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Alteration in dendritic morphology of pyramidal neurons from the prefrontal cortex of rats with renovascular hypertension
Brain Research 1021 (2004) 112 – 118 www.elsevier.com/locate/brainres
Research report
Alteration in dendritic morphology of pyramidal neurons from the prefrontal cortex of rats with renovascular hypertension Elenia Vegaa, Maria de Je´sus Go´mez-Villalobosb, Gonzalo Floresb,* b
a Escuela de Biologı´a. Universidad Auto´noma de Puebla, Puebla, Me´xico Laboratorio de Neuropsiquiatrı´a, Instituto de Fisiologı´a. Universidad Auto´noma de Puebla, 14 Sur 6301, San Manuel, Puebla 72570, Me´xico
Accepted 27 June 2004
Abstract We have studied, in the rat, the dendritic morphological changes of the pyramidal neurons of the medial part of the prefrontal cortex induced by the chronic effect of high blood pressure. Renovascular hypertension was induced using a silver clip on the renal artery by surgery. The morphology of the pyramidal neurons from the medial part of the prefrontal cortex was investigated in these animals. The blood pressure was measured to confirm the increase in the arterial blood pressure. After 16 weeks of increase in the arterial blood pressure, the animals were sacrificed by overdoses of sodium pentobarbital and perfused intracardially with a 0.9% saline solution. The brains were removed, processed by the Golgi–Cox stain method and analyzed by the Sholl method. The dendritic morphology clearly showed that the hypertensive animals had an increase (32%) in the dendritic length of the pyramidal cells with a decrease (50%) in the density of dendritic spines when compared with sham animals. The branch-order analysis showed that the animals with hypertension exhibit more dendritic arborization at the level of the first to fourth branch order. This result suggests that renovascular hypertension may in part affect the dendritic morphology in this limbic structure, which may implicate cognitive impairment in hypertensive patients. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neuropsychiatric disorders Keywords: Renovascular hypertension; Dendrite; Golgi–Cox stain; Medial part of the prefrontal cortex; Pyramidal neuron
1. Introduction Hypertension is an important risk factor for cerebrovascular disease causing brain damage with the development of vascular cognitive impairment and vascular dementia [2,3,5,15,28,41,44,45,51]. Disruption of the blood–brain barrier is thought to contribute to these disorders [1,10]. Several studies in animal models of hypertension have demonstrated that chronic elevated blood pressure may produce brain changes such as brain atrophy, loss of nerve cells in cerebrocortical areas, and
* Corresponding author. Tel.: +522 244 1657; fax: +522 233 4511. E-mail address: [email protected] (G. Flores). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.06.042
glial reaction [2,3,5,15,28,35,41,44,51]. In-vivo imaging studies in patients with essential hypertension have corroborated these brain changes [28,56]. In addition, antihypertensive treatment with Ca2+ antagonists showed a protective effect on brain damage caused by hypertension [2,15]. However, the progressive decline in the cognitive function associated with the hypertension is not well understood. Several studies suggest that the prefrontal cortex is involved with the cognitive processes, particularly learning and memory [9,16,22,56]. Lesions of the supralimbic area of the medial part of the prefrontal cortex may alter the memory and learning [16,22]. The medial part of the prefrontal cortex is interconnected via glutamatergic projections [24,49] with the ventral hippocampus and with various other limbic cortexes via intracortical projections
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[21,24]. Hippocampal neuronal activity exerts an important regulatory control on the medial part of the prefrontal cortex cells [14,18,34]. Synapses from the hippocampus to the medial part of the prefrontal cortex are modifiable synapses and may express different forms of plasticity in all cognitive processes [10,22,52]. Recent studies related the synaptic plasticity in the hippocampal–prefrontal cortex pathway with two specific aspects of learning and memory, i.e. the consolidation of information and working memory [7,22,31]. Morphological studies of the pyramidal neurons of medial part of the prefrontal cortex using a hypertensive animal model, caused by using a clip in the thoracic aorta, may in part help to understand the cognitive changes resulting from chronic high blood pressure. Our investigation was designed to assess whether chronic hypertension affects the dendritic length and spine density on pyramidal neurons of layer 3 of the medial part of the prefrontal cortex. The dendritic morphology clearly showed that animals with renovascular hypertension had an increase in the dendritic length of the pyramidal neurons with a decrease in the density of dendritic spines when compared to sham animals. The results suggest an interesting and important effect of chronic hypertension on these pyramidal cells.
