Age-related alterations in noradrenergic input to the hippocampal formation: structural and functional studies in intraocular transplants

269

Brain Research, 478 (1989) 269-280 Elsevier BRE 14196

Age-related alterations in noradrenergic input to the hippocampal formation: structural and functional studies in intraocular transplants Maria Eriksdotter-Nilsson 1, Greg Gerhardt 2,/~ke Seiger 3, Lars Olson 1, Barry Hoffer 2 and Ann-Charlotte Granholm 2 1Departmentof Histology and Neurobiology, Karolinska Institutet, Stockholm (Sweden), ZDepartmentof Pharmacology and Psychiatry, Universityof Colorado Health Sciences Center, Denver, CO (U.S.A.) and 3Departmentof NeurologicalSurgery, University of Miami School of Medicine, Miami, FL (U.S.A.) (Accepted 12 July 1988)

Key words: Hippocampal formation; Intraocular transplantation; Aging; Immunohistochemistry; Electrophysiology; Electrochemistry

Intrinsic versus extrinsic determinants of age-related alterations in hippocampal noradrenergic transmission were investigated using intraocular allografts in rats. Three groups of animals were examined: young hippocampal transplants in young hosts, old transplants in old hosts and young transplants in old hosts. Postsynaptic sensitivity to noradrenaline (NA) v;as measured by extracellular recordings of spontaneous activity and superfusion with known concentrations of catecho!anfines in the anterior chamber of the eye. Hill plots demonstrated that the dose-response relationships of NA-induced depressions were linear and parallel in the 3 groups. Aged hippocampal grafts displayed a highly significant subsensitivity to NA of one order of magnitude. The ECs0 for this group was 203.1 gM as compared to 29.2 in young grafts. Young intraocu!a~ grafts in old hosts responded similarly to transplants in young hosts, with an ECs0 of 32.4/~M for the depressant actions cf NA. Collaterals of the host iris sympathetic ground plexus invaded the hippocampal grafts. The density of this noradrenzrgic innervation was estimated by immunohistochemistry for tyrosine hydroxylase. A slightly increased density and fluorescence intensity of the noradrenergic fibers were observed in the old transplants as compared to the young transplants in young and old hosts. This was correlated with a significantly (P < 0.01) increased content of NA in old transplants, as measured with high performance liquid chromatography. The old transplants also contained a large number of autofluorescent lipofuchsin granules, which were absent in the young traasp!ants, regardless of the recipient age. Taken together, these results suggest the existence of alterations in pre- as well as postsynaptic noradrenergic mechanisms in the aging hippocampus. These changes were dependent on transplant age rather than host age, thus suggesting an involvement of intrinsic rather than extrinsic determinants in this model system. INTRODUCTION The hippocampal f,Jrmation is known to play a critical role in m em o r y and learning processes 73' 74,sL87,Bs. In behavioral studies it has been shown by several investigators that also aged rats show memory deficits 7'31'4~'6°'8°. There is evidence that age-related deficits in m emo r y are closely related to electrophysiological changes in the rat hippocampal for° mation 5,6. In particular, pyramidal neurons seem to be important for m e m o r y processing 74'7s. In aged

rats, a significant cell loss has been reported in the pyramidal layer of the hippocampal formation 49 and hippocampal astrocytes increase in size with age n. Recently, Bickford-Wimer et al.~° reported a significant reduction of the inhibitory potency of locally applied noradrenaline ( N A ) on hippocampal pyramidal neurons in aged Fischer 344 rats. The noradrenergic system has been suggested to be involved in learning and memory modulation 33,4s'57'Ts, although a clear relationship of noradrenergic transmission to memory is still a controversial issue. A n u m b e r of studies

Correspondence: M. Eriksdotter-Nilsson, Department of Histology and Neurobiology, Karolinska Institutet, Box 60400, S-104 Stockholm, Sweden. 0006-8993/89/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

