez-Clausell, 2004). However, in spite of the fact that neurons die in the MEXA, in the literature there are no reports showing synaptic plasticity in the amygdala similar to that seen in the hippocampus. A way of evaluating these changes is by studying Timm histochemistry, the expression of synaptophysin and microtubule-associated protein 2 (MAP2). The Timm method (Danscher, 1981) a histochemical technique that selectively labels synaptic terminals of rich zinc content, have been used to show synaptic reorganization mossy fiber in the hippocampus (Cross and Cavazos, 2007; Kienzler et al., 2009; Sloviter et al., 2006). Synaptophysin is the major integral membrane protein in pre-synaptic vesicles thought to be involved in vesicle formation and exocytosis (Valtorta et al., 2004). It is used as a marker for synaptic activity (Chambers et al., 2005; Derksen et al., 2007; Smith et al., 2000). MAP2 is dendrite specific and

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holds the microtubule-based cytoskeleton together (Hering and Sheng, 2001). MAP2 expression has been extensively used as a tool of synaptic plasticity (Briones et al., 2006; Derksen et al., 2007; Jalava et al., 2007). In this study, we used these techniques to determine whether after kainic acid (KA) induced neurodegeneration there exists a synaptic reorganization in neurons of the medial extended amygdala.

2.

Results

2.1. Development of status epilepticus after systemic injection of kainic acid With the dose used in this study, all animals showed severe limbic seizures, reaching level 4 of the scale described (full limbic convulsions with salivation, rearing, bilateral clonus of the upper extremities and loss of balance); and showed severe neuronal death in MEXA (Fig. 1).

2.2.

Timm histochemistry

Normal Timm staining was observed in control animals of all subdivisions of MEXA (Figs. 2 and 3). Ten days after KA injection a dramatic loss of staining was detected, also observable in the 1 month post injection group. A slow but persistent recovery of Timm reaction density was thereafter observed. It was clearer in experimental groups from the second to the fourth month of survival, with peak levels of intensity at 3 months after injection of KA, as compared with control animals. Differences between MeA subdivisions could also be detected. Thus, although in all subdivisions of MeA the peak of Timm staining is at 3 months, in MePD and MePV this staining is greater than in the other subdivisions and when compared with control animals the differences are statistically significant. Significant differences were found in the two-way ANOVA by comparisons between control vs experimental animals, with peak levels at 3 months after KA injection in all subdivisions of MEXA (Table 1). In the MeA, two-way ANOVA showed significant treatment and time effects after KA injections for Timm staining. In the MeAV there were significant effects of treatment group F(1,50) = 273.49, P < 0.001 and time after KA injection F(4,50) = 95.94, P < 0.001, as well as significant treatment × time interaction F(4,50) = 77.70, P < 0.001. The MeAD showed significant effects of treatment F(1,50) = 475.80, P < 0.001, time after KA injection F(4,50) = 143.03, P < 0.001 and interaction F(4,50) = 154.61, P < 0.001. The MePD showed significant effects of treatment group F(5,70) = 883.90, P < 0.001 and time after KA injection F(4,50) = 268.88, P < 0.001, as well as significant treatment × time interaction F(4,50) = 316.49, P < 0.001. ANOVA for the MePV also showed significant effects of treatment group F(1,50) = 158.56, P < 0.001 and time after KA injection F(4,50) = 106.30, P < 0.001, as well as significant treatment × time interaction F(4,50) = 67.89, P < 0.001. The results for the BSTM subdivisions showed a similar pattern of Timm staining in the MeA nucleus at the different survival times after KA injection (Fig. 3). However, differences

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Fig. 1 – Neuronal death in the rat medial amygdaloid nucleus and medial bed nucleus of the stria terminalis. Cresyl violet staining of a coronal brain section (5×) exemplifying the location of the rat MePV and MePD (panel A). Panel B: histological sections (5×) of the same view staining with the amino-cupric-silver technique showing neuronal death after SE. The insert shows high magnification (20×) of a coronal section of experimental rats. Cresyl violet staining of a coronal brain section (5×) exemplifying the location of the rat BSTMa (panel C). Panel D: histological sections (5×) of the same view staining with the amino-cupric-silver technique showing neuronal death after SE. The insert shows high magnification (20×) of a coronal section of experimental rats. Scale bar in B = 500 µm. Scale bar in insert = 200 µm. BSTMa: anterior medial bed nucleus of the stria terminalis; MePD: medial amygdaloid nuclei, posterodorsal division; MePV: medial amygdaloid nuclei, posteroventral division; SE: status epilepticus.