2. Material and methods 2.1. Animals Male Wistar rats (300–350 g) were obtained from our animal facility. Animals were individually housed in a temperature- and humidity-controlled environment in a 12– 12 h light–dark cycle with free access to food and water. Renovascular hypertension was induced by a 0.2-mm internal diameter, silver clip. Under chloral hydrate anesthesia (360 mg/kg, i.p.), the left renal artery was occluded by the silver clip. Sham rats underwent a similar procedure with manipulation of the left renal artery but without permanent attachment of the clip. All surgical procedures described in this study are in accordance with the bGuide for the Care and Use of Laboratory AnimalsQ of the Mexican Council for Animal Care as approved by the BUAP Animal Care Committee. All efforts were made to minimize animal suffering and to reduce the number of animals used.
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cuff method (XBP1001 Rat tail, Blood Pressure system, Kent Scientific). Systolic blood pressures (meanFS.E., mm Hg) for the sham rats and age-matched renovascular hypertensive rats were measured as previously described [39]. 2.3. Golgi–Cox stain Immediately after the last blood pressure measurement (16 weeks after the clip attachment), the rats (n=10 per group) were deeply anesthetized with sodium pentobarbital and perfused intracardially with 0.9% saline solution. The brains were removed and processed by Golgi–Cox staining, by using procedures described previously [42,43,52]. The brains were first stored in the dark for 14 days in Golgi–Cox solution then 3 days in 30% sucrose. The brains were cut into 200-Am-thick sections on the coronal plane at the level of the medial part of the prefrontal cortex [37] by using a vibratome. Sections were collected on cleaned, gelatin-coated microscope slides (four sections/ slide) and stained with ammonium hydroxide for 30 min, followed by Kodak Film Fix for another 30 min, and then washed with water, dehydrated, cleared, and mounted using a resinous medium. The Golgi-impregnated pyramidal neurons of the medial part of the prefrontal cortex were readily identified by their characteristic triangular soma shape, apical dendrites extending toward the pial surface, and numerous dendritic spines. The criteria used to select neurons for reconstruction were essentially as was described previously [43,52]; location of the cell soma in layer 3 of the medial prefrontal cortex; full impregnation of the neurons, presence of at least three primary, basilar dendritic shafts, each of which branched at least once, and no morphological changes attributable to Golgi–Cox stain. Five neurons in each hemisphere (10 neurons per animal) were drawn using camera lucida at a magnification of 250 (DMLS, Leica Microscope) by a person who was not
2.2. Measure of the blood pressure One week before and after use of the clip on the left renal artery, the blood pressure was measured in the renovascular hypertensive (n=10) and sham (n=10) animals. From the second week, the blood pressure was measured every 2 weeks for 16 weeks. Systolic and diastolic blood pressures were measured by the tail-
Fig. 1. Graph of the systolic pressure of the renovascular hypertension animal. The systolic pressure was increased in the animals with hypertension. Closed circles indicate the meanFS.E.M. (n=10) from hypertensive animals and the open circles correspond to the mean and FS.E.M. (n=10) from sham rats. (*P b 0.01).
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Fig. 4. Total dendritic length from medial–prefrontal–cortex–layer 3 pyramidal neurons of renovascular hypertensive animals (n=100 neurons per group). The dendritic length was increased in the hypertensive animals (*P b 0.01).
Data from the Sholl analyses and the spine densities were analyzed using a two-tailed Kruskal–Wallis and Mann– Whitney tests ( P b 0.05 was considered significant).
3. Results 3.1. Blood pressure Fig. 2. Photomicrograph illustrating Golgi–Cox-impregnated dendrite arborization and dendrite spines on medial–prefrontal–cortex–layer 3 pyramidal neurons of sham rats (A,B) and animals with renovascular hypertension (C,D).
knowledgeable of the surgery conditions. For each neuron, the dendritic tree, including all branches, was reconstructed and the dendritic tracing was quantified by Sholl analysis [50]. The dendritic surface was quantified by counting the number of branches at each order from the cell body by Sholl analysis [43,52], and by counting the number of ring intersections using an overlay of concentric rings (10 Am between rings). The density of dendritic spines was measured from the basal dendrites by drawing at least 10-Am-long segments from close to the cell body and from the terminal tips at high power (1000) and counting the number of spines.
Fig. 3. Spine density of medial–prefrontal–cortex–layer 3 pyramidal neurons of renovascular hypertensive (n=100 neurons) or sham animals (n=100 neurons). Both proximal and distal spine density were decreased in the hypertensive animals when compared with the sham controls (*P b 0.01).
