Declined learning and memory ability is a common phenomenon in aged individuals from humans to rodents. Such age-related cognitive decline has been shown to be associated with the dysfunction of the medial temporal lobe, consisting of the hippocampus and its adjacent entorhinal cortex (EC), perirhinal (PRC) and parahippocampal (postrhinal (POR) in rodents) cortices (the parahippocampal region), and the prefrontal cortex (PFC) (for reviews see Gallagher and Rapp, 1997; Tisserand and Jolles, 2003; Burke and Barnes, 2006; Greenwood, 2007). In the adult brain, neurogenesis has been found in the dentate gyrus (DG) of the hippocampus (Altman and Bayer, 1990; Gage, 2002). Newly generated granule cells can mature into functional neurons and the normal rate of neurogenesis in the DG has been shown to be important for maintaining hippocampal function (Markakis and Gage, 1999; Van Praag et al., 2002). During aging, however, the rate of hippocampal neurogenesis is dramatically decreased (Kuhn et al., 1996; Bizon et al., 2004). Several recent studies have demonstrated a link between hippocampal neurogenesis and learning and memory (Driscoll and Sutherland, 2005; Wati et al., 2006). Polyamines putrescine, spermidine and spermine are positively charged aliphatic amines present in all tissues of almost all species (Wallace, 2000; Wallace et al., 2003). Tissue polyamine levels are mainly regulated by the activities of ornithine decarboxylase (ODC; the rate-limiting enzyme that converts ornithine to putrescine) and spermidine/spermine N1 -acetyltransferase (the key enzyme in polyamine interconversion) (Wallace et al., 2003). 

Declined learning and memory ability is a common phenomenon in aged individuals from humans to rodents. Such age-related cognitive decline has been shown to be associated with the dysfunction of the medial temporal lobe, consisting of the hippocampus and its adjacent entorhinal cortex (EC), perirhinal (PRC) and parahippocampal (postrhinal (POR) in rodents) cortices (the parahippocampal region), and the prefrontal cortex (PFC) (for reviews see Gallagher and Rapp, 1997; Tisserand and Jolles, 2003; Burke and Barnes, 2006; Greenwood, 2007). In the adult brain, neurogenesis has been found in the dentate gyrus (DG) of the hippocampus (Altman and Bayer, 1990; Gage, 2002). Newly generated granule cells can mature into functional neurons and the normal rate of neurogenesis in the DG has been shown to be important for maintaining hippocampal function (Markakis and Gage, 1999; Van Praag et al., 2002). During aging, however, the rate of hippocampal neurogenesis is dramatically decreased (Kuhn et al., 1996; Bizon et al., 2004). Several recent studies have demonstrated a link between hippocampal neurogenesis and learning and memory (Driscoll and Sutherland, 2005; Wati et al., 2006). Polyamines putrescine, spermidine and spermine are positively charged aliphatic amines present in all tissues of almost all species (Wallace, 2000; Wallace et al., 2003). Tissue polyamine levels are mainly regulated by the activities of ornithine decarboxylase (ODC; the rate-limiting enzyme that converts ornithine to putrescine) and spermidine/spermine N1 -acetyltransferase (the key enzyme in polyamine interconversion) (Wallace et al., 2003). 

It has been well documented that polyamines play important roles in cell proliferation and differentiation, synthesis of DNA, RNA and proteins, protein phosphorylation, signal transduction, as well as the regulation of neurotransmitter receptors (for reviews see Williams, 1997; Wallace, 2000; Oredsson, 2003). Previous studies have reported spermidine-induced memory facilitation and spermine-induced enhancement in synaptic plasticity (Rubin et al., 2000, 2001; Berlese et al., 2005; Camera et al., 2007), and the underlying mechanism may be through the interaction with the polyamine binding site at the N-methyl-D-aspartate (NMDA) receptors (Rock and Macdonald, 1995; Williams, 1997). Recently, Malaterre et al. (2004) has demonstrated that the reduction in putrescine levels (induced by difluoromethylornithine, a potent specific and irreversible inhibitor of ODC) significantly impairs adult neurogenesis in the DG in young rats, suggesting an important role of putrescine in hippocampal neurogenesis. Since aging impairs hippocampal neurogenesis dramatically, we hypothesize that the putrescine level decreases with age in the DG. A number of studies have investigated age-related changes in ODC activity and polyamine levels in the CNS in humans and experimental animals. Morrison et al. (1995), for example, reported decreased spermine, but not putrescine or spermidine, level in the aged occipital cortex. Vivo et al.(2001) demonstrated significantly increased ODC activity and putrescine and spermidine content in the spinal cord, but not cerebellum, cortex or hippocampus, in aged rats relative to the young adult controls. Collectively, this earlier research suggests that the ODC/polyamine system is potentially involved in the normal process of aging.

