SYNAF’SE 23~1-9 (1996)

Enhanced Synaptophysin Immunoreactivity in Rat Hippocampal Culture by 5=HTlA Agonist, SlOOb, and

Corticosteroid Receptor Agonists MAYUMI NISHI, PATRICIA M. WHITAKER-AZMITIA, AND E F “ C. AZMITIA

Department of Biology, New York University, New York, New York 10003 (M.N., E.C.A.) and Department of Psychiatry, State University ofNew York, Stony Brook, New York 11794 (l?M.W.-A.)

KEY WORDS Synaptogenesis, Differentiation, Varicosity, Aldosterone, Ru28362, Ipsapirone

ABSTRACT Serotonin (5-HT) has been shown to modulate brain maturation during development and adult plasticity. This effect in the whole animal may be due to activation of 5-HTIA receptors and a corresponding increases in SlOOb and corticosterone. Synapto- physin, an integral protein of the synaptic vesicle membrane that correlates with synaptic density and neurotransmitter release, is reduced by depletion of 5-HT in the cortex and hippocampus of the adult rat. Injections of a 5-HTl~ agonist or dexamethasone can reverse the loss of synaptophysin immunoreactivity (IR). In this study we used morpho- metric analysis of synaptophysin-IR to study the effects of the 5-HTu agonist, ipsapirone, and the neuronal extension factor, SlOOb on hippocampal neurons grown in a serum and steroid free media. Both compounds increased the synaptophysin-IR at doses previously established to be highly specific. Ipsapirone ( M) was more effective on neuronal cell bodies staining and SlOOb (10 ng/ml) was more effective in increasing the number of synaptophysin-IR varicosities on neuronal processes. In addition both types of corticoste- roid receptor agonists, a t previously established specific doses, Ru28362 M) and aldosterone ( M) produced smaller increases compared to control groups in both the cell body staining and the number of varicosities. The effect of these differentiating factors on the expression of synaptophysin-IR suggests multiple regulation sites for producing and maintaining pre-synaptic elements in the brain. o 1996 Wiley-Liss, Inc.

INTRODUCTION During development, serotonin (5-HT) has been pro-

posed to be a neurotrophic factor since removal of 5- HT during development produces a delay in forebrain maturations as demonstrated by neuronal labeling (Lauder and Krebs, 1978). The appearance of vibrasse somatosensory area is also delayed when 5-HT is re- duced (Bennett-Clarke et al., 1995; Blue et al., 1991). In addition, removal of 5-HT produces evidence of neu- ronal “dematuration” in adults. There is a loss of synap- tic profiles and decreased synaptophysin and microtu- bule-associated protein 2 immunoreactivity (IR) in adult hippocampus after para-chlorophenylalanine (PCPA) or parachloroamphetamine (PCA) (Azmitia et al., 1995; Cheng et al., 1994). There is also behavioral evidence of a loss of habituation and a decreased capac- ity for active response (Carlton and Advokat, 1973; Con- ner et al., 1970).

The mechanism of action of 5-HT as a neurotrophic 0 1996 WILEY-LISS, INC.

factors may involve the 5-HTlA receptor which is located on neurons and glial cells (Azmitia et al., 1990a; Whi- taker-Azmitia et al., 1994); 5-HTIA receptor located on hippocampal neurons has been shown to open K’ chan- nels and regulate cyclic-AMP levels (Andrade et al., 1986). Addition of a 5-HTU agonist dramatically en- hances the maturation of choline acetyltransferase-IR neurons (Riad et al., 1994) and glial fibrillary acidic protein-IR glial cells (Whitaker-Azmitia et al., 1990) in culture, and forebrain neurons and S100b-IR glial cells in the brains of cocaine-pretreated pups (Akbari et al., 1994).

The hippocampal glial5-HTu receptor regulates the expression and secretion of SlOOb (a serotonergic and cortical maturation factor) (Azmitia et al., 1990a, 1995; Haring et al., 1993; Whitaker-Azmitia et al., 1990).

Received April 9, 1995; accepted in revised form August 7, 1995.

2 M. NISHI ET AL

DIFFERENTIATING FACTORS AND SYNAPTOGENESIS 3

SlOOb tested in culture at doses between 0.1 and 100 ng/ml has been shown to function as a neuronal matura- tional factor by increasing process outgrowth on sero- tonergic (Azmitia et al., 1990b) and cortical (Kligman and Marshak, 1985) neurons. In fact, in a mutant strain of mouse, lacking the SlOOb protein, the cortex, hippo- campus and 5-HT fibers are stunted (Ueda et al., 1993). Therefore, glial derived SlOOb, possibly under the regu- lation of a 5-HTlA receptor, can induce neuronal matu- ration.