Control blood pressure was measured before the attachment of the clip by surgery and no differences in the systolic blood pressure were measured between the sham and hypertensive rats (Fig. 1). Two weeks after the application of the clip, there was an increase the systolic blood pressure in the renovascular hypertensive rats when compared with the sham animals ( P b0.01) (Fig. 1). 3.2. Dendritic morphology Dendritic branching and density of dendritic spines of neurons (100 neurons per group) of the medial part of the prefrontal cortex were measured by Golgi–Cox stain for both hypertensive and sham rats. Maximum branch order, spine density, and total dendritic length obtained were similar to our previous report [52]. The Golgi–Cox impregnation procedure clearly filled the dendritic shafts
Fig. 5. Sholl analysis of dendrites of medial–prefrontal–cortex–layer 3 pyramidal neurons. Closed circles indicate the meanFS.E.M. (n=100 neurons) of hypertensive animals and the open circles correspond to the meanFS.E.M. (n=100 neurons) of sham rats. The group of animals that developed hypertension showed an increase in the dendritic length when compared with the sham-control (*P b 0.01).
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Fig. 6. Graphs of branch order of pyramidal neurons of layer 3 of the medial prefrontal cortex from renovascular hypertensive animals. Closed circles indicate the meanFS.E.M. (n=100 neurons from 10 rats) from animals with hypertension and the open circles correspond to the meanFS.E.M. (n=100 neurons from 10 rats) from sham rats. The group of animals that developed hypertension showed an increase in the dendritic arborization at the level of the first to fourth branch order compared to the sham rats (*P b 0.01 to first to third order; **P b 0.05 to fourth order).
and spines of layer 3 of the pyramidal medial part of the prefrontal cortex neurons (Fig. 2). Comparisons between hypertensive and sham animals showed that the mean spine density of the dendrites of pyramidal neurons of layer 3 of the medial part of the prefrontal cortex in the hypertensive animals were lower than their controls (48% and 51% decrease in the proximal and distal dendritic spines from the body of the neuron, P b0.001) (Figs. 2 and 3). As measured by Sholl analysis, total dendritic length of the medial part of the prefrontal cortex neurons differed significantly ( P b 0.001) between hypertensive and sham rats (Fig. 4). Interestingly, the hypertensive animals showed an increased in the dendritic length ( P b0.001). The analysis of intersection per radius of shell shows that the hypertensive animals had more intersections per shell or more dendritic arborization than the sham rats ( P = 0.03) (Fig. 5). In addition, the branch-order analysis also suggests that the hypertensive rats had more dendritic arborization with an increase in the first to the fourth branch order in comparison with the sham animals ( P b0.01 to the first to third order; P b0.05 to the fourth order) ( P b 0.01) (Fig. 6).
4. Discussion Our aim was to investigate the consequences of 16 weeks of high blood pressure, induced by the occlusion of a renal artery by a silver clip, on the basilar, dendriticstructural morphology of layer 3 pyramidal cells of the prefrontal cortex. We found that hypertension causes major reductions in dendritic spine density with increases in the dendritic length in layer 3 pyramidal neurons of the medial part of the prefrontal cortex and these data may be linked in part with the cognitive impairment seen in hypertension. The high levels of systolic pressure caused by the occlusion of a renal artery, reported here, are consistent with previous reports [2,25,39,55] using the same procedure. In those studies, hypertensive rats showed a clear
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increase in the systolic blood pressure with an alteration in cognition [41]. Several reports support a causal role of hypertension in the cognitive decline in hypertensive patients [2,3,28,41]. Hypertension produces changes in the brain, such as vascular remodeling, impaired cerebral autoregulation, white-matter lesions, and cerebral microbleed [2,3,28]. Furthermore, hypertension has been implicated in vascular dementia [2,41]. Evidence has demonstrated that the cognitive functions are regulated by the prefrontal cortex [7,8,9,16,56]. Participation of the medial prefrontal cortex in cognition is well recognized when its connections are analyzed. The medial prefrontal cortex receives and sends excitatory projections to the CA1 region from the hippocampus [21,24,49], a critical structure in memory [7,16]. The layer 3 of the medial prefrontal cortex may be regulated by hippocampal projections [12,21,24,48,49] and its activity may be modulated by synaptic inputs from the hippocampus [17,18,33,34]. The nucleus accumbens sends signals to the ventral pallidum [58], a critical structure in emotions [42,43], and the ventral pallidum sends signals to the thalamus [58]. Our results clearly show that renovascular hypertension produces alteration in the morphology of the dendrites of the pyramidal neurons. Exactly how renovascular hypertension came to enhance the dendritic arborization at the level of the first to fourth branch orders of the pyramidal neurons of the medial part of the prefrontal cortex is not clear. However, as mentioned, renovascular hypertension is associated with vascular remodeling, and physiological studies have shown that renovascular hypertension is associated with a dysfunctional endothelium caused by deficient production of nitric oxide (NO) derived from the endothelium [29], which alters the vasodilatation in this model [25,55]. At the level of the neurons, the activity of the enzyme that catalyzes the production of NO, nitric oxide synthase, is decreased in animals with renovascular hypertension when compared to sham animals [20]. Interestingly, a recent report analyzed the activity of the nitric oxide synthase in different regions of the brain, before and after establishing hypertension in rats [40], and the authors suggest that when hypertension exists the activity of the nitric oxide synthase is enhanced in the hypothalamus and brainstem. The hypothalamus is a critical structure in the control of hormones, e.g. the production of corticosterone is controlled by an adrenocorticotropic-hormone, which is regulated by a corticotropinereleasing hormone [26]. The corticotropine-releasing hormone is produced by the hypothalamus. The corticosterone per se also may affect the density of dendritic spines [26]. High levels of corticosterone may produce a decrease in the density of dendritic spines in pyramidal neurons of the hippocampus [26]. In addition, several recent studies have shown that the nitric oxide per se may in part be regulating the dendritic spines and branching in the pyramidal cells of the cortex [4,30,32,38,46,47]. An increase in the activity of the NO may explain the increase in the dendritic length [4,30,38].