between BSTM subdivisions could also be observed. Thus, the BSTMp showed more Timm staining. A two-way ANOVA for Timm staining in the BSTMa also detected significant effects of treatment group F(1,50) = 447.69, P < 0.001 and time after KA injection F(4,50) = 128.07, P < 0.001, as well as significant treatment × time interaction F(4,50)=149.10, P < 0.001. In the BSTMp, there were significant effects of treatment group F(1,50) = 134.80, P < 0.001 and time after KA injection F(4,50) =

155.18, P < 0.001, as well as a significant treatment × time interaction F(4,50) = 142.21, P < 0.001.

2.3.

Synaptophysin and MAP2 immunostaining

Controls were virtually unstained with synaptophysin. However, weak labeling was recognized occasionally (Figs. 4A and D). Synaptophysin expression was strongly induced in

Fig. 2 – Timm staining in medial amygdaloid nucleus. Histological sections of the posterodorsal medial amygdaloid and posteroventral medial amygdaloid nucleus of male rats (5×) of a coronal section of control and experimental at different survival times staining with the Timm technique. Scale bar = 500 µm. MePD: medial amygdaloid nuclei, posterodorsal division; MePV: medial amygdaloid nuclei, posteroventral division.

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Fig. 3 – Timm staining in medial bed nucleus of the stria terminalis. Histological sections of the anterior medial bed nucleus of the stria terminalis of male rats (5×) of a coronal section of control and experimental at different survival times staining with the Timm technique. Scale bar = 500 µm. BSTMa: anterior medial bed nucleus of the stria terminalis.

animals at 10 days after KA injection and it was maintained until the 2 month group. This expression was abundant and in this survival times synaptophysin is observed like nuclear marcation (Figs. 4B and E). At 3 and 4 months, synaptophysin return to baseline levels. Differences between MeA and BSTM subdivisions could also be observed. Synaptophysin expression was greater in MePD, revealing a distribution pattern located ventrally to the fibers of the stria terminalis (Fig. 4B). Moreover, BSTMp also showed more synaptophysin expression than BSTMa (Fig. 4E), especially at 1 and 2 months. Weak staining for MAP2 was observed in control animals (Figs. 4A and D). MAP2 expression was greater in experimental animals at 10 days and it was augmented until 3 to 4 months (Figs. 4C and F). No differences between MeA or BSTM subdivisions could be observed to MAP2 expression.

3.

Discussion

The present experiments were designed to provide a detailed view of the temporal and spatial distribution of status epilepticus-induced Timm stained, synaptophysin and MAP2 expression in regions of the rat MEXA. Towards these efforts, SE was induced with kainate and the differently stained were analyzed 10 days to 4 months thereafter.

There were four major findings in this study. First, a dramatic loss of staining was detected in animals from 10 days to 2 months after KA injection, also observable in the 1 month post injection group. Second, a slow but continuous process of recovery of the Zn-rich terminals at the longer survival times (3 to 4 months) is indicative of an active mechanism of neuroplasticity in this TLE model. Third, a stronger induction of synaptophysin was observed from 10 days to 2 months, whereas MAP2 expression was evident from 10 days with peak levels at 3 to 4 months. Fourth, those stained for Timm and synaptophysin varied substantially between the different nuclei and their subdivisions.

3.1.

Neuronal death-induced axonal sprouting

Axonal sprouting is a fundamental characteristic in the developing brain and is an essential cellular process for the establishment of connections and neural circuits, but the idea prevailed until a few decades ago that this mechanism did not continue in the adult brain. This was challenged by a series of experimental observations by Steward et al. (1974), who demonstrated that some nervous fibers in the hippocampus had the capability of axonal sprouting and rearrangement of synaptic connectivity in response to injury of the entorhinal cortex (Sutula, 2002).