270 have demonstrated age-related alterations in the noradrenergic system, including: (1) synthesis and activity of synthesizing enzymes26'ss; (2) number of locus coeruleus noradrenergic neurons79'sa; (3) NA levels3,25,34,36,Tl.82; and (4) electrophysiological sensitivity of target neurons tc i~xIA9'10'45"46'56. An age-related decline in postsynaptic NA responsiveness has also been reported in a number of brain areas such as cerebellum s,37,55and cortex cerebri 45'46. A major issue in aging research is the differentiation of direct vs indirect determinants of senescence. By varying the host and the transplant age the intraocular grafting model can be utilized to defineate extrinsic and intrinsic factors regulating development and aging in defined brain regions 24'37. Thus, if the age-related biological properties of a graft maintain the donor age timetable, intrinsic determinism would be postulated; in contrast, if the biological properties shifted to the host age time table, extrinsic determinism would be suggested. Recently we found that Purkinje neurons in old cerebellar grafts manifested a marked postsynaptic subsensitivity to superftlsed NA 37. This finding has also been reported for cerebellum in situs,9,55"56. The hippocampus is a well characterized area (for review see ref. 76), receiving noradrenergic innervation 13'29 from the ipsilateral locus coeruleus 2'44'51'7s. Hippocampal grafts in oculo have been shown to mature in an organotypic manner 64 and to receive a functional adrenergic innervation from the sympathetic ground plexus of the host iris2s,77. Such hippocampal grafts also manifest many physiological and pharmacological properties resembling hippocampus in situ 2s'40"77, such as the ability to produce and sustain epileptic activity. This study focuses on age-related pre- and postsynaptic alterations in noradrenergic transmission in hippocampal transplants. We have examined postsynaptic sensitivity of superfused NA on glutamate-induced pyramidal cell discharge rate. The NA innervation was evaluated with postmortem high performance liquid chromatography and with immunohistochemistry. Three experimental groups were used to allow independent manipulation of graft and host age and thus examine intrinsic vs extrinsic determinants of any possible age-related noradrenergic changes.

MATERIALS AND METHODS

Experimental groups Three groups of rats were examined. In the first group (young/young) the grafts were examined 2-3 months after traasplantation into the eyes of young adult (150 g) rats. The second group (old/old) was studied at a graft age of 22-23 months and a host age of 24-25 months. The third group (young/old) consisted of fetal hippocampal tissue transplanted to the anterior eye chamber of 20-month-old rats. Thus, at the time of recording, the grafts were 2-3 months old and the hosts were 22-23 months old (Fig. 1).

Homologous hippacampal grafts Pieces of fetal hippocampal formation, taken at gestational day 1~, were transplanted bilaterally into the anterior chamber of the eye of adult female Sprague-Dawley rats (Alab, Stockholm, Sweden) as previously described 6s. Growth, vascularization and survival of the grafts were observed at regular intervals through the cornea of ether-anesthetized recipients using a stereomicroscope. The transplant size was derived from measurements of the length and the width of the usually ovoid grafts as described 12. Transplants were allowed to mature for at least 8 weeks in oculo before recording.

Electrophysiological recordings Host animals were anesthetized with urethane (1.25 g/kg i.p.) and the cornea overlying the transplant was removed. A plexiglass chamber was placed over the eye for superfusion with 37 °C Earle's balanced salt solution as previously described 39. Extracellular recordings from intraocular hippocampal grafts were performed using a single-barrel glass micropipette filled with 5 M NaCI. After amplification, single action potentials were displayed on an oscilloscope, separated from background activity, converted to constant voltage pulses, and integrated at 1 s intervals to indicate discharge rate 39. Electroencephalographic (EEG) activity was also amplified and displayed directly on a strip chart recorder together with the ratemeter output. NA (1/zM-1 mM) and sodium penicillin-G (1000-1600 U/ml) were administered in the superfusate for a minimum of 10 min to allow for the drug concentration in the perfusion chamber to reach equilibrium. Recovery periods