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The pyramidal neurons of the medial prefrontal cortex used glutamate as the neurotransmitter. Spine creation and destruction at glutamatergic synapses is largely controlled by glutamate itself [36]. Some types of glutamate receptors, such as N-methyl-d-aspartic acid (NMDA) or metabolitropic receptors, activate phosphorylation of skeletal microtubular protein and influence synaptic maturation, spine morphology, and possibly the growth of new spines [19,36,54]. Perhaps dendritic development is dependent on NMDA receptor activity, and then the asymmetric synapse density in striatal neurons (formed by glutamatergic input) dramatically declines by NMDA blockade in neonatal rats [22,23,54], whereas the dendritic spines of the cortical neurons are sites of the majority of excitatory synapses and are associated with long-term synaptic plasticity and are inhibited by the activation of the NMDA receptors [13]. Glutamate is also a potent neurotoxin and may, under certain circumstances, produce neural damage and possible spine elimination [53,54]. Some studies have demonstrated that the NMDA receptors may mediate the spinal sympathetic reflexes, which initiate episodic hypertension after a spinal cord injury [27,59], consistent with this relation between the glutamate transmission and hypertension. In addition, the rostral ventrolateral medulla neurons of animals with renovascular hypertension exhibited an increased response to glutamate actions [6]. Rilmenidine, a second-generation, centrally acting, antihypertensive drug, with a hypotensive effect, is dependent on functional NMDA receptors [59]. All this data, taken together with a recent report, showed the activation of NMDA receptors and subsequent release of nitric oxide may in part trigger the growth of presynaptic phylopodia, which play an important role in synaptogenesis and spine formation [32]. Pisu et al. [38] have demonstrated the NO-glutamate interactions via NMDA receptors in the development of the dendritic tree of the Purkinje neurons. One can hypothesize that the alteration of the activity of the nitric oxide, together with altered response to glutamate, especially via an NMDA receptor, may in part participate in the morphological changes found in the dendritic pyramidal neurons of the medial part of the prefrontal cortex from the rats with renovascular hypertension. There is a need for further studies, to relate the NO and glutamate activity in the medial part of the prefrontal cortex at different times after establishment of hypertension in rats, to clarify all these explanations. Another possibility is that the loss of the inputs to the medial part of the prefrontal cortex may produce a decrease in the spine dendrites in the pyramidal neurons by loss of the synapse on spines [11,23]. However, under some physiological conditions this is not true, e.g. during the estrous cycle there is a decrease of the spine density of the pyramidal neurons of the area CA1 of the hippocampus [57] without loss of the inputs. Another possibility is that chronic hypertension may alter the integrity of the blood–CSF barrier in addition to the vascular alteration in the brain [1], and several toxins may cross this barrier with a toxic effect
on the dendritic morphology [11]. It is tempting to speculate that the damage of the blood–CSF barrier may result in a decrease in the dendritic spine density. The increase in the total dendritic length with the decrease in the dendritic spine is especially intriguing, because this may be caused by an ineffective mechanism of the synaptic connectivity in the medial prefrontal cortex. In summary, our findings provide evidence for a decrease in dendritic spines with an increase in the arborization of the pyramidal neurons of the medial part of the prefrontal cortex as a result of chronic renovascular hypertension. Given the functional role and the interconnection of the medial part of the prefrontal cortex with other cognitive structures such as hippocampus and nucleus accumbens, these morphological changes reported here may contribute to the explanation of some cognitive data reported in hypertensive patients.
Acknowledgements This study was supported in part by grants from CONACyT-Mexico (No. 40664) and VIEP-BUAP (No. IV34-04/SAL/G to GF). We are grateful to Dr. Carlos Escamilla for his help and suggestions related to keeping of animals. EV is a student of BUAP. MJG and GF are members of the Researcher National System of Mexico. Thanks to Dr. Ellis Glazier for editing the English-language text.
