HZPPOCAMPUS, VOL. 1, NO. 4, PAGES 391-398, OCTOBER 1991

Decreased Glutamate Release Correlates With Elevated Dynorphin Content in the Hippocampus of Aged Rats With Spatial Learning Deficits Wan-Qin Zhang," William R. Mundy," Linda Thai,* Pearlie M. Hudson, * Michela Gallagher ,t Hugh A. Tilson," and J. S. Hong* *Laboratory of Molecular and Integrated Neuroscience, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 U.S.A. a n d tDepartment of Psychology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514 U.S.A.

ABSTRACT The effects of aging on extracellular glutamate and tissue dynorphin content in the hippocampus were examined in Fischer-344 rats. Young adult (4-month-old) and aged (24-month-old) rats were trained to find a hidden platform in the Morris water task. Aged rats were unable to acquire the spatial learning task as rapidly as young controls. Following behavioral testing, an in viiw microdialysis perfusion method was used to determine extracellular glutamate levels in the hippocampus. There was a 2S-35% reduction in extracellular glutamate concentration in both dorsal and ventral hippocampus of aged rats compared to young rats, in the absence of any change in tissue glutamate levels. Radioimmunoassay showed an increase in dynorphin A(1-8)-like immunoreactivity [DYN-A( 1-8)LII in both dorsal and ventral hippocampus, but not striatum, of aged rats. Immunocytochemistry indicated that this increase was localized to the dentate granule cells and mossy fibers. Furthermore, among the aged rats the increase in DYN-A(1-8)LI was inversely correlated with the decrease in extracellular glutamate. These results suggest that the disregulation of dynorphin observed in cognitively impaired aged rats is related to reduced excitatory transmission within the hippocampal formation. Key words: extracellular glutamate, dynorphin, hippocampal formation

There is considerable evidence that learning and memory deficits in aged animals are related to changes in the neurobiology of the hippocampus. In particular, several studies have identified morphological and functional correlates specifically related to the decline in spatial learning and memory in aged rats. In a study of regional glucose metabolism in aged rat brains, Gage et al. (1984) found a significant correlation between diminished 2-deoxyglucose in hippocampus, septum, and cortex and the degree of spatial learning impairment. Geinisman et al. (1986) reported that aged rats with spatial memory impairments show a loss of axospinous synapses in the dentate gyrus compared to unimpaired aged and young rats. Several electrophysiological correlates of spatial memory deficits in aged rats have been found, including a more rapid rate of decay of long-term enhancement in the hippo~ _ _ _ Correspondence and reprint requests to William R. Mundy, U.S. Environmental Protection Agency, Neurotoxicology Division, MD-74B, Research Triangle Park, NC 27711 U.S.A. -

campus (Barnes and McNaughton, 1985)and loss of spatially selective firing in hippocampal complex spike cells (Barnes et al., 1983). Changes that coincide with spatial learning deficits have also been observed in cholinergic function in the aged hippocampus, including loss of acetylcholinesterase (Biegon et al., 1986) and a decrease in muscarinic receptor binding (Gallagher et al., 1990). Dynorphin is an endogenous opioid peptide that is localized in the dentate gyrus granule cells and their mossy fiber projections within the hippocampal formation (McGinty et al., 1983; Zamir et al., 1985). In a recent study, Jiang et al. (1989) found a significant elevation of DYN-A( 1-8) and dynorphin mRNA in the aged hippocampus compared to that in young adults, and this elevation was associated with a deficit in spatial learning ability. Those findings suggested that elevated levels of hippocampal dynorphin are related to the behavioral impairment observed in aged rats. This suggestion is consistent with the results of a study by McDaniel et al. (1990) that found that microinjection of dynorphin into the hippocampus of young rats produced a similar impairment in spatial learn-