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Fig. 1. Illustration of the experimental design. Th~'eeexperimental groups were studied. The group named y/y (young grafts in young hosts) consisted of fetal hippocampal tissue transplanted (TP) to 2 month-old host rats and were thereafter allowed to mature 2-3 months before recording from the graft. The group y/o (younggrafts in old hosts) consisted of fetal hippocampal tissue transplanted to 20 month-old host rats, then remaining in the eye 2-3 months before recording. Similarly, the group o/o (old grafts in old hosts) consisted of fetal hippocampal grafts, grafted to 2 month-old rats, and then remaining in the host eye for 22-23 months before recording. were also 10 min or longer. Sodium glutamate (50 mM) was usually added to the glass micropipette solution to augment firing of the transplanted hippocamoal pyramidal neurons. Data from cells which showed a decrease in action potential amplitude during drug superfusion or did not show a recovery after cessation of drug application were discarded. Doseresponse curves were constructed from the ratemeter records and ECs0 values with 95% confidence limits were computed by linear transformation using Hill plot analysis.

Louis, MO, U.S.A.). Fourteen/~M thick cryostat sections were incubated with antisera to tyrosine hydroxylase (TH), raised in rabbits (Eugene Tech International, N J), diluted 1:50 in phosphate-buffered saline and processed according to the indirect immunofluorescence technique ~9. Details of the immunohistochemical procedure have been described elsewhere 24. Sections were examined with a fluorescence microscope using epi-illumination. Consecutive sections were stained with Cresyl violet and examined with a light microscope. The TH-positive fiber density of the grafts was evaluated semiquantitatively on a blind basis usi.ng a scale of arbitrary units between 0 and 3+ with 7 steps. RESULTS

Intraoculargrowth The hippocampal grafts became rapidly vascularized and grew extensively in oculo in all 3 groups. After the first few months in oculo, the transplants ceased to grow and the graft size thereafter remained unchanged. The aged grafts that remained in oculo for 23 months did not decrease in size with age. The young transplants in the old hosts reached a smaller final size than young grafts in young rat hosts.

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Morphology

Transplants were rapidly removed from the eyes, weighed, frozen on dry ice and stored at -70 °C. Tissue content of NA was measured by high-performance liquid chromatography (HPLC) with electrochemical detection (EC) using the method of McKay et al. 59. Dihydroxybenzylamine was used as an internal standard to calculate recovery. Monoamine and metabolite values were calculated as total ng/g wet weight of tissue.

Young transplants in young adult hosts (Fig. 2a,b) and old transplants in the old host animals (Fig. 2c,d) had a similar histological appearance with a typical well organized neuron-rich layer resembling the pyramidal layer. These densely packed neurons were large with a prominent nucleolus. Superficial to this layer, a cell-poor zone resembling the stratum oriens was observed. Beneath the neuron-rich layer another cell-poor area was found, reminiscent of the stratum radiatum (Fig. 2b,d). In the group of young transplants grafted to old hosts, the grafts did not appear as well organized as in the other groups. A layer reminiscent of a pyramidal layer was usually present, but not as denseiy packed as in the other two groups and often only seen in o~e part of the transplant (Fig. 2e,f). TH-positive fibers, originating from the sympathetic ground plexus of the host iris, were observed throughout the transplants in all 3 groups (Fig. 3).

Immunohistochemicalanalysis Transplants to be processed for immunohistochemical analysis were immersion-fixed in a formalin/picric acid mixture (4% paraformaldehyde and 0.4% picric acid in 0.16 M sodium phosphate buffer, pH 6.9) according to Zamboni and deMartino 86 for 2 h and then immersed in a solution containing 10% sucrose, 0.01% sodium azide (Merck, Darmstadt, IF.R.G.) and 0.02% Bacitracin (Sigma Chemicals, St.

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Fig. 2. Cresyl violet-stained sections o f intraocular h i p p o c a m p a l grafts. A clearly visible p y r a m i d a l layer is o b s e r v e d in a y o u n g g,raft in a y o u n g host (a,b) and in a 23 m o n t h - o l d graft (¢,d). A s o m e w h a t less organized p y r a m i d a l layer is seen in a y o u n g transplant in an old host ( e , 0 . Note the low cell densities above and b e n e a t h the pyramidal layer. T h e b o x e d areas in a, c and e are e n l a r g e d in the corresponding figures to the right; b, d and f respectively. Scale bar: a,c,e, 100pro; b,d,f, 100pro.