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Research report
Alteration in dendritic morphology of pyramidal neurons from the prefrontal cortex of rats with renovascular hypertension Elenia Vegaa, Maria de Je´sus Go´mez-Villalobosb, Gonzalo Floresb,* b
a Escuela de Biologı´a. Universidad Auto´noma de Puebla, Puebla, Me´xico Laboratorio de Neuropsiquiatrı´a, Instituto de Fisiologı´a. Universidad Auto´noma de Puebla, 14 Sur 6301, San Manuel, Puebla 72570, Me´xico
Accepted 27 June 2004
Abstract We have studied, in the rat, the dendritic morphological changes of the pyramidal neurons of the medial part of the prefrontal cortex induced by the chronic effect of high blood pressure. Renovascular hypertension was induced using a silver clip on the renal artery by surgery. The morphology of the pyramidal neurons from the medial part of the prefrontal cortex was investigated in these animals. The blood pressure was measured to confirm the increase in the arterial blood pressure. After 16 weeks of increase in the arterial blood pressure, the animals were sacrificed by overdoses of sodium pentobarbital and perfused intracardially with a 0.9% saline solution. The brains were removed, processed by the Golgi–Cox stain method and analyzed by the Sholl method. The dendritic morphology clearly showed that the hypertensive animals had an increase (32%) in the dendritic length of the pyramidal cells with a decrease (50%) in the density of dendritic spines when compared with sham animals. The branch-order analysis showed that the animals with hypertension exhibit more dendritic arborization at the level of the first to fourth branch order. This result suggests that renovascular hypertension may in part affect the dendritic morphology in this limbic structure, which may implicate cognitive impairment in hypertensive patients. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neuropsychiatric disorders Keywords: Renovascular hypertension; Dendrite; Golgi–Cox stain; Medial part of the prefrontal cortex; Pyramidal neuron
1. Introduction Hypertension is an important risk factor for cerebrovascular disease causing brain damage with the development of vascular cognitive impairment and vascular dementia [2,3,5,15,28,41,44,45,51]. Disruption of the blood–brain barrier is thought to contribute to these disorders [1,10]. Several studies in animal models of hypertension have demonstrated that chronic elevated blood pressure may produce brain changes such as brain atrophy, loss of nerve cells in cerebrocortical areas, and
* Corresponding author. Tel.: +522 244 1657; fax: +522 233 4511. E-mail address: [email protected] (G. Flores). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.06.042
glial reaction [2,3,5,15,28,35,41,44,51]. In-vivo imaging studies in patients with essential hypertension have corroborated these brain changes [28,56]. In addition, antihypertensive treatment with Ca2+ antagonists showed a protective effect on brain damage caused by hypertension [2,15]. However, the progressive decline in the cognitive function associated with the hypertension is not well understood. Several studies suggest that the prefrontal cortex is involved with the cognitive processes, particularly learning and memory [9,16,22,56]. Lesions of the supralimbic area of the medial part of the prefrontal cortex may alter the memory and learning [16,22]. The medial part of the prefrontal cortex is interconnected via glutamatergic projections [24,49] with the ventral hippocampus and with various other limbic cortexes via intracortical projections
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[21,24]. Hippocampal neuronal activity exerts an important regulatory control on the medial part of the prefrontal cortex cells [14,18,34]. Synapses from the hippocampus to the medial part of the prefrontal cortex are modifiable synapses and may express different forms of plasticity in all cognitive processes [10,22,52]. Recent studies related the synaptic plasticity in the hippocampal–prefrontal cortex pathway with two specific aspects of learning and memory, i.e. the consolidation of information and working memory [7,22,31]. Morphological studies of the pyramidal neurons of medial part of the prefrontal cortex using a hypertensive animal model, caused by using a clip in the thoracic aorta, may in part help to understand the cognitive changes resulting from chronic high blood pressure. Our investigation was designed to assess whether chronic hypertension affects the dendritic length and spine density on pyramidal neurons of layer 3 of the medial part of the prefrontal cortex. The dendritic morphology clearly showed that animals with renovascular hypertension had an increase in the dendritic length of the pyramidal neurons with a decrease in the density of dendritic spines when compared to sham animals. The results suggest an interesting and important effect of chronic hypertension on these pyramidal cells.