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ing. Thus, a disregulation of dynorphin expression during aging may contribute to behavioral impairments in aged animals. There is evidence that dynorphin expression in the hippocampus is negatively regulated by neuronal activity. For example, increased neuronal activity induced by electroconvulsive shock or electrical kindling decreases the level of DYN-A(1-8) and dynorphin mRNA in dentate granule cells (Kanamatsu et al., 1986; McGinty et al., 1986). Similar results were obtained when the dentate granule cells were stimulated directly (Morris et al., 1988). Stimulation of the perforant path, a glutamate-containing pathway providing a major excitatory input to the hippocampus, also decreases DYN-A( 18) and dynorphin mRNA (Mitchell et al., 1987; Xie et al., 1990a). During normal aging, there is a loss of perforant path synaptic connections with granule cells (Geinisman and Bondaroff, 1976) and inputs become more sparse, as indicated by a decrease in the perforant path afferent volley (Barnes, 1979; Barnes and McNaughton, 1980).Thus, the elevated dynorphin content observed in aged rats may be a consequence of age-related decreases in perforant path function. In the present study, the release of glutamate, the excitatory amino acid transmitter in the perforant path, was examined in aged rats that were impaired in a spatial learning task. Glutamate in the hippocampus was measured using in vivo microdialysis, which allows for the continual monitoring of extracellular neurotransmitter levels in freely moving animals. The relationship between glutamate release and dynorphin content in the hippocampus was examined.

MATERIALS A N D METHODS Subjects Male Fischer-344 rats at 4 months (young) and 24 months (aged) of age at the time of testing were used as subjects. All animals were obtained from the aging colony at the National Institutes of Health (NIH) and allowed to acclimate to the National Institute of Environmental Health Sciences (NIEHS) colony room for I month prior to the experiments. The rats were housed separately in a room controlled for temperature (22" t 2°C) and humidity (50 2 10%) with a 12 h/ 12 h lightidark cycle. Food (NIH diet 31) and water were provided ad libitum.

Behavioral testing Young (n = 20) and aged (n = 40) animals were tested for spatial learning ability in a Morris water task. Seven aged rats that were unable to complete behavioral testing were dropped from the study. The rats were trained to swim to a platform hidden in a large circular pool (148-cm diameter x 60-cm high) located in a test room containing numerous extramaze cues. The pool was filled with water (28" 2 2°C) to a depth of 40 cm. A transparent platform 10 cm in diameter was submerged 1.5 cm below the water surface. Four equally spaced locations around the edge of the pool served as start points and divided the pool into quadrants. The rats received five daily sessions consisting of four trials. On each day all four starting points were used once in a random sequence without replacement. A trial began by

placing the rat into the pool facing the wall at one of the starting points. The latency to find the escape platform was recorded up to a maximum of 60 seconds. If a rat did not escape onto the platform within that time, it was placed on the platform where it remained for 15 seconds. For each rat, the platform was fixed in the center of one of the quadrants and remained in that location for the duration of the experiment. On day 6 each rat was tested for the extent of spatial learning in a 60-second probe trial. During this trial the platform was removed and the time spent swimming in each of the quadrants was recorded for each rat.

Microdialysis In vivo microdialysis was determined on the day after completion of behavioral testing. An additional seven aged rats not tested behaviorally were used in the neurochemical studies. Dialysis probes were constructed as described previously (Zhang et al., 1988) and were stereotaxically implanted into the dorsal or ventral hippocampus. The coordinates were 3.3 mm posterior to bregma, 2.5 mm lateral to the midline, and 6.0 mm vertical below dura for the dorsal hippocampus, and 5.8 mm posterior to bregma, 5.0 mm lateral to the midline, and 7.5 mm vertical below dura for the ventral hippocampus. The rats were housed in plastic cages in the laboratory and allowed to habituate for 2 days before beginning the perfusion. The dialysis probe was connected by polyethylene tubing (PE-10) through a swivel (which allowed the rats to move freely) to a Harvard infusion pump, which continually perfused the dialysis probe with artificial cerebrospinal fluid (ACSF) at a flow rate of 2.0 pL/min. Twenty hours after continuous perfusion with ACSF, three consecutive 1-hour perfusates were collected to determine the basal levels of extracellular amino acids. Data from rats with cannulae that were not patent or found outside of the target area were excluded. Thus, data used for statistical analysis include young (dorsal, n = 10; ventral, n = 8) and aged (dorsal, n = 17; ventral. n = 18) rats.