Steroids may represent another route whereby 5- HT~A agonist can result in enhanced neuronal and glial maturation. Hypothalamic 5-HTlA receptor activation can induce the release of corticosterone (a potent differ- entiating factor) by activating ACTH pituitary secretion (Owens et al., 1990). Corticosterone can bind to a high- affinity receptor [type I, mineralocorticoid receptor (MR)] and a low-affinity receptor [type 11, glucocorticoid receptor (GR)] which present on both neurons and glial cells (Bohn et al., 1994; Sutano and DeKloet, 1991). Selective agonists to these receptors exist, aldosterone at M binds selectively to GR (ref. Sutano and DeKloet, 1991, review). Both of these receptors have been re- ported to stimulate neuronal maturation (see Jacob- son, 1991).

Are serotonin, SlOOb, and steroids capable of inde- pendently regulating neuronal maturation? Does the stimulation of synaptophysin occur in a similar manner by all these factors? We investigated the short-term effects of these agents on rat hippocampal primary cul- tures from 18-19-day-old fetal rat hippocampi grown in the absence of serum and steroids. In the present study, by using morphometric analyses, we detected the change of synaptophysin-IR in cell clusters of cultured hippocampal cells and synaptophysin-IR varicosities on neuronal processes. Our results indicate that 5-HTlA

M binds selectively to MR and Ru28362 at

Fig. 1. Synaptophysin-IR in cell cluster region of dissociated hippo- campal culture. A Photographs showing cell clusters region with syn- aptophysin-IR cell bodies and processes surrounding the cell bodies. The cultures were grown for 48 hr in the presence of fetal bovine serum, then for 24 h in the absence of serum and steroid, and finally treated with the following substances. a: Control culture was grown in the absence of serum and steroid for the last 48 h before fxation. b: Ipsapirone (W9 M) was added for the last 48 h before fixation. c: SlOOb (10 ng/ml) was added for the last 48 h before fixation. d: Ru28362

M) was added for the last 48 h before fxation. e: Aldosterone M) was added for the last 48 h before fixation. In Figure lAa-e,

occasional whole cell bodies are stained darkly (asterisk). More cells are stained darker in b-e compared with a. Scale bar, 20 pm. B: Effects of ipsapirone, SlOOb, Ru28362, and aldosterone on synaptophysin-IR density in cell clusters. The gray values obtained from the computer- aided densitometric software have been subtracted from 100 (back- ground setting). The means of three individual micro-culture dishes with two replicate wells per dish (Sampling area = 5 X lo5 pm2/each well) & SE were as follows; Control: 39.4 2 1.25, ipsapirone: 50.1 2 0.957**, S100b: 44.5 t 1.37*, Ru28362: 43.6 & 0.989, aldoste- rone: 44.2 2 1.20. The results were analyzed using ANOVA followed by Student’s t-test. *P < 0.05, **P < 0.01: compared to control. In Figure lB, these values are plotted as a percentage of the control value.

receptor activation in cultures increases the synapto- physin-IR in cell bodies while addition of SlOOb pro- duced its largest effect on the neuronal processes. Both corticosterone receptors are also active in stimulating synaptophysin expression, but in a reduced capacity.

MATERIALS AND METHODS Cell culture

Dissociated hippocampal primary cultures were pre- pared from 18- to 19-day-old Sprague-Dawley rat fe- tuses, according to the method of Azmitia and Hou (1994). Whole brains were removed and transferred to ice-cold D1 solution (0.8% NaC1, 0.04% KC1, 0.006%, Na2HP04 * 12 HzO, 0.003% KH2P04, 0.5% glucose, 0.00012% phenol red, 0.0125% penicillin G, and 0.02% streptomycin). Hippocampi were isolated and mechani- cally dissociated by triturating through a fire-polished glass pipette. The use of trypsin was avoided since a recovery of large neurons following trypsin dissociation was reduced (Azmitia and Whitaker-Azmitia, 1987). The cells were plated at an initial plating density of 1 x lo6 cells/cm2 by adding 200 ~1 of the cell suspension to each well (area of 0.28 cm2, Lab-Tek Chamber Slide; Nunc, Inc., Naperville, IL) precoated with 15 mg/ml poly-D-lysine (MW = 70,000-150,000; Sigma Chemical Co., St. Louis, MO). The cultures were maintained in complete neuronal medium (CNM), consisting of 92.5% (v/v) Eagle’s minimum essential medium (MEM, Sigma Chemical Co., St. Louis, MO), 1% (w/v) non-essential amino acids (GIBCO-Life Technologies, Inc., Gaithers- burg, MD), 0.16% (w/v) glucose and 5% (v/v) fetal bovine serum (Sigma Chemical Co., St. Louis, MO) for 48 h in a COz incubator at 37°C with 5% COd95% air.