273 The fibers were either thin and varicose resembling central noradrenergic neurites or smooth and relatively thick. A few of the TH-positive fibers were found in blood vessel walls but fibers also appeared within the transplant neuropil without any obvious contact with vascular structures. The density of THpositive fibers was higher in the old grafts (Table I, Fig. 3c) as compared to the density of the fibers in the young grafts in young hosts (Table I, Fig. 3a) and in young grafts in old hosts (Table I, Fig. 3e), but this difference was not significant. The aged transplants also had an abundance of lipofuchsin granules which were not present in the young transplants in young or in old hosts (Fig. 4). The granules were distributed throughout the old transplants and displayed a strong auto-fluorescence intensity.

TABLE I

High-performance liquid chromatography The noradrenergic innervation of the hippocampal grafts was quantitated using HPLC-EC. Whole tissue levels of NA, expressed as ng/g wet weight of tissue were as follows: 847 _+ 130 (n = 16) in young/young grafts; 3042 + 852 (n = 10) in old/old grafts and 1094 + 201 (n = 12) in young/old grafts. The NA content in the old grafts was significantly increased as compared to the young/young group (P < 0.01) and the young/old group (P < 0.05, Table I).

29.2/~M and a 95% confidence limit of 21.8-39.1 /~M. Pyramidal neurons in old hippocampal grafts (23 months) were significantly less sensitive than neurons in the young/young group with a clear shift to the right in the dose-response curve for NA-induced depressions. The EC50 was 203.1 ~M with a 95% confidence limit of 162-254/~M. The NA-induced depressions in young grafts in old hosts were almost identical to the inhibitions in the young/young group. The EC50 value was 32.4/~M with a 95% confidence limit of 25.5-41.3/~M (Table I, Figs. 5 and 6). Superfusion of penicillin (1000-1600 U/ml) elicited interictal spiking of variable frequency and/or seizures in all 3 groups. There seemed to be no difference in the interictal spiking between the old and the young transplants.

Electrophysiology Extracellular recordings were performed from transplanted pyramidal cells, identified by their slow firing rate and spontaneous complex spike discharge 27. Sodium glutamate (50 raM) in the recording pipette elicited increased firing rates in all 3 groups. Discharge rates of individual neuroas, using glutamate in the pipette, did not significantly (statistically tested with analysis of variance) differ between the 3 group,s. The discharge rate averaged in young grafts in young hosts 4.4 + 0.6 Hz (n = 15), in old grafts in old hosts 5.6 + 0.5 Hz (n = 16) and in young grafts in old hosts the discharge rate was 4.1 + 0.4 I-Iz (n = 12, Table I). The glutamate-induced increased discharge rate permitted examination of the depressant effects of NA in all 3 groups. Grafted pyramidal neurons in young grafts in young hosts responded to superfused NA with depressions in firing rates with an ECs0 of

Summary of the morphological, biochemicaland electrophysiological data obtainedfrom the intraocularhippocampal grafts in the 3 groups studied

y/y (younggrafts in younghosts), y/o (younggraftsin old hosts) and o/o (old grafts in old hosts), n is in text. Group NA-innervation Histochemistry HPLC-EC (density of TH- (nglg) positive fibers)

y/y y/o o/o

1.4 + 0.1 1.0 + 0.2 1.6 + 0.2

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847 _+130 4.4 _+0.6 1094 _+201" 4.1 _+0.4 3042 _ 852** 5.6 + 0.5

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* The NA-content in the grafts in the o/o group was significantly higher (P < 0.05) than the content in younggrafts in old hosts. ** The NA-content in old grafts was significantlyhigher (P < 0.01) than the content in younggrafts in younghosts.