2. Material and methods 2.1. Animals Male Wistar rats (300–350 g) were obtained from our animal facility. Animals were individually housed in a temperature- and humidity-controlled environment in a 12– 12 h light–dark cycle with free access to food and water. Renovascular hypertension was induced by a 0.2-mm internal diameter, silver clip. Under chloral hydrate anesthesia (360 mg/kg, i.p.), the left renal artery was occluded by the silver clip. Sham rats underwent a similar procedure with manipulation of the left renal artery but without permanent attachment of the clip. All surgical procedures described in this study are in accordance with the bGuide for the Care and Use of Laboratory AnimalsQ of the Mexican Council for Animal Care as approved by the BUAP Animal Care Committee. All efforts were made to minimize animal suffering and to reduce the number of animals used.
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cuff method (XBP1001 Rat tail, Blood Pressure system, Kent Scientific). Systolic blood pressures (meanFS.E., mm Hg) for the sham rats and age-matched renovascular hypertensive rats were measured as previously described [39]. 2.3. Golgi–Cox stain Immediately after the last blood pressure measurement (16 weeks after the clip attachment), the rats (n=10 per group) were deeply anesthetized with sodium pentobarbital and perfused intracardially with 0.9% saline solution. The brains were removed and processed by Golgi–Cox staining, by using procedures described previously [42,43,52]. The brains were first stored in the dark for 14 days in Golgi–Cox solution then 3 days in 30% sucrose. The brains were cut into 200-Am-thick sections on the coronal plane at the level of the medial part of the prefrontal cortex [37] by using a vibratome. Sections were collected on cleaned, gelatin-coated microscope slides (four sections/ slide) and stained with ammonium hydroxide for 30 min, followed by Kodak Film Fix for another 30 min, and then washed with water, dehydrated, cleared, and mounted using a resinous medium. The Golgi-impregnated pyramidal neurons of the medial part of the prefrontal cortex were readily identified by their characteristic triangular soma shape, apical dendrites extending toward the pial surface, and numerous dendritic spines. The criteria used to select neurons for reconstruction were essentially as was described previously [43,52]; location of the cell soma in layer 3 of the medial prefrontal cortex; full impregnation of the neurons, presence of at least three primary, basilar dendritic shafts, each of which branched at least once, and no morphological changes attributable to Golgi–Cox stain. Five neurons in each hemisphere (10 neurons per animal) were drawn using camera lucida at a magnification of 250 (DMLS, Leica Microscope) by a person who was not
2.2. Measure of the blood pressure One week before and after use of the clip on the left renal artery, the blood pressure was measured in the renovascular hypertensive (n=10) and sham (n=10) animals. From the second week, the blood pressure was measured every 2 weeks for 16 weeks. Systolic and diastolic blood pressures were measured by the tail-
Fig. 1. Graph of the systolic pressure of the renovascular hypertension animal. The systolic pressure was increased in the animals with hypertension. Closed circles indicate the meanFS.E.M. (n=10) from hypertensive animals and the open circles correspond to the mean and FS.E.M. (n=10) from sham rats. (*P b 0.01).
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Fig. 4. Total dendritic length from medial–prefrontal–cortex–layer 3 pyramidal neurons of renovascular hypertensive animals (n=100 neurons per group). The dendritic length was increased in the hypertensive animals (*P b 0.01).
Data from the Sholl analyses and the spine densities were analyzed using a two-tailed Kruskal–Wallis and Mann– Whitney tests ( P b 0.05 was considered significant).
3. Results 3.1. Blood pressure Fig. 2. Photomicrograph illustrating Golgi–Cox-impregnated dendrite arborization and dendrite spines on medial–prefrontal–cortex–layer 3 pyramidal neurons of sham rats (A,B) and animals with renovascular hypertension (C,D).
knowledgeable of the surgery conditions. For each neuron, the dendritic tree, including all branches, was reconstructed and the dendritic tracing was quantified by Sholl analysis [50]. The dendritic surface was quantified by counting the number of branches at each order from the cell body by Sholl analysis [43,52], and by counting the number of ring intersections using an overlay of concentric rings (10 Am between rings). The density of dendritic spines was measured from the basal dendrites by drawing at least 10-Am-long segments from close to the cell body and from the terminal tips at high power (1000) and counting the number of spines.
Fig. 3. Spine density of medial–prefrontal–cortex–layer 3 pyramidal neurons of renovascular hypertensive (n=100 neurons) or sham animals (n=100 neurons). Both proximal and distal spine density were decreased in the hypertensive animals when compared with the sham controls (*P b 0.01).