Drug treatment After 48 h the CNM was removed and the cultures

were rinsed two times with serum-free media (MEM with 0.16% glucose, 1% non-essential amino acids, 20 mM putrescine, 15 nM sodium selenite, 5 Fg/ml insulin, and 100 Fg/ml transferrin). After incubation in the se- rum free medium for 24 h, the cultures were treated with lo-@ M Ru28362 (a synthetic GR agonist, Roussel Uclaf, Romainville, France), M aldosterone (Ald, Sigma Chemical Co., St. Louis, MO), M ipsapirone (IPS, Miles, Inc., Naperville, IL), or 10 ng/ml SlOOb (East Acres Biologicals, Southbridge, MA) for 48 h be- fore fixation. Control culture was treated in the same way except that no drug was added. The concentrations of the four agonists were selected according to extensive dose-response studies ( to M).

Immunoc ytochemistry Cultured cells were fixed for 30 min at room tempera-

ture (RT) in 4% of paraformaldehyde in phosphate buffer (PB, pH 7.4) containing 0.12 M sucrose. After blocking with 5% bovine serum in P3 for 1 h at RT, the primary mouse anti-synaptophysin monoclonal anti-

4 M. NISHI ET AL.

DIFFERENTIATING FACTORS AND SYNAFTOGENESIS 5

body was applied (1/400 dilution; Sigma Chemical Co., St. Louis, MO) and incubated for 48 h at 4°C. The speci- ficities of this antibody has been already studied (Az- mitia et al., 1995). After rinsing with PB, the cultures were reacted with biotinylated goat anti-mouse anti- body (1/250 dilution; Boehringer Mannheim, Indianap- olis, IN) for 1 hr at RT. The cultures were rinsed with PB and incubated in streptavidin-peroxidase conjugate (1/8,000 dilution; Boehringer Mannheim, Indianapolis, IN) for 1 h at RT. The cells were visualized with 0.02% 3,3’-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and 0.006% HzOz in 0.1 M Tris-HC1 buffered (pH 7.6) saline. Slides were dehydrated with ethanol, fol- lowed by a citrus-oil based clearing agent, Histo-Clear, and immediately cover-slipped in Permount. As a nega- tive control, the entire immunocytochemical procedure was replicated, eliminating primary antibody. Nonspe- cific staining was not observed (see Fig. 20. Slides were subjected to morphometric analysis as follows.