Amino acid determination Contents of amino acid transmitters in perfusates or brain tissue were determined by high-performance liquid chromatography (Hudson et at., 1986). Samples were injected onto an ion exchange column (Aminex 9, 250 x 4 rnm, Bio-Rad Laboratories, Richmond, CA) maintained at 62°C. Amino acids were eluted at a flow rate of 0.6 mL/min with programmed gradients of solvents A (0.2 M sodium citrate, pH = 3.2) and B (0.2 M sodium borate, pH = 10.0). O-phthaldialdehyde reagent was then combined with the column eluent at a flow rate of 0.3 mL/min. Detection of fluorescent isoindol derivatives of sample amino acids (excitation wavelength of 340 nm with an emission filter at 420 nm; fluorescence detector model 420E, Waters Associates, Milford, MA) were compared with those of amino acid standards for taurine, aspartate, glutamate, glutamine, glycine, and leucine (internal standard). The data for amino acid content were based on integrated peak areas and calculated using simple linear interpolation.

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DECREASED GLUTAMATE RELEASE AND DYNORPHIN CONTENT / Zhang et al.

Tissue content of dynorphin The rats were sacrificed after the last collection, and their brains were removed and dissected into dorsal and ventral hippocampus and striatum. The tissue content of DYN-A( I 8)-like immunoreactivity [DYN-A(l-8)LI] was determined by radioimmunoassay as described previously (Kanamatsu et al., 1986). Briefly, tissue was homogenized in 2 M acetic acid and immersed in boiling water for 5 minutes. After centrifugation at 15,000 x g for 20 minutes, the supernatant was lyophilized. Aliquots of the reconstituted sample were used for radioimmunoassay. Iodinated DYN-A( 1-8) was used as a tracer. An antiserum against DYN-A(l-8) was used that does not cross-react with [Met’]-enkephalin, [Leu5]-enkephalin, or DYN-A(1-17). To determine the location of dynorphin in the hippocampal formation, five aged and five young rats not tested behaviorally were anesthetized and perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4), followed by overnight postfixation in the perfusion medium. Vibratome sections !50 p n thick were then incubated in a rabbit antiserum against DYN-A(]-8) for 48 hours at 4°C. Tissues were then transferred into the secondary antibody (goat antirabbit IgG serum) for 1 hour, with another hour of incubation in the avidin-biotin peroxidase complex, followed by 10 minutes of diaminobenzidine for the peroxidase reaction. Tissue from young and aged rats was run concurrently. For a detailed description, see McGinty et al. (1983).

Statistical analysis For repeated observations on the same animal, a repeatedmeasures analysis of variance (ANOVA) was used to test for overall statistical significance. If a significant overall effect was observed, post hoc comparisons were made using Fisher’s least significant difference test. comparison of two means was made using a two-tailed Student’s t-test for statistical significance. Correlations were examined using the Pearson product-moment correlation.

RESULTS The spatial learning ability of young and aged rats was tested in the Morris water task. Figure 1A shows the latency to escape onto the hidden platform averaged for each test day. A two-way repeated-measures ANOVA (age x day) indicated a significant effect of age ( P < .001), day ( P < .001), and a significant age X day interaction (P < .001). Young rats learned to swim directly to the hidden platform over the 5-day training period, with escape latencies decreasing to 10 seconds on day 5. In contrast, aged rats were significantly slower to locate the platform and improved little with training. The impairment of spatial learning in the aged animals was confirmed by the results of the probe trial on day 6. Figure IB shows the percentage of time spent swimming in the training quadrant (which formerly contained the platform) and the opposite quadrant. A two-way repeated-measures ANOVA (age X quadrant) indicated a significant effect of quadrant (P < .001) and a significant age x quadrant interaction ( P < .001). Post hoc analysis showed that aged rats spent significantly less time in the training quadrant (P < .01) and significantly more time in the opposite quadrant ( P < .05)

