Alterations in the dendritic morphology of prefrontal pyramidal neurons in adult rats after blockade of NMDA receptors in the postnatal period

Brain Research 1062 (2005) 166 – 170 www.elsevier.com/locate/brainres

Short Communication

Alterations in the dendritic morphology of prefrontal pyramidal neurons in adult rats after blockade of NMDA receptors in the postnatal period Krzysztof We˛dzony*, Katarzyna Fijaa, Marzena Mac´kowiak Institute of Pharmacology Polish Academy of Sciences, Department of Pharmacology, Laboratory of Pharmacology and Brain Biostructure, Sme˛tna 12 Street, 31-343 Krako´w, Poland Accepted 25 September 2005 Available online 28 October 2005

Abstract The present study assessed whether the blockade of NMDA receptors in the postnatal period, used to model the symptoms of schizophrenia altered morphology of pyramidal neurons in the medial prefrontal cortex of rats. CGP 40116, an antagonist of NMDA receptors, was given postnatally (days 1 – 21 after birth). The analysis of the morphology of pyramidal neurons visualized by the Golgi-Cox technique revealed that the exposure to an antagonist of NMDA receptors in the postnatal period diminished the length of basilar dendrites, while that of apical dendrites remained unchanged. The number of dendritic branches and the spine density remained unchanged. It is concluded that the blockade of NMDA receptors in the postnatal period only partially models morphological changes in pyramidal neurons of the medial prefrontal cortex, which are observed in some cases of schizophrenia. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Excitatory aminoacids: anatomy and physiology Keywords: Postnatal period; NMDA receptor; Animal models of schizophrenia; Medial prefrontal cortex; Golgi-Cox impregnation

Cytoarchitectural alterations affecting cortical connectivity have been observed in the brains of schizophrenic patients [1,27]. In particular, an atrophy of the basilar dendrites of cortical pyramidal neurons [3,16], a decrease in the density of dendritic spines [13], reduction in the volume of neuropil [27], alterations in the expression of markers of GABA-ergic interneurons [18] and a decrease in the density of tyrosine hydroxylase-positive fibers [1] have been reported. According to a current hypothesis, aberrant neural development underlies the above-mentioned morphological features of schizophrenia [34]. Since NMDA receptors are engaged in several developmental events such as proliferation, migration, cell death, survival and formation of neural circuits (for review, see [6]) their pharmacological blockade has been used to model schizophrenia in experimental Abbreviations: MPC, medial prefrontal cortex * Corresponding author. Fax: +48 12 6374500. E-mail address: [email protected] (K. We˛dzony). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.09.012

animals. Typical schizophrenia-like alterations in the behavior of adult animals treated postnatally with NMDA antagonists are hypermotility, impaired learning, deficits of sensorimotor gating [8,30]. Changes in the metabolism of dopamine, the density of dopaminergic receptors [7,8] and the decrease in the density of dopaminergic fibers in the medial prefrontal cortex (MPC) [31] have also been observed. It is not known, however, whether the abovementioned behavioral changes are accompanied by any alteration in brain morphology. Thus, the main aim of the present study was to find out whether the blockade of NMDA receptors in the postnatal period by administration of CGP 40116 – a competitive antagonist of NMDA receptors – [9] alters the morphology of the dendritic tree of pyramidal neurons in layer III of the MPC. In contrast to previous studies modeling schizophrenia by postnatal administration of psychotomimetic NMDA receptor antagonists like phencyclidine [30] or MK-801 [17], for the present study, we selected plus enantiomer of CGP 37849, i.e., CGP 40116