Morphometric methods The Optimas (ver.4.0) image acquiring software pro-

gram (Bioscan, Inc., Seattle, WA) was used for all mor- phometric analyses. Using a Leitz Microscope equipped with a video camera, the cell cluster areas including processes surrounding cell bodies were selected ran- domly and the mean area gray-value (ArGV) of each cell cluster was measured (magnification for this procedure: 25x objective; Sampling area = 5 X lo5 pm2/each well). The background light was standardized to a ArGV of 100 5 1. Since a higher ArGV corresponds to a lighter area, the measurements were subjected from 100 in order to facilitate graphic analysis. The mean value of the 100-ArGV obtained from three separate micro- culture dishes with two replicate wells per dish was calculated for each well. For varicosity analysis detected on processes running between the cell clusters, a differ- ent method was employed. First, a region of interest was selected and then a standard threshold was set at 4% ArGV > 85 for counting the number of the synapto- physin-IR varicosities. The magnification for this proce- dure was increased (40X objective). Numbers of the varicosities per 60 pm of each process selected in the above threshold condition were counted. The ratio of

~~~ ~

Fig. 2. Photographs showing processes with synaptophysin-IR vari- cosities. a: Control culture was grown in the absence of serum and steroid for the last 48 h before fixation. b: Ipsapirone M) was added for the last 48 h before fixation. c: SlOOb (10 ng/ml) was added for the last 48 h before furation. d Ru28362 M) was added for the last 48 h before fxation. e: Aldosterone M) was added for the last 48 h before fixation. f: An example of negative control treated with Ru28362 in the same manner as d. After fixation, the immunocy- tochemical procedure was replicated, eliminating primary antibody as described in Materials and Methods. This photograph includes both cell clusters regions and processes running between the cell clusters. More synaptophysindR varicosities (triangle) are seen in b-e com- pared with a. Scale bar, 20 pm.

300 processes from three experiments (100 processes from each experiment) were calculated for making his- togram. All measurements were exported to Microsoft Excel spreadsheet, where statistical analyses were per- formed (i.e., mean, standard deviation, and standard error of the mean). Statistical differences were deter- mined using ANOVA followed by Student’s t-test.

RESULTS Dissociated cell bodies migrated to form cell clusters

within 5 days in fetal hippocampal cultures (Fig. 1Aa- e). Synaptophysin-IR staining densities observed in cell clusters were heterogeneous. The surface of the cell was stained in some cells, but in other cells the whole cell body was darkly stained. Morphometric measurements of average gray values of synaptophysin-IR densities were analysed in three separate micro-culture dishes with two replicate wells per dish (sampling area = 5 X lo5 pm2/each well) and plotted as a percentage of the control value (Fig. 1B). In addition, varicosities were seen on neuronal processes running between the cell clusters (Fig. 2a-e). The number of discrete synaptophy- sin-IR varicosities per 60 pm of processes was obtained by counting 100 processes per well (n = 3) and ex- pressed as a histogram (Fig. 3). Only 40% of the pro- cesses had more than 2 varicosities per unit length in control culture.

Effects of ipsapirone M) on the synaptophy-

sin-IR cell clusters were morphometrically analyzed. Synaptophysin-IR staining intensities observed in cell clusters were increased with ipsapirone treatment (Fig. 1Ab). As can be seen, not only was the intensity of staining increased but also the number of neurons showing whole cell labeling was increased. In addition, the number of synaptophysin-IR varicosities seen on neuronal processes was increased in the ipsapirone treated group (Fig. 2b) compared with control (Fig. 2a). Morphometric studies supported the descriptive find- ings as shown in Figure 1B. Ipsapirone showed the highest increase (27.1%, P < 0.01) in the synaptophy- sin-IR density in cell clusters compared with that of control. Figure 3 shows the histogram of synaptophysin- IR-varicosity number per 60 pm of each process. As seen in Figure 3, ipsapirone showed a right shift, indi- cating an increase in numbers of synaptophysin-IR vari- cosity. Nearly 70% of the processes had more than 2 varicosities per unit length. The viability of the cultured cells was not affected.

The effects of ipsapirone

Effects of SlOOb Cells were densely stained in SlOOb treated group

compared to control culture and there was an apparent increase in the number of whole cell stained neurons (Fig. 1Ac). Also, there appeared to more synaptophysin- IR varicosities than observed in control cultures (Fig.

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If .i 2 0 - v) % c n 2 1 5 - P a C h

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M. NISHI ET AL.

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Number of varicosity

Fig. 3. Histogram of synaptophysin-IR varicosity number per 60pm of process. The values are the mean (%) obtained from 300 processes.