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than young rats. However, both young and aged rats showed a spatial bias, preferring the training quadrant to the opposite quadrant (P < .001 for young and aged). To determine the relationship between levels of DYN-A(l8)LI and glutamate in the hippocampus, rats were implanted with microdialysis probes in the dorsal or ventral hippocampus, and the level of extracellular glutamate release was measured over a 3-hour period. A two-way (age x time) ANOVA indicated that extracellular glutamate levels remained stable over the 3-hour time period (effect of time not significant). The level of extracellular glutamate was lower in the aged animals in both the dorsal and ventral hippocampus (Fig. 2, top). However, this effect was significant only in the ventral hippocampus (effect of age, P < .005). The extracellular levels of other amino acids in dorsal and ventral hippocampus is given in Table 1. No effect of age on amino acid release was observed (all P > .l) except for aspartate in the ventral hippocampus (P < .05). Tissue levels of glutamate were also determined for dorsal and ventral hippocampus. Glutamate levels were similar in both young and aged rats (mean ? SEM = 10.5 ? 1.3 and 9.3 2 0.3 nmolimg tissue in dorsal hippocampus and 9.2 ? 0.2 and 9.5 ? 0.4 nmol/mg tissue in ventral hippocampus for young and old, respectively). Tissue levels of DYN-A( 1-8) were determined upon completion of the microdialysis procedure. The levels of DYNA(1-8)LI in dorsal and ventral hippocampus are shown in Figure 2 (bottom). There was a significant age-related elevation in both dorsal and ventral hippocampus (P < .001, Student’s t-test). As previously reported (Jiang et al., 1989), we found no effect of age on striatal dynorphin content (mean

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sures in the young group. The correlation between DYNA(l-8) and aspartate in the ventral hippocampus in aged rats was also not significant (Pearson’s r = 0.33, P > .1).

DISCUSSION

In the present study, aged rats that were impaired in a spatial learning task were shown to have an elevation of DYNf A(1-8)LI in the hippocampus compared with that of young rats. Immunocytochemistry revealed that this increase was 0 localized in the dentate granule cells and mossy fibers. This elevation was not observed in the striatum, another neuroanatomical region that contains high levels of opioid peptides including dynorphin. The present results in aged Fischer-344 rats confirm the age-related increase in hippocampal DYN-A(1-8)LI observed by Jiang et al. (1989) in Long-Evans rats and support the observation that increased levels of hippocampal dynorphin are associated with spatial d learning and memory deficits. It is of interest that the outbred strain of Long-Evans rats showed a much greater variability Fig. 2. (Top) Extracellular level of glutamate in dorsal and in the effects of aging on spatial learning ability, with a subset ventral hippocampus measured by in vivo microdialysis in freely moving rats. Data are means 2 SEM for three con- of aged animals learning the spatial task as rapidly as younger secutive hourly determinations (young: dorsal, n = 10, ven- controls (Jiang et al., 1989). In contrast, the aged Fischer-344 tral, n = 8; aged: dorsal, n = 17, ventral, n = 18). *Signif- rats (an inbred strain) used in the present study were conicantly different from young ( P < .005, ANOVA). (Bottom) sistently poor learners. Tissue content of DYN-A(l-8)LI in dorsal and ventral hipThe perforant path, which originates in the entorhinal corSEM (young: n = 18; aged: n tex, provides a major cortical input to the hippocampal forpocampus. Data are mean = 35). **Significantly different from young (P < .001, Stumation in rats (Hjorth-Simonsen and Jeune, 1972; Segal and dent’s t-test). Landis, 1974; Steward, 1976) and uses the amino acid glutamate as an excitatory neurotransmitter. Thus, stimulation 2 SEM = 16.9 +- 0.8 and 15.0 1.0 pmol/g tissue for young of the perforant path leads to the increased release of gluand aged, respectively). Immunocytochemical staining of tamate (Nadler et al., 1976; White et al., 1977). Conversely, DYN-A( 1-8) in the dorsal hippocampus indicated that im- decreased activity in the perforant path would result in a demunoreactivity was greatly enhanced in the dentate gyrus crease in glutamate release. In the present study, glutamate granule cells and mossy fibers of aged rats as compared to release in the hippocampus was examined using an in vivo young (Fig. 3). microdialysis procedure that allows for the continuous monIn order to examine more fully the relationship between itoring of neurotransmitter levels in the extracellular space dynorphin levels and amino acids in aged rats, correlations (Beneveniste, 1989).Extracellular glutamate levels were found were made between DYN-A(1-8)LI and extracellular amino to be stable over the 3-hour collection period, although there acid levels in dorsal and ventral hippocampus (Fig. 4). There was considerable variation in glutamate levels between inwas a significant negative correlation in both dorsal (Pear- dividual animals. In aged rats there was a 35% reduction in son’s r = -0.76, P < .05) and ventral hippocampus (Pear- basal levels of extracellular glutamate in both dorsal and venson’s r = -0.55, P < .05) for glutamate, indicating that de- tral hippocampus, as compared to young controls. This recreased glutamate release in the hippocampus was associated duction occurred in the absence of a comparable decrease in with increased levels of DYN-A(1-8)LI among the aged rats. the tissue concentration of glutamate in dorsal or ventral hipIn contrast, there was no significant correlation for these mea- pocampus. In addition, there was a small (14%) but significant 0-