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In this study we have demonstrated pre- and postsynaptic alterations in noradrenergic mechanisms in old hippocampal grafts in oculo. We found an age-related decline in noradrenergic receptivity of intraocular pyramidal neurons as well as an increased content of NA and a slightly increased adrenergic fiber density in aged grafts as compared to young grafts regardless of host age. The alterations of NA transmission seen here thus appear to be due to transplant age and not host age, suggesting an intrinsic determinism of NA mechanisms. These data are in line with a pre-

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Fig. 4. Micrographs of autofluorescent lipofuchsin granules in a 23 month-old hippocampai graft (a). Note the strong fluorescence intensity of the pigments (arrows). No lipofuchsin granules are found in a young graft in a young host animal (b). Scale bar, 100 ~m.

vious report from our laboratories where a subsensitivity to N A of Purkinje neurons was found in aged cerebellar grafts 37. This age-related decline in N A receptivity has also been demonstrated in hippocampus 1°, as well as in cerebellum, cingulate cortex and neocortex s'9'45'46'55'56. Thus, a postsynaptic subsensitivity to N A may be a general p h e n o me n o n in the brain during aging. Not all characteristics of a transplant in oculo are independent of host age at grafting. Host age does seem to play a role regulating at leae,t partly transplant growth. In this study the grafts in the old hosts grew less well than young grafts transplanted to young adult hosts. A similar p h e n o m e n o n has recently been demonstrated also for cortex cerebri grafts 24. Recent studies have s'lggested a significant relationship between loss of central noradrenergic func-

tion and senescent m e m o r y decline 3°'5°. Leslie et al. 5° showed a clear correlation between cell loss in locus coeruleus and retention latency on an inhibitory avoidance task in aged mice. Clinical studies have also shown a relationship between noradrenergic impairment and senescent m e m o r y decline ~4"42. Thus, although age-related neuronal loss has been demonstrated in several transmitter systems in the aged brain 15,17,23,49, degeneration of the locus coeruleus noradrenergic neurons has been specifically associated with senescent m e m o r y loss. A decreased activity or loss of neurons displaying higher firing rates has also been reported in the locus coeruleus of aged rats 63,79,83. Prolonged electrical stimulation of the locus coeruleus or disinhibition with a-adrenergic receptor antagonists can prevent age-related m e m o r y deficits without affecting behavioural response in

Fig. 3. TH-immunoreactive fibers in cryostat sections of hippocampal transplants in oculo. The fibers grow into the transplants from the host irides. The irides, shown as a thin rim below the transplants, manifest a denser adrenergic innervation than the transplant neuropil (a,c,e). Some fibers are found in close proximity to blood vessels whereas others do not have any association with vascular structures (arrows). Both varicose fibers and thicker fibers or bundles without visible varicosities are found (b,d,f). a: TH-immunoreactive fibers ~re seen scattered in a transplant after 3 months in the eye of a young rat. b: a varicose fiber is shown at a higher magnification, c: a 23 month-old transplant. The density of TH-positive fibers and the intensity of the immunoreactivity seem to be slightly higher than in (a) and (e). d: an enlargement of a smooth and thick fiber is shown, e: a young hippocampal graft in an old host animal is seen. The density of the TH-immunofluorescence is similar to that in (a). The arrow points to a varicose fiber enlarged in (f). Scale bar: a,c,e, 100 /~m; b,d,f, 50/~m.

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LOG DOSE NE Fig. 5. Log dose-response curves for the depressant effects of superfused NA on the discharge rate of pyramidal neurons in y/y (triangles), o/o (squares) and y/o (circles) hippoc~mpal transplants. Individual data points represent an n of 3-8 per point (mean + S.E.M.). The ECs0 values and 95% confidence limits are calculated from linear (Hill) transformations.