 To the best of our knowledge, there is no systematic investigation of the effects of aging on polyamine levels in memory-associated brain structures. In the present study, we measured the putrescine, spermidine and spermine levels in the hippocampus and the EC, PRC, POR and PFC, as well as the temporal cortex (TE) (an auditory cortex), in young, middle-aged and aged rats. Because the CA1, CA3 and DG areas of the hippocampus have differential contributions to memory processing (Rolls and Kesner, 2006), age-related changes in polyamines in the hippocampus were investigated at the sub-regional level.

EXPERIMENTAL PROCEDURES

In the present study, 24-month-old Sprague–Dawley (SD) rats were termed aged rats (Cha et al., 1998, 2000; La Porta and Comolli, 1999; Liu et al., 2003a,b,c, 2004a,b, 2005, 2008a,b; Kollen et al., 2008). Significant behavioral and electrophysiological impairments were observed in 24-month-old SD rats as compared with the young adults (4-month-old) (Liu et al., 2004b, 2005; Kollen et al., 2008). Groups of aged (24-month-old, n9), middle-aged (12-month-old, n9) and young (4-month-old, n9) male SD rats were housed three to five per cage (533326 cm3 ), maintained on a 12-h light/dark cycle (lights on 7 a.m.) and provided ad libitum access to food and water. 

The health condition (e.g. body weight, eyes, teeth, fur, skin, feet, urine and general behavior) of aged and middle-aged animals was regularly monitored by animal technicians and a consultant veterinarian. Only animals showing good health were used for the study. All experimental procedures were carried out in accordance with the regulations of the University of Otago Committee on Ethics in the Care and Use of Laboratory Animals. Every attempt was made to limit the number of animals used and to minimize their suffering. Tissue preparation All rats were killed by decapitation without anesthesia. The brains were rapidly removed and left in cold saline (4 °C) for 45 s. The sub-regions of the hippocampus (CA1, CA2/3 and DG), and the PFC, EC, PRC, POR and TE were dissected freshly on ice. There has been some debate as to whether the rat has an area of cortex that could be considered analogous to the PFC of primates. In the present study, we treated the anterior cingulate cortex (Zilles’s Cg1, Cg2, and Cg3; 9) as equivalent to at least a portion of the primate PFC that receives afferents from the medialis dorsalis of the thalamus. The frontal association cortex, prelimbic cortex, cingulate cortex (area 1) and the most anterior part of the secondary motor cortex were freshly dissected out as defined in Paxinos and Watson (1998) on ice (see Fig. 1 of Liu et al., 2004a). After the PFC was dissected out, the anterior portion of the brain, cerebellum and brain stem were cut off and the remaining parts of the brain were separated by making a cut along the longitudinal fissure. The ventro-medially located white matter was then care fully removed to expose the hippocampus.

The HPLC system consisting of a programmed solvent delivery system at a flow rate of 1.5 ml/min, an autosampler, a reversed-phase C18 column, and a fluorescence detector set at the excitation wavelength of 252 nm and emission wavelength of 515 nm. Identifications of spermidine and spermine were accomplished by comparing the retention times of samples with the known standard. Assay validation showed that the analytical method was sensitive and reliable with acceptable accuracy (88 –112% of true values) and precision (intra- and inter-assay CV 15%). The concentrations of spermidine and spermine in tissue were calculated with reference to the peak area of external standards and values were expressed as
g/g wet tissue. Because of its low level in the brain tissues, putrescine concentrations in brain tissue samples were measured by a highly sensitive liquid chromatography/mass spectrometric (LC/MS/MS) method (Zhang et al., 2007). After adding internal standard to 20
l of the perchloric acid extracts, the samples were alkalized with saturated sodium carbonate and derivatized with dansyl chloride. Putrescine and its internal standard were extracted with toluene. The toluene phase was evaporated to dryness, reconstituted and injected onto the LC/MS/MS system. The samples were analyzed by a reversed-phase C18 column (1502.0 mm, 5
m, Phenomenex) with 80% acetonitrile: 20% water containing 0.1% formic acid as mobile phase at a flow rate of 0.2 ml/min. The retention time of putrescine and the internal standard were 4.0 and 4.8 min, respectively. The total run-time was 15 min.Detection by MS/MS used an electrospray interface (ESI) in positive ion mode. 