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Table 1 . Basal Extracellular Level of Amino Acids From Dorsal and Ventral Hippocampus in Young and Aged Rats Amino Acid Release (pmolih)

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Dorsal Young(n = 10) Aged(n = 17) Ventral Young (n = 8) Aged (n = 18)

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73.3 (6.91) 76.1 (4.34)

2754 (233) 3257 (326)

204.1 (30.4) 220.0 (23.2)

99.7 (4.96) 86.2* (2.841

2298 (317) 2388 (258)

230.0 (22.4) 280.6 (42.7)

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296.0 (41.3) 309.0 (29.6) 370.8 (53.9) 365.9 (27.9)

Samples collected over a 3-hour period. Tau, taurine; Asp, aspartate; Gln, glutamine; Gly, glycine * Significantly different from young ( P < .05).

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Fig. 3 . Immunocytochernical staining of DYN-A(l-8) in dorsal hippocampus from a typical (A) young and (B) aged rat. Note enhanced dynorphin immunoreactivity in dentate granule cells and mossy fibers in the aged rat.

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by an increase in dynorphin mRNA (Jiang et al., 1989). The mechanism underlying a possible glutamate control of dynorphin mRNA expression is unclear. Inhibitory GABAergic interneurons are located in the dentate gyrus in association with granule cells (Ribak et al., 1978), and activation of the inhibitory interneurons may alter dynorphin mRNA. Alternatively, glutamate-activated second messengers in granule cells may act directly to suppress dynorphin gene expression.

References r = -0.55 p ( 0.05

Tissue content of DYN A (1-8) ( pmol/g tissue ) Fig. 4. Correlation of extracellular glutamate level and DYNA(1-8)Ll in aged rats. (Top) Dorsal hippocampus (n = 17). (Bottom) Ventral hippocampus (n = 18). There was a significant correlation between extracellular glutamate and tissue level of DYN-A(1-8)LI in both dorsal (Pearson’s r = -0.76, P < .05) and ventral (Pearson’s r = -0.55, P < .05) hippocampus.

decrease in extracellular aspartate. These results suggest that there may be a reduction in neuronal release of excitatory transmitters within the aged hippocampus and are consistent, in particular, with previous work indicating that the perforant path innervation of the dentate granule cells becomes more sparse with age (Geinisman and Bondareff, 1976; Barnes, 1979; Barnes and McNaughton, 1980). Previous work has demonstrated that stimulation of the perforant path decreases DYN-A( 1-8)LI content in the hippocampus (Mitchell et al., 1987), indicating that dynorphin expression may be negatively regulated by neuronal activity. The present results show a significant negative correlation between glutamate release and dynorphin levels in the hippocampus, with increased DYN-A(1-8)LI in aged rats associated with decreased glutamate release. The technique used to monitor glutamate efflux in the present study lacks sufficient anatomical resolution to determine the precise sites of glutamate release. However, the inverse correlations noted between the degree of glutamate reduction and the magnitude of dynorphin elevation is consistent with a model developed from other studies indicating that dynorphin is negatively regulated by inputs onto granule cells. This converging evidence suggests a regulatory role for glutamate on dynorphin levels in the hippocampus and is consistent with the observation that glutamate antagonist administration prevents the decrease in hippocampal dynorphin content induced by perforant path stimulation (Xie et al., 1990b). The regulation of dynorphin appears to be at the level of the genome, since recent studies using in situ hybridization and RNA blot analysis have shown a decrease in dynorphin mRNA after stimulation of the perforant path or dentate gyrus (Morris et al., 1988; Xie et al., 1990a). In addition, the elevation in hippocampal dynorphin in aged rats is accompanied

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Decreased glutamate release correlates with elevated dynorphin content in the hippocampus of aged rats with spatial learning deficits.

The effects of aging on extracellular glutamate and tissue dynorphin content in the hippocampus were examined in Fischer-344 rats. Young adult (4-mont...
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