Table 1 – Effect of seizure activity induced by the injection of kainic acid on Timm staining in the medial extended amygdala of adult male rats when compared against control animals. Date are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. MEAX regions

Time after KA injection Control

10 D

1M

2M

3M

MeA MeAV MeAD MePV MePD

76.83 ± 3.43 75.55 ± 1.95 74.16 ± 1.12 80.28 ± 2.54

15.66 ± 0.65*** 21.48 ± 1.2*** 20.75 ± 1.7*** 18.23 ± 1.1***

33.5 ± 2.1*** 45.45 ± 1.5*** 43.26 ± 1.9*** 29.5 ± 2.3***

62.66 ± 1.54** 75.16 ± 2.2* 72.5 ± 3.39* 69.5 ± 1.38***

79.5 ± 1.05 78.5 ± 1.91 85.16 ± 1.71** 84.16 ± 0.43*

BSTM BSTMa BSTMp

78.5 ± 3.43 77.5 ± 1.61

15.66 ± 0.92*** 13.5 ± 1.8***

36.83 ± 2.04*** 39.33 ± 3.15***

66.66 ± 1.74** 80 ± 1.85**

76.83 ± 2.46 86.66 ± 0.46**

4M 76.84 ± 2.22 72.16 ± 0.77 73.83 ± 2.14 82.5 ± 0.83

73.5 ± 1.54 86.8 ± 3.05**

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Fig. 4 – Synaptophysin and MAP2 expression. Histological sections of the posterodorsal medial amygdaloid nucleus and anterior medial bed nucleus of the stria terminalis of male rats (5×) of a coronal section of control and experimental at different survival times staining with immunohistochemistry for synaptophysin and MAP2. The inserts show high magnification (20×) of a coronal section of experimental male rats. Panels A–D: control animals; panels B–E: 1 month survival; panels C–F: 3 month survival. Scale bar in A (for 5×) = 500 µm. Scale bar in high magnification (for 20×) = 200 µm.

Moreover, it has been demonstrated that seizures induce neuronal death which is followed, in non-lesioned neurons, by the triggering of plastic phenomena (Bouilleret et al., 1999; Jiao and Nadler, 2007; Lothman and Bertram, 1993). Numerous reports of these phenomena have been communicated from studying the mossy fibers in the hippocampus (Jiao and Nadler, 2007; Kienzler et al., 2009; Rakhade and Jensen, 2009; Sutula and Dudek, 2007). The reinnervation of the mossy fibers was detected by applying Timm histochemistry, due to its facility to detect Zn changes in the hippocampus. The results obtained in this study showed that there are important changes in Timm staining, not only in the hippocampus but also in the MEXA. The first result obtained in this study agrees with previous reports, in which the MEXA presents numerous Zn-rich neurons (Cunningham et al., 2007; Riba-Bosch and PérezClausell, 2004). The second result was observed in experimental animals from 10 days to 2 months of survival after the KA induction of SE. In these animals, an important reduction of

staining of pre-synaptic terminals rich in Zn was observed. It has been suggested that the homeostasis of Zn in the brain may be affected by the KA-induced SE, with a marked decrease of synaptic Zn being found in animals 24 h after KA injection, due to an excessive liberation of Zn together with glutamate induced by intense brain activity during SE (Takeda et al., 2003). No study was found in the current bibliography measuring Zn levels after SE in animals with longer survival periods, although it has been suggested that the recovery of Zn in the synaptic vesicle occurs in a few hours (López-García et al., 2001). Thus, the loss of staining observed in this study seems to be due to the massive neuronal death produced by SE, and therefore, the important loss of pre-synaptic terminals degenerating after SE. The third result obtained is that, in animals from 2 months to 4 months, there is a slow but marked recovery of Zn-rich pre-synaptic terminals. Based on these results, it is suggested that in the MEXA there is a process of reactive synaptogenesis (at least the presynaptic element). However, it is not possible to conclude that

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this reactive synaptogenesis is pathological or aberrant since this technique shows non-affected as well as new putative glutamate terminals. Further studies are necessary on this topic. The results here obtained reinforce the hypothesis that, after SE with an already established epileptic syndrome characterized by recurrent epileptic seizures, the neural circuits affected change through diverse mechanisms of neuroplasticity, of which reactive synaptogenesis is the one that this study was able to determine. Whether the plastic changes observed in our experimental animals in the MEXA and in areas of the hippocampus underlie the process of epileptogenesis and/or drug-resistant epilepsy is more than what is possible to evaluate in the present study. Interestingly, dramatic neuronal death occurs in the MEXA after KA-induced SE. It is followed by a latency period where the animals do not exhibit any seizures, and, from the second month, the animals begin to show spontaneous recurrent seizures. At histological levels, this is matched by an increase of Timm staining as a marker of pre-synaptic terminals and therefore of synaptic reorganization in the amygdala. These changes might be the basis not only of a process of epileptogenesis, but also of symptoms of TLE.