 A one-way ANOVA revealed significant differences between groups in the putrescine/spermidine molar ratios in CA1 (F(2,24)5.86; P0.0085), DG (F(2,24)6.87; P0.004), PFC (F(2,24)4.28; P0.026) and EC (F(2,24)4.22; P0.027), but not CA2/3 (F(2,24)2.85; P0.75), PRC (F(2,24)1.71; P0.20), POR (F(2,24)2.68; P0.09) and TE (F(2,24)0.49; P0.62). Post hoc tests revealed that the putrescine/spermidine ratios were significantly decreased in the aged CA1 (P0.05), DG (P0.01) and PFC (P0.05), and the middle-aged CA1 and DG (all P0.01). There was also a significant difference between the aged and middle-aged PFC (P0.05) with the lower ratio in the former. In EC, the putrescine/spermidine ratios were significantly increased in the aged and middle-aged groups relative to the young one (all P0.05) with no difference between the former two. For the spermidine/spermine molar ratio, a one-way ANOVA revealed highly significant differences between groups in CA1 (F(2,24)16.31; P0.0001), CA2/3 (F(2,24) 19.49; P0.0001), PFC (F(2,24)13.24; P0.0001) and PRC (F(2,24)18.39; P0.0001), but not DG (F(2,24) 2.05;P0.15),EC (F(2,24)0.90;P0.42),POR (F(2,24) 1.90; P0.17) and TE (F(2,24)0.42; P0.66). Post hoc tests revealed that the spermidine/spermine ratios were significantly increased in the aged CA1, CA2/3 and PRC (all P0.001), and the middle-aged CA1 (P0.01), CA2/3 (P0.01) and PRC (P0.001). There were also significant differences between the aged and middle-aged groups in CA1 (P0.05) and CA2/3 (P0.01). For PFC, a significantly increased spermidine/spermine ratio was found in the middle-aged group relative to the aged and young groups (all P0.001) and there was no difference between the latter two. 

 It has been shown that aging results in hypofunction in the DG due to reduced synaptic contacts from the EC and hyperfunction in the CA3 due to reduced cholinergic inputs and/or GABA receptor expression (for a review see Wilson et al., 2006). In the present study, spermine did not change with age in any sub-regions examined. There were age-associated decreases in putrescine and the putrescine/spermidine ratios in the CA1 and DG, and increased putrescine level with age in the CA2/3. Significantly increased levels with age in spermidine and spermidine/spermine ratios were noticed in the CA1 and CA2/3, and there were no differences between groups in the DG. Collectively, these results suggest that the aging process affects polyamines differently across the three sub-regions of the hippocam pus. Virgili et al. (2001) compared the putrescine, spermidine and spermine levels in the whole hippocampus between aged and young rats, and failed to detect agerelated alterations for all three polyamines. Given the differential effects of aging on polyamines across the subregions of the hippocampus described above, the examination of the whole hippocampus may contribute greatly to their negative results. The EC, PRC and POR (parahippocampal in primates) (the parahippocampal region) are the gateway to the hippocampus. The hippocampus receives spatial information through the POR (parahippocampal) and medial entorhinal area, and non-spatial information through the PRC and lateral entorhinal area (Manns and Eichenbaum, 2006). It has been shown that the parahippocampal region is critically involved in complex functions, such as memory, object recognition, sensory representation and spatial orientation (Witter and Wouterlood, 2002). 