K. We˛dzony et al. / Brain Research 1062 (2005) 166 – 170

because of its selectivity toward NMDA receptors [9] and lack of psychotomimetic effects in paradigm evaluating efficacy of sensorimotor gating [33]. In order to accomplish that task we used Golgi-Cox impregnation procedure which clearly visualized pyramidal neurons of layer III, their dendritic processes and spines (Fig. 1). Pregnant dams (Wistar) were used. The day of parturition was regarded as postnatal day 0. Rat pups of either sex were injected with increasing doses of CGP 40116 (Ciba-Geigy). On days 1, 3, 6 and 9 a dose of 1.25 mg/kg was given, followed by a dose of 2.5 mg/kg on days 12, 15, 18 and finally, on day 21 a dose of 5 mg/kg was injected (for further details see [31]). The volume of drug solution was 0.01 ml per 1 g of the body weight. On day 22, the rats were separated from mothers and females were removed. All the following experiments were performed on adult male rats weighing approximately 300 g at the time of final experiment, i.e., on day 60 of life. Control pups received vehicle only. The Golgi-Cox impregnation was applied according to the procedure described by Gibb and Kolb [12]. In brief, the rats under deep pentobarbital anesthesia (100 mg/kg i.p.) were perfused transcardially with a solution of a 0.9% NaCl—250 ml per rat; then their brains were removed and placed in the Golgi-Cox solution (20 ml per brain, the composition as follows: potassium dichromate—1.25%, mercuric chloride sublimate—1.25% and potassium chromate—1%, in distilled water) and left in total darkness for 2 weeks. Then, the brains were stored for additional 3 days in a solution of a 30% sucrose. 200 Am thick sections were cut using a vibratome (Leica VT 1000 S) at a level of MPC [21]. Sections were rinsed in distilled water for 60 s and incubated in the darkness in a fresh solution of ammonium hydroxide (14%) for 30 min; then they were washed again in distilled water (60 s) and incubated again in the darkness in a solution of the Kodak fixer for 30 min. For quantitative analysis, we selected pyramidal neurons with somata in

Fig. 1. Photomicrographs and reconstructions of Golgi-Cox impregnated, typical layer II/III pyramidal cell in rat medial prefrontal cortex. A— photomicrograph of typical layer II/III pyramidal cell of control animal. Upper and lower insets show spines on apical and basilar dendrites respectively. B, C—reconstructions of Golgi-Cox impregnated pyramidal neurons of layer II/III of control (B) and treated in the postnatal period with CGP 40116 (C).

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layer III of MPC according to the common criteria established by Golgi-Cox studies [19,25]. In brief, the selected neurons had the following features (1) clear localization of the cell body in the layer III approximately 350 Am from the pia, and clear characteristics of pyramidal neurons, defined by the presence of a basilar dendritic tree and a single apical dendrite (2) uniformly and completely stained with undisturbed processes, (3) localization in the middle of the section in order to avoid analysis of the neuron whose processes extend largely into adjacent sections (4) relative separation from neighboring neurons and blood vessel, which could obscure their morphology (5) similar size of neuronal body [19]). The morphology of the pyramidal neurons (Fig. 1) was reconstructed under a light microscope using the Leica DMLB microscope (Plan Apo objective 20, the aperture values 0.7) and a drawing tube (Camera Lucida drawings). The camera Lucida drawings were digitized along with calibration bars and were exported into the Image-Pro Plus software (Media Cybernetics). The length of dendritic segments was measured manually using morphometric subroutines of the Image-Pro Plus by tracing lines on dendritic processes. The total length of dendrites is given in Am. For a spine density analysis (Fig. 1) branches (second order) were visualized under a light microscope. In order to obtain a high optical resolution, objective Plan Apo 1.4, (magnification 100) and an oil condenser were used. A segment of the dendrite longer than 10 Am was traced, and the spines were counted. Spine density = the number of spines/length (10 Am). Two segments of basilar and two segments of apical dendrites per neuron on branch of the second order have been used for analysis, approximate distances of the segments from cell body were 35 Am and 145 Am, respectively for basilar and apical dendrites. The morphology of neurons from seven adults, CGP 40116treated rats in the postnatal period and seven respective controls were analyzed, i.e., number of neurons under analysis totaled 112. Four sections per rats and approximately two neurons per section were studied. The number of dendritic branches was calculated manually from the Camera Lucida drawings. The dendritic branches originating directly from cell bodies were designated as a first-order segments until they branched off into second-order segments from which stemmed into third-order ones, etc. Values obtained from individual animal were averaged, and calculated with one-way ANOVA (treatment as independent variable for total length of dendrites and density of spines). The branch orders were calculated using ANOVA for repeated measures (treatment as independent variable and branch order as a repeated measure). Each group of animals has been analyzed by an observer unaware of the treatment. Postnatal treatment with CGP 40116 does not change the thickness of the cortex [32], the number and size of the cell body or distribution of Golgi-Cox-positive neurons. It was found that the total length of apical dendrites did not significantly differ between postnatally treated rats and