3.2. Synaptophysin and MAP2 expression as a marker of neural plasticity Synaptophysin has been widely used for the visualization of the pre-synaptic membranes, as functional marker of synapses and as sign of synaptogenesis in animal models and in human beings (Chambers et al., 2005; Derksen et al., 2007; Lambert et al., 2005; Smith et al., 2000). In the present study synaptophysin expression increases dramatically from 10 days up to 2 months after the induction of SE. It might hypothesized that the massive synaptophysin expression would be a compensatory mechanism of the neurons that, in the massive loss of synapses, upregulates the production of this protein in order that rapidly new synapses are formed. Further studies are necessary with other techniques than immunohistochemistry (for example in situ hybridization). In the present study the results show that the neurons of the MEXA are capable of an expression of novo of synaptophysin between 10 days and 2 months of survival. It is possible to hypothesize that the presence of this protein is a transitory phenomenon that is related to the growth of new dendrites, axons and the formation of new synapses. The neural activity in the MEXA of animals that suffered a SE evidently exceeds that of animals that did not suffer this stress. In this case, the increase of synaptophysin would be the direct result of this high neural activity. Nevertheless, the way of expression of this protein also can reflect the presence of new synapses. It is possible that in the period of 10 days up to 2 months, the neurons that survived the SE produce elements of synaptic connections pre and postsynaptic looking for new connections. More connections can be created than those that are necessary and only some to remain established. This would result in a decrease of synapses from 2 months, which is reflected in the decrease of synaptophysin to baseline levels, being that this model of

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synaptogenesis is similar to the natural process of pruning of synapses that happens during the development. It is evident that other proteins and factors start intervening for the stabilization of the new created synapses, corresponding to this fact is what was obtained by the Timm technique, where it found that the peak is up to 3 months. Similar results were obtained by the MAP2 expression, which was in a gradual increase with the peak at 3 to 4 months. Further studies including the observation of the tissue after an SE at electronic microscopy will give indications of these hypotheses. On the other hand, MAP2 expression has been used in many animal models, physiological and pathological (Benice et al., 2006; Li et al., 1998). The changes shown in this study can be related to the synaptic reorganization that happens after the induction of the SE with KA. Thus, many dendrites and synaptic terminals degenerate in the MEXA from the first hours and presumably until several days. Then, the neurons that survived, in a compensatory attempt post-injury, upregulated synaptophysin and MAP2 expression. However, it's not possible to rule out other proteins involved in the synaptogenesis such as the efrinas, Syn-CAM and neuroligina (McAllister, 2007). If MAP2 is important for the dendritic growth, it is hoped that its levels increase during the plastic changes that exist after damage such as SE. Thus, from the month after KA injection, there appears an increase of MAP2 expression greater than in control animals, supported in the time with peak levels in animals of 3 to 4 months after SE. Strikingly, the peak of MAP2 expression is coincident with the peak of Timm stained. These observations suggest that in the MEXA of epileptic animals, the protein MAP2 contributes to the stabilization of dendrites of neurons of this structure after the axonal denervation and, eventually it favors the development of dendrites of neurons that resisted the SE, new dendrites and dendritic spine that will contribute to the reorganization of new cerebral circuits by means of the establishment of new synapses. Advances in our knowledge of the intrinsic mechanisms of brain plasticity and reactive synaptogenesis will help us to understand the process of epileptogenesis and the recovery of damaged or lost functions in the brains of epileptic people, and thus allow us to implement favorable clinical and pharmacological interventions.

4.

Experimental procedures

4.1.

Animals

Male Wistar rats weighing 230–250 g were used in this study. All rats were housed in individual cages under a controlled environment (temperature 20 °C, humidity 50–60%, lights on 07:00–19:00). Standard food pellets and tap water were available ad libitum. All procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised 1996 and efforts were made to minimize the number of animals used and their suffering.

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Induction of status epilepticus with kainic acid

Kainic acid (Sigma K-250) was dissolved in 0.9% NaCl and injected i.p. (9 mg/kg). Control animals received an equal volume of 0.9% NaCl; n=36. The development and duration of seizure activity after KA injection was observed and video filmed for 3 h. The behavioral development of status epilepticus was classified using the scale described by Lothman and Collins (1981). Only those animals which reached the fourth stage of the scale were considered for the study.

4.3.