Control blood pressure was measured before the attachment of the clip by surgery and no differences in the systolic blood pressure were measured between the sham and hypertensive rats (Fig. 1). Two weeks after the application of the clip, there was an increase the systolic blood pressure in the renovascular hypertensive rats when compared with the sham animals ( P b0.01) (Fig. 1). 3.2. Dendritic morphology Dendritic branching and density of dendritic spines of neurons (100 neurons per group) of the medial part of the prefrontal cortex were measured by Golgi–Cox stain for both hypertensive and sham rats. Maximum branch order, spine density, and total dendritic length obtained were similar to our previous report [52]. The Golgi–Cox impregnation procedure clearly filled the dendritic shafts
Fig. 5. Sholl analysis of dendrites of medial–prefrontal–cortex–layer 3 pyramidal neurons. Closed circles indicate the meanFS.E.M. (n=100 neurons) of hypertensive animals and the open circles correspond to the meanFS.E.M. (n=100 neurons) of sham rats. The group of animals that developed hypertension showed an increase in the dendritic length when compared with the sham-control (*P b 0.01).
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Fig. 6. Graphs of branch order of pyramidal neurons of layer 3 of the medial prefrontal cortex from renovascular hypertensive animals. Closed circles indicate the meanFS.E.M. (n=100 neurons from 10 rats) from animals with hypertension and the open circles correspond to the meanFS.E.M. (n=100 neurons from 10 rats) from sham rats. The group of animals that developed hypertension showed an increase in the dendritic arborization at the level of the first to fourth branch order compared to the sham rats (*P b 0.01 to first to third order; **P b 0.05 to fourth order).
and spines of layer 3 of the pyramidal medial part of the prefrontal cortex neurons (Fig. 2). Comparisons between hypertensive and sham animals showed that the mean spine density of the dendrites of pyramidal neurons of layer 3 of the medial part of the prefrontal cortex in the hypertensive animals were lower than their controls (48% and 51% decrease in the proximal and distal dendritic spines from the body of the neuron, P b0.001) (Figs. 2 and 3). As measured by Sholl analysis, total dendritic length of the medial part of the prefrontal cortex neurons differed significantly ( P b 0.001) between hypertensive and sham rats (Fig. 4). Interestingly, the hypertensive animals showed an increased in the dendritic length ( P b0.001). The analysis of intersection per radius of shell shows that the hypertensive animals had more intersections per shell or more dendritic arborization than the sham rats ( P = 0.03) (Fig. 5). In addition, the branch-order analysis also suggests that the hypertensive rats had more dendritic arborization with an increase in the first to the fourth branch order in comparison with the sham animals ( P b0.01 to the first to third order; P b0.05 to the fourth order) ( P b 0.01) (Fig. 6).
4. Discussion Our aim was to investigate the consequences of 16 weeks of high blood pressure, induced by the occlusion of a renal artery by a silver clip, on the basilar, dendriticstructural morphology of layer 3 pyramidal cells of the prefrontal cortex. We found that hypertension causes major reductions in dendritic spine density with increases in the dendritic length in layer 3 pyramidal neurons of the medial part of the prefrontal cortex and these data may be linked in part with the cognitive impairment seen in hypertension. The high levels of systolic pressure caused by the occlusion of a renal artery, reported here, are consistent with previous reports [2,25,39,55] using the same procedure. In those studies, hypertensive rats showed a clear
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increase in the systolic blood pressure with an alteration in cognition [41]. Several reports support a causal role of hypertension in the cognitive decline in hypertensive patients [2,3,28,41]. Hypertension produces changes in the brain, such as vascular remodeling, impaired cerebral autoregulation, white-matter lesions, and cerebral microbleed [2,3,28]. Furthermore, hypertension has been implicated in vascular dementia [2,41]. Evidence has demonstrated that the cognitive functions are regulated by the prefrontal cortex [7,8,9,16,56]. Participation of the medial prefrontal cortex in cognition is well recognized when its connections are analyzed. The medial prefrontal cortex receives and sends excitatory projections to the CA1 region from the hippocampus [21,24,49], a critical structure in memory [7,16]. The layer 3 of the medial prefrontal cortex may be regulated by hippocampal projections [12,21,24,48,49] and its activity may be modulated by synaptic inputs from the hippocampus [17,18,33,34]. The nucleus accumbens sends signals to the ventral pallidum [58], a critical structure in emotions [42,43], and the ventral pallidum sends signals to the thalamus [58]. Our results clearly show that renovascular hypertension produces alteration in the morphology of the dendrites of the pyramidal neurons. Exactly how renovascular hypertension came to enhance the dendritic arborization at the level of the first to fourth branch orders of the pyramidal neurons of the medial part of the prefrontal cortex is not clear. However, as mentioned, renovascular hypertension is associated with vascular remodeling, and physiological studies have shown that renovascular hypertension is associated with a dysfunctional endothelium caused by deficient production of nitric oxide (NO) derived from the endothelium [29], which alters the vasodilatation in this model [25,55]. At the level of the neurons, the activity of the enzyme that catalyzes the production of NO, nitric oxide synthase, is decreased in animals with renovascular hypertension when compared to sham animals [20]. Interestingly, a recent report analyzed the activity of the nitric oxide synthase in different regions of the brain, before and after establishing hypertension in rats [40], and the authors suggest that when hypertension exists the activity of the nitric oxide synthase is enhanced in the hypothalamus and brainstem. The hypothalamus is a critical structure in the control of hormones, e.g. the production of corticosterone is controlled by an adrenocorticotropic-hormone, which is regulated by a corticotropinereleasing hormone [26]. The corticotropine-releasing hormone is produced by the hypothalamus. The corticosterone per se also may affect the density of dendritic spines [26]. High levels of corticosterone may produce a decrease in the density of dendritic spines in pyramidal neurons of the hippocampus [26]. In addition, several recent studies have shown that the nitric oxide per se may in part be regulating the dendritic spines and branching in the pyramidal cells of the cortex [4,30,32,38,46,47]. An increase in the activity of the NO may explain the increase in the dendritic length [4,30,38].