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respective controls ( F (1,12) = 0.05; p = 0.82) (Figs. 1, 2) whereas the total length of basilar dendrites was significantly diminished by nearly 22% in GGP 40116 treated rats ( F (1,12) = 11.3; p < 0.01) (Figs. 1, 2). The postnatal treatment with CGP 40116 did not influence dendritic arborization, i.e., the number of branches measured on branch orders I –VIII and I– VII for apical and basilar dendrites, respectively ( F (1,12) = 0.47, p = 0.5; F (1,12) = 1.1, p = 0.3) (Table 1 and Fig. 1). The density of dendritic spines (Table 2) located on both basilar and apical parts of the dendritic tree did not significantly differ between groups ( F (1,12) = 0.03, p = 0.86; F (1,12) = 0.59, p = 0.46, respectively). The present study showed that postnatal administration of CGP 40116 differentially affected the morphology of basilar and apical parts of layer III pyramidal neurons in MPC of adult rats. The length of basilar dendrites was markedly and selectively reduced in postnatally treated rats, while apical segments remained intact. Hence, the changes observed in the MPC are consistent with the results of other studies showing that the blockade of NMDA receptors at early stages of development leads to a loss of neuronal dendrites of various types of neurons in the different parts of central and peripheral nervous system [15,24,29], however, in some cases, an opposite effect is also observed [26]. Dendritic structure (geometry) is dynamically shaped by synaptic activity and the size of the dendritic arbor is proportional to excitatory innervation [23]. For example, diminution of neuronal activity by sensory deprivation results in an atrophy of dendrites [10]. Among others, glutamatergic excitatory neurotransmission is crucial for the shaping of the dendritic arbor [15,24]. Thus, it is conceivable that changes in the morphology of pyramidal neurons, observed in the MPC may result from diminished gluta-

Table 1 The impact of NMDA receptor blockade in the postnatal period on the number of dendritic branches as a function of the branch order of apical and basilar dendritic systems of layer III pyramidal neurons in the medial prefrontal cortex Branch order

Order Order Order Order Order Order Order Order

I II III IV V VI VII VIII

Apical dendrites

Basilar dendrites

Controls

Controls

1 2 2.6 2.4 2.3 1.9 1 0.3

T T T T T T T T

0 0.04 0.1 0.1 0.2 0.2 0.2 0.1

CGP 1 2 2.3 2.1 1.8 1.6 1 0.3

T T T T T T T T

0 0.02 0.1 0.2 0.2 0.3 0.2 0.1

5.5 6.8 4.2 1.3 0.5 0.1 0.04 –

T T T T T T T

0.2 0.3 0.4 0.3 0.2 0.06 0.04

CGP 5.6 6.4 3.8 0.9 0.3 0.1 0 –

T T T T T T T

0.2 0.4 0.3 0.2 0.1 0.01 0

Controls—animals treated with saline; CGP—animals treated with CGP 40116 in the postnatal period. The values represent the means T SEM, n = 7 rats per group. ANOVA for repeated measures.