2c). As shown in Figure lB, the synaptophysin-IR den- sity in cell clusters was increased with treatment of SlOOb (12.8%, P < 0.05). The results presented in Fig- ure 3 illustrates that SlOOb showed the greatest right shift, indicating that SlOOb was the most effective in increasing the numbers of synaptophysin-IR varicosity per unit length. Almost 85% of the processes had more than 2 varicosities and almost 40% had 5 or more after exposure to S100b. The viability of the cultured cells was not affected.

Effects of steroids The cell clusters showed more staining in Ru28362

(lo-@ M) and aldosterone M) treated groups (Fig. 1A d and e, respectively) in comparison with control (Fig. 1A a). Synaptophysin-IR varicosities were more abundant on neuronal processes after Ru28362 and al- dosterone treatment (Fig. 2 d and e, respectively) com- pared with control (Fig. 2a). Morphometric studies in Figure 1B showed that the synaptophysin-IR density in cell clusters was increased with treatments of Ru28362 (10.6%) and aldosterone (12.2%) in comparison with that of control. As seen in Figure 3, both steroids pro- duced a shift to the right in the histogram, indicating an increase in the numbers of synaptophysin-IR varicosity. The percentage of processes with more than 2 varicosi-

ties per unit length was 60% for aldosterone and 70% for Ru28362. The viability of the cultured cells was not affected.

DISCUSSION In the present study, we have employed morphomet-

ric analyses to investigate the short-term effects of a number of factors known to produce neuronal differenti- ation on the synaptophysin-IR. Synaptophysin is an integral protein of the synaptic vesicle membrane which correlates with synaptic density and neurotransmitter release (Rehm et al., 1986). Primary cultures of hippo- campal cells were grown for 48 h in the presence of fetal bovine serum and then for 24 h in the absence of serum and steroid. The substances tested were ipsapirone (a selective 5-HTIA agonist), SlOOb (neurite extension fac- tor), and Ru28362 and aldosterone (GR and MR agonist, respectively). This study was based upon the need to understand the direct relationship between change of synaptophysin-IR and the various differentiating fac- tors functioning in the intact central nervous system.

In the present study, ipsapirone significantly in- creased the synaptophysin-IR in cell clusters and vari- cosities, with the highest effect on cell cluster synapto- physin-IR; 5-HTIA agonist can enhance neuronal

DIFFERENTIATING FACTORS AND SYNAPTOGENESIS 7

Interactions of Differentiating Factors on Synaptophysin-expression

S100b

HIPPOCAMPAL

SYNAPTOPHY SIN Receptor

CORTICOSTERONE

Fig. 4. A schematic diagram of the interactions of the differentiating factors. 5-HTU agonist, SlOOb, and corticosteroid receptor agonists (GR and MR) have connection among each other and on the expression of synaptophysin in cultured hippocampal neurons.

maturation after prenatal expression to cocaine (Akbari et al., 1994). In adult rats, the short-term treatment with ipsapirone reversed the loss of synaptophysin-IR after PCA injection (Azmitia et al., 1995); 5-HTIA recep- tors and their agonists may be involved in promoting synaptogenesis both in culture and in vivo. One pro- posed mechanism for this stimulation may be 5-HTlA induced release of SlOOb (Fig. 4).

SlOOb itself when added to the hippocampal cultures produced a large increase in numbers of synaptophysin- IR varicosities on neuronal processes. SlOOb is a neurite extension factor for cortical and serotonergic neurons (Azmitia et al., 1990b; Kligman and Marshak, 1985). Synaptophysin is Ca-binding protein and may play an important role in Ca2+-dependent neurotransmitter re- lease. Recently it has been suggested that SlOOb might increase intracellular free calcium concentration ([Ca2+]J in both glial and neuronal cells (Barger and VanEldik, 1992). Thus, there is a possibility that the increase of ICa2+li caused by SlOOb induces the synapto- physin expression. Future work will examine this possi- bility.

Finally, this study showed that both GR and MR ago- nists increased synaptophysin-IR in cell clusters and varicosity numbers. Corticosterone is a potent differ- entiating steroid that can act on two receptor systems in the brain, mineralocorticoid (type I) and glucocorticoid (type 11). Aldosterone is a mineralocorticoid that acts as a specific agonist on MR. MR are largely confined to the hippocampus and involved primarily in salt homeo- stasis (Sutanto and DeKloet, 1991). Ru28362 is a syn- thetic steroid that acts as a specific agonist on GR (Damm et al., 1994). Ru28362 and aldosterone are po- tent differentiating steroids during early neuronal de- velopment (Bohn, 1980, Jacobson, 1991). Hippocampal neurons and astrocytes contain both GR and MR (Bohn

et al., 1994; Cintra et al., 1994). Both receptors appear to function through common glucocorticoid response el- ements in the nucleus, although different effects be- tween these two receptor types have been reported (Pearce and Yamamoto, 1993). In the present study, both steroids, at low concentration (10-s-10-9 M), appear to produce a similar increase in synaptophysin-IR, al- though it is not clear if the mechanisms of action are identical.