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The pyramidal neurons of the medial prefrontal cortex used glutamate as the neurotransmitter. Spine creation and destruction at glutamatergic synapses is largely controlled by glutamate itself [36]. Some types of glutamate receptors, such as N-methyl-d-aspartic acid (NMDA) or metabolitropic receptors, activate phosphorylation of skeletal microtubular protein and influence synaptic maturation, spine morphology, and possibly the growth of new spines [19,36,54]. Perhaps dendritic development is dependent on NMDA receptor activity, and then the asymmetric synapse density in striatal neurons (formed by glutamatergic input) dramatically declines by NMDA blockade in neonatal rats [22,23,54], whereas the dendritic spines of the cortical neurons are sites of the majority of excitatory synapses and are associated with long-term synaptic plasticity and are inhibited by the activation of the NMDA receptors [13]. Glutamate is also a potent neurotoxin and may, under certain circumstances, produce neural damage and possible spine elimination [53,54]. Some studies have demonstrated that the NMDA receptors may mediate the spinal sympathetic reflexes, which initiate episodic hypertension after a spinal cord injury [27,59], consistent with this relation between the glutamate transmission and hypertension. In addition, the rostral ventrolateral medulla neurons of animals with renovascular hypertension exhibited an increased response to glutamate actions [6]. Rilmenidine, a second-generation, centrally acting, antihypertensive drug, with a hypotensive effect, is dependent on functional NMDA receptors [59]. All this data, taken together with a recent report, showed the activation of NMDA receptors and subsequent release of nitric oxide may in part trigger the growth of presynaptic phylopodia, which play an important role in synaptogenesis and spine formation [32]. Pisu et al. [38] have demonstrated the NO-glutamate interactions via NMDA receptors in the development of the dendritic tree of the Purkinje neurons. One can hypothesize that the alteration of the activity of the nitric oxide, together with altered response to glutamate, especially via an NMDA receptor, may in part participate in the morphological changes found in the dendritic pyramidal neurons of the medial part of the prefrontal cortex from the rats with renovascular hypertension. There is a need for further studies, to relate the NO and glutamate activity in the medial part of the prefrontal cortex at different times after establishment of hypertension in rats, to clarify all these explanations. Another possibility is that the loss of the inputs to the medial part of the prefrontal cortex may produce a decrease in the spine dendrites in the pyramidal neurons by loss of the synapse on spines [11,23]. However, under some physiological conditions this is not true, e.g. during the estrous cycle there is a decrease of the spine density of the pyramidal neurons of the area CA1 of the hippocampus [57] without loss of the inputs. Another possibility is that chronic hypertension may alter the integrity of the blood–CSF barrier in addition to the vascular alteration in the brain [1], and several toxins may cross this barrier with a toxic effect
on the dendritic morphology [11]. It is tempting to speculate that the damage of the blood–CSF barrier may result in a decrease in the dendritic spine density. The increase in the total dendritic length with the decrease in the dendritic spine is especially intriguing, because this may be caused by an ineffective mechanism of the synaptic connectivity in the medial prefrontal cortex. In summary, our findings provide evidence for a decrease in dendritic spines with an increase in the arborization of the pyramidal neurons of the medial part of the prefrontal cortex as a result of chronic renovascular hypertension. Given the functional role and the interconnection of the medial part of the prefrontal cortex with other cognitive structures such as hippocampus and nucleus accumbens, these morphological changes reported here may contribute to the explanation of some cognitive data reported in hypertensive patients.
Acknowledgements This study was supported in part by grants from CONACyT-Mexico (No. 40664) and VIEP-BUAP (No. IV34-04/SAL/G to GF). We are grateful to Dr. Carlos Escamilla for his help and suggestions related to keeping of animals. EV is a student of BUAP. MJG and GF are members of the Researcher National System of Mexico. Thanks to Dr. Ellis Glazier for editing the English-language text.
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