Moreover, the EC and PRC are affected initially and severely in Alzheimer’s disease, a neurodegenerative disease characterized by the progressive memory loss (Braak and Braak, 1991; Van Hoesen et al., 2000). Our recent research has demonstrated that the parahippocampal region is also affected greatly by aging (Liu et al., 2003a,b, 2004b, 2008a,b, in press). In the present study, increased level of putrescine with age was found in the EC and aging did not alter the spermidine levels in the parahippocampal region structures. For spermine, age-related increase was seen in the POR, whereas decreased level with age was observed in the PRC. Furthermore, significantly increased putrescine/ spermidine and spermidine/spermine ratios with age were found in the EC and perirhinal cortices, respectively. The PFC is involved in a variety of memory functions and is vulnerable to age-associated deterioration (Shimamura, 1995; Greenwood, 2007). Interestingly, age-related changes in all three polyamines were found in the PFC with decreased putrescine level and putrescine/spermidine ratio, and increased spermidine and spermine levels and spermidine/ spermine ratio. By contrast, all three polyamines in the TE (an auditory cortex) were not affected by aging. These results clearly demonstrate that the aging process has differential effects on polyamines across the five cortical regions examined. Das and Kanungo (1982) reported agerelated decrease in spermidine and spermine, but not putrescine, content in the rat cerebral cortex, whereas Virgili et al. (2001) failed to detect age-associated changes in all three polyamines in the cortex.

 Since the sub-regions of the cortex were not examined in these two studies, a direct comparison of age-related changes in polyamines cannot be made with the present one. The underlying mechanisms and functional significance of age-related region-specific alterations in polyamines observed in this study are not fully understood at present. It has been well documented that the physiological concentrations of polyamines are essential for cells to grow and/or function in an optimal manner (for reviews see Oredsson, 2003; Wallace et al., 2003). Because of their nature as polycations, putrescine, spermidine and spermine can interact with negatively charged DNA, RNA, proteins and phospholipids to stabilize their structure (Carter, 1994). Therefore, decreased polyamine levels could potentially affect DNA, RNA and protein synthesis and other metabolic function, and lead to the neuronal dysfunction. Previous studies have demonstrated that aging impairs neurogenesis in the DG dramatically and that reduced rate of hippocampal neurogenesis correlates to age-associated memory impairments (Driscoll and Sutherland, 2005; Wati et al., 2006). Given the role of polyamines in neurogenesis (Malaterre et al., 2004), decreased putrescine levels in the DG in both aged and middle-aged rats (see Fig. 1) may contribute significantly to age-related impairments in hippocampal neurogenesis. It has been shown that GABA, the major inhibitory neurotransmitter in vertebrate brain, can be formed from putrescine and there are close regulatory interrelations between GABA and putrescine (Seiler and Al-Therib, 1974; Seiler et al., 1979). 

Thus, altered putrescine levels could potentially affect the neuronal excitability. Given the hyperactivity of CA3 neurons in the aged rats (for a review see Wilson et al., 2006), it is likely that increased putrescine levels in the CA2/3 with age observed in this study may be a compensatory mechanism to normalize the excitability of CA3 neurons. Polyamines modulate learning and memory by interacting with the polyamine binding site at the NMDA receptors (Rock and Macdonald, 1995; Williams, 1997). The NMDA receptor contains at least two families of subunits: NMDAR1 (NR1) and NMDAR2 (NR2A-D). NR1 is necessary and sufficient for the formation of functional channels of the NMDA receptor, whereas NR2 enhances the activity of the receptor when coupled with the NR1 subunit (Magnusson, 1998). Considerable research suggests that aging affects NMDA receptors in the hippocampus (Clayton and Browning, 2001; Magnusson et al., 2006). We have recently observed that NR1 and NR2A protein levels significantly decreased in the aged parahippocampal region and CA2/3 region of the hippocampus (Liu et al., 2008b, in press). The protein expression deficit of NR2B has been shown to be correlated with spatial learning and memory impairments tested in the water maze task and increased expression of NR2B improves learning and memory function in the aged brain (Clayton et al., 2002; Cao et al., 2007). It is of interest to note that NMDA receptors having NR2B subunit are highly sensitive to polyamines (Williams et al., 1994; Williams, 1997) and that polyamines can also be tightly regulated by the NMDA receptor (Fage et al., 1992).