young animals sg. It is not known to date, if the degeneration and/or slowing down of locus coeruleus neurons in the senescent rats leads to the postsynaptic NA subsensitivity reported here and by numerous other investigators (see above). It might be expected that the opposite would occur; i.e. a disuse supersensitivity. Even though both NA content and turnover have been shown to be decreased in brains of aged rodents and primates 3,25,26,34,36,5s,s2, plasma NA levels of aged Fisher 344 rats are increased TM. This may be indica-

tive of an increased activity of the adrenals and the sympathetic ganglia during senescence. Thus, the increased content of NA in aged hippocampal transplants in oculo reported here may reflect ar~ increased superior cervical ganglionic activity rather than a direct effect of aging in the hippocampus. Also, it is possible that the collaterals from the host sympathetic ground plexus continue to invade the hippocampal transplants throughout the whole time period in oculo, thus resulting in a slow hyperinnervation. The increased density of noradrenergic ~erminals in the old transplants may have contributed to the observed NA subsensitivity via an increased reuptake. Howe~er, we have recently found that old hippocampal grafts in adrenergically denervated eTes are still subsensitive to NA, thus eliminating the p ~ sibility of an increased reuptake (Granholm and Eriksdotter-Nilsson, unpublished). Radioligand binding studies have demonstrated the presence of both a- and/~-adrenergic receptors in the hippocampus in situ and in other brain areas 4, 20,43,66,?0,s4 as well as in intraocular grafts ss. Recent studies have shown that NA elicits at least two different, dose-dependent responses in the hippocampal pyramidal neurons; low doses induce excitations while high doses cause inhibitions of discharge rates s2, 62.6? In the present study, low doses (1-5 ~tM) of NA elicited excitations of variable magnitude, which were difficult to quantitate, but did not differ markedly between the 3 groups. However, higher doses of

+

NE56 pM

Y/Y

ii

' l

Y/O NE 56 pM '

j

~t~llL .

,

NE 560 )uM 0/0

!1

'

Fig. 6. Ratemeter records showing the depressant effects of superfused NA on hippocampal transplants. In the two young transplants (y/y and y/o) 56/aM NA elicits a depression in neuronal activity. To obtain a similar degree of inhibition in the old graft (o/o), a concentration of 560/aM of superfused NA is needed.

277 NA elicited depressions of pyramidal neuron firing rate. The aged hippocampal transplants were clearly subsensitive to NA in their inhibitory response, with dose-response curves shifted to the right by one order of magnitude. These data extend biochemical studies, which indicate that both a-and fl-adrenergic receptor affinity and/or density is decreased in the brain during aging 3s'5°,Sa'61'6s. However, the reduced receptor density may in part be due to cell loss. In hippocampus the number of pyramidal cells is reduced with age 49. This cell loss has been found to be correlated with extensive lipopigment accumulation t5. The intraneuronal amount of lipofuchsin increases with age t6'~2. Accumulation of residual bodies is considered a sign of oxidative damage to the neurons and may, eventually, lead to cell death 22"32'54.In the aged hippocampal grafts studied here, a high number of lii~.afuchsin granules was observed. The lipofuchsin granules were not found in young grafts. This finding further supports intrinsic determinism of aging in our mode!~ since no lipofuchsin granules were found in the young transplants in old hosts. Because of its low seizure threshold t, the hippocampus has been extensively used in epilersy research. Interictal spikirtg and overt seizures can easily be induced in the isolated hippocampus, either by electrical stimulation or convulsive drugs, such as penicillin or cobalt 21,4°,69. Anticonvulsant drugs, such as diazepam and barbiturates antagonize penicillininduced seizure discharge 4°'47. Diazepam, barbiturates and penicillin are suggested to act through GABAergic mechanisms. Several lines of evidence suggest that the GABAergic system is essentially un-

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ACKNOWLEDGEMENTS This study was supported by the Swedish Medical Research Council 14X-03185, 14X-06555, Loo and Hans Ostermans Foundation, Swedish Society of Medicine, Magnus Bergwall Foundation, USPHS Grants AG04418, AG0634, ES02011, the Veterans Administration Research Service and the Miami Project Foundation. The skillful technical assistance of Lena Holmberg, Karin Lundstr6mer, Carina Ohlsson and Barbro Standwerth is gratefully acknowledged.

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