Timm histochemistry

Several survival times were chosen to study synaptic plasticity over time. The animals were perfused starting 10 days after KA injections. The remaining animals were fixed after successive 1 month survival periods until the 4-month period of the study was completed. At the appropriate time, rats from experimental and control groups were deeply anesthetized with a dose of pentobarbital (40 mg/kg, i.p.). The brains were perfused and fixed by transcardial slow perfusion with the following solutions: 50 mL sodium sulfide solution (Na2S, 11.7 g; NaH2PO4, 11.9 g in 1000 mL distilled water) followed by 50 mL glutaraldehyde (3% in 0.1 M phosphate buffer) and followed again by 300 mL sodium sulfide solution. The brains were removed and post fixed in glutaraldehyde for 1 h and left overnight in a 30% (w/v) solution of sucrose in fixative; they were then cut on a cryostat (30 µm thick) and sections were placed on glass slides and stored at −70 °C. Sagittal frozen sections were developed in the dark for 70– 100 min in a 12:6:2 mixture of gum arabic (20% w/v), hydroquinone (5.6% w/v; H7148; Sigma, St. Louis, MO, USA), citric acid–sodium citrate buffer with 1.5 mL of a silver lactate solution (17% w/v; Fluka, Buchs, Switzerland) at 26 °C (Danscher, 1981). They were then rinsed in 5% thiosulphate for 12 min, immersed in xylene and coverslipped in DPX (Fluka, Buchs, Switzerland).

4.4.

4.6.

Silver staining

The sections were stained following the protocol of the aminocupric-silver technique. This method was chosen as being the latest and most sensitive version of the technique, considered most specific for neurotoxicological analysis of the nervous system (de Olmos et al., 1994).

4.7.

Statistical analysis

For the densitometric analysis, data were measured with a twoway ANOVA followed by a post-hoc LSD Fisher test, and the results were expressed as the mean ± SEM, with P < 0.05 as the limit of significance of differences between groups.

Quantitative analysis of Timm histochemistry

Brain coronal sections were analyzed with an Axioplan microscope with a Leica video camera attached and connected to a PC. Densitometric analysis was made using the Scion Image program for Windows (Scion Corp. 2000, based on NIH, USA), with a magnification of 10× and the resulting measures were expressed as gray percentage units and were statistically analyzed. Using the Paxinos and Watson stereotaxic Atlas (2007) for the rat brain, selected sections in the following planes with respect to the bregma were used for densitometric analysis and synaptophysin and MAP2 expression: −2.30 for MeAD and MeAV; −3.14 for MePD and MePV. For the BSTM we selected sections in the following planes with respect to the bregma: −0.30 for BSTMa and −0.80 for BSTMp. Densitometric analysis was performed by tracing the borders of each subregion of the MeA and BSTM nucleus, using the Scion Program.

4.5.

of 0.8% sucrose, 0.8% ClNa, and 0.4% glucose followed by 4% paraformaldehyde in 0.2 M borate buffer pH 7.4. The brains were kept in the skull overnight at 4 °C, and then removed and placed in 30% sucrose until sunk. Brains were then cut coronally at 40 µm with a freezing microtome and sections were collected serially in the same fixative solution. To visualize the possible colocalization of MAP2 and synaptophysin in the same cells, dual immunohistochemistry staining was used. A series of tissue sections was stained using the peroxidase–antiperoxidase method followed by the avidin–biotin procedure. Tissue sections were incubated with the primary antisynaptophysin antibody (1:1000; Santa Cruz Biotechnology) for 72 h, followed by biotinylated goat antirabbit IgG (1:200, Vector Laboratories, Burlingame, CA, USA) for 2 h at room temperature and avidin–biotin complex reagent for 2 h (1:200, Vector Laboratories). The sections were developed in 3′3′-diaminobenzidine (0.2 mg/mL; Sigma, USA) with 0.02% cobalt chloride (Fisher Scientific, USA) for a black color. After that, the same sections were incubated with the primary anti-MAP2 antibody (1:200; Calbiochem) for 72 h, followed by the same protocol described above and developed in 3′3′-diaminobenzidine without cobalt chloride for a brown color.

Perfusion and histology for immunohistochemistry

The rats were anesthetized i.p. with 30% chloral hydrate and perfused transcardially with a blood-washing solution consisting

Acknowledgments We thank Soledad de Olmos for providing technical support. This study was supported by grants from SECyT and CONICETArgentina.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.brainres.2010.01.087.

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