matergic neurotransmission due to the blockade of NMDA receptors in the postnatal period, i.e., the phase of rapid dendritic growth and arborization of rat cortical pyramidal neurons [22]. In the postnatal period between days 1– 20 after parturition, cortical neurons are particularly vulnerable to such pharmacological insults as the blockade of NMDA receptors since above receptors are transiently overexpressed in that period [20]. The diminished length of basilar dendrites concomitant the lack of alterations in spine density observed in rats treated with an NMDA receptor antagonist in the postnatal period may reflect a decrease in the total number of spines and a loss of synapses. The above conclusion is based on the assumption that a reduction of the dendritic length decrease the available surface required for the formation of synaptic contacts. The results indicating that treatment with either a competitive or a non-competitive antagonist of NMDA receptors, respectively, in the postnatal period leads to a reduction of the number of synaptic contacts, as well as to changes in synaptic morphology [4] are in line with the above suggestion. The functional analysis showing that postnatal administration of NMDA receptor antagonists reduces the synaptic efficacy of hippocampal slices [2] supports such a possibility. The changes in the morphology of pyramidal neurons may have important consequences for brain functioning. Available data suggest that pyramidal neurons in layer III of the MPC are engaged in the processing of cognitive Table 2 The impact of NMDA receptor blockade in the postnatal period on the density of the dendritic spines of layer III pyramidal neurons in rat medial prefrontal cortex

Fig. 2. The scatter plot illustrating the impact of NMDA receptor blockade in the postnatal period on the total length of the basilar and apical dendritic tree of layer III pyramidal neurons in the medial prefrontal cortex. Filled symbols illustrate individual values for each animal, while open symbols represent the mean T SEM, n = 7. An asterisk indicates statistically significant effects of the administration of CGP 40116 (CGP) in the postnatal period in comparison with the respective controls (Veh). One-way ANOVA, and Dunnett test for post hoc comparison.

Region of analysis

Apical dendrites Basilar dendrites

Spine density (number/10 Am) Controls

CGP

6.9 T 0.5 5.8 T 0.4

6.1 T 0.3 6.0 T 0.4

Controls—animals treated with saline; CGP—animals treated with CGP 40116 in the postnatal period. The values represent the mean T SEM, n = 7 rats per group. One-way ANOVA.

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functions [14]. First, they are activated during working memory tasks [11]. Second, the basilar part of cortical layer III neurons receives excitatory inputs originating in anterior thalamic nuclei [28].This projection is engaged in the feedback loop reducing the transfer of sensory input to the cortex and possibly protects the cortex against the informational overload [5]. It is speculated that deregulation of this thalamic filter may cause hyperarousal and psychosis due to the informational overload of the cerebral cortex. The reduction of the length of dendrites (neuronal elements receiving thalamo-cortical input) may suggest a dysfunction of a protective thalamic filter and in consequence lead to psychotic-like behavior. It is noteworthy that the morphological changes observed after postnatal blockade of NMDA receptors in rats are to a certain extend similar to the effects described in schizophrenic brains. It was reported that the basilar dendritic system of layer III prefrontal neurons was markedly reduced in a group of schizophrenic patients [3,16]. The changes in the length of basilar dendrites observed in schizophrenia were accompanied with a reduction in spine density [13]. Thus, CGP 40116 given in the postnatal period modeled only some aspects of the pathology typical of schizophrenia. Summing up, our findings are consistent with the reduced neuropil hypothesis [27] in which diminished amount of neuropil is regarded as a significant feature of anatomical pathology in the schizophrenic brain due to a decrease in the lengths of dendrites. In conclusion, the above data indicate that long-lasting changes in prefrontal anatomy may occur as a result of the blockade of NMDA receptors in the postnatal period and that they seem to imitate some anatomical changes observed in some cases of schizophrenia.

Acknowledgments This study was supported by grants from Polpharma Foundation, grant number 008/2002 to KW and statutory activity of Institute of Pharmacology PAS.

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