In a previous study, it was indicated that cultured photoreceptor cells produced an increase in synapto- physin-IR varicosities as they differentiate (Mandell et al., 1993). These synaptophysin-IR varicosities are not always apposed to a postsynaptic cell. These authors concluded that differentiated photoreceptors are capa- ble of synaptic renewal and the regeneration of presyn- aptic-like terminals is an intrinsic ability of neurons. In our studies, we demonstrate that substances which influence neuronal maturations are capable of altering this “intrinsic ability” to generate presynaptic-like ter- minals that stain positive for synaptophysin.

The expression of synaptophysin-IR is of interest in studies of neurodevelopment and in the brain’s response to injury or diseases. Synaptophysin-IR increases dur- ing synaptogenesis (Devoto and Barnstable, 1987; Knaus et al., 1986; Leclere et al., 1989) and following cerebral ischemia (Stroemer et al., 1992). On the other hand, synaptophysin-IR is decreased in aging (Masliah et al., 1993a1, Alzheimer’s disease (Hamos et al., 1989; Honer et al., 1992; Lassmann et al., 1992; Masliah et al., 1989, 1991) and other dementias (Masliah and Terry, 199313). Furthermore, synapse loss in Alzheimer’s dis- ease has been found to be particularly severe in the hippocampus (Lippa et al., 1992).

In conclusion, we have demonstrated that 5-HTlA re- ceptor agonist, SlOOb and corticosteroid receptor ago- nists can all increase synaptophysin-IR in cultured hip- pocampal neurons. There are many points at which these substances can interact in the intact brain. For example, 5-HTu receptor agonists stimulate the pitu- itary-adrenal axis (Chaouloff, F., 1993) and can increase SlOOb in brain (Azmitia et al., 1990a, 1995; Haring et a]., 1993). 5-HTIA receptor agonist in culture stimulates the release of SlOOb from astrocytes and promotes their maturation (Whitaker-Azmitia et al., 1990). Corticoste- roids can, in turn, increase 5-HT synthesis, turnover and stimulate the growth of the somatodendritic area of serotonergic neurons (Azmitia and McEwen, 1969, Azmitia et al., 1993; DeKloet et al., 1982). Adrenal ste- roids can selectively downregulate hippocampal ~ - H T ~ A receptors a t the level of 5-HT1~ receptor mRNA expres- sion (Chalmers et al., 1993). Furthermore, electrophysi- ological study indicated that 5-HT-induced hyperpolar- ization of the membrane depends on the relative MW GR occupation (Joels et al., 1994). Finally, SlOOb is a potent 5-HT neurite extension factor in primary cul- tures of raphe neurons (Azmitia et al., 1990b). Mutant

8 M. NISHI ET AL

mice expressing low levels of SlOOb have reduced ~ - H T but normal catecholamine innervation of a narrower

activity: Primary microcultures of midbrain raphe and hippocam- pus. In: Methods in Neuroscience 22; Neurobiology of Steroids. De- Kloet E.R. and Sutanto w.. eds. Academic Press. San Dieeo. CA.

I

cortex (Ueda et al, 1994). This study of these factors in culture indicate that serotonin, SlOOb, and corticoste-

pp. 359-371.

tAer-Azmitia, P.M. (1995) 5-HTIA agonist and dexmethasone re- Azmitia, E.C., Rubinstein, V.J., Strafaci, J.A., Rios, J.C., and Whi-

versal of parachloroamphetamine induced loss of MAP-2 and synap- tophysin immunoreactivity in adult rat brain. Brain Res., 677: 181-192.

Barger, S.W., and VanEldik, L.J. (1992) SlOOb stimulates calcium fluxes in glial and neuronal cells. J. Biol. Chem., 267:9689-9694.

Bennett-Clarke, C.A., Lane, R.D., and Rhoades, R.W. (1995) Fenflura- mine deDletes serotonin from the develoDine cortex and alters thala-

raids may all have convergent actions on neuronal mat- uration. Future studies will examine the sequential and combined use of these drugs to explore their CO-OP-

eration. The in ViVO experiments indicate that these factors

may also be involved in synaptic maintenance. Evidence from in vivo immunocytochemical studies after PCA treatment suggests that serotonin, SlOOb, and steroids may be involved in maintaining the normal expression of synaptophysin and microtubule-associated protein 2 in cortex, hippocampus, and hypothalamus of adult rats (Azmitia et al., 1995; Whitaker-Azmitia et al., 1994). The ability of these three substances to stimulate syn- aptophysin-IR in fetal hippocampal cultures strength- ens this association and supports the idea that the same differentiating factors are active in both the developing and mature brain. A schematic diagram of the interac- tions of the differentiating factors is shown in Figure 4. The role of serotonin, SlOOb, and steroids in the loss of synaptophysin during Alzheimer’s disease is intriguing. Although these compounds are all affected by Alzhei- mer’s disease, a consistent trend is not apparent. SlOOb, steroids, and serotonin levels are increased, but 5-HTIA and steroid receptors are decreased (Azmitia et al., 1992). Resolution of these interactions may provide new insights into the etiology of synaptic loss associated with Alzheimer’s disease.

ACKNOWLEDGMENTS We thank Dr. Aoki for critical reading of the manu-

script. This work has been supported by funds from N U program project 1 PO1 AG10208.

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