European Journal of Neuroscience, Vol. 6, pp. 1720-1 728, 1994 0 European Neuroscience Association

Differential Effects of Serotonin and Raphe Grafts in the Hippocampus and Hypothalamus: A Combined Behavioural and Anatomical Study in the Rat

G. Richter-Levin’, L. Acsady2, T. F. Freund2 and M. Sega13 ’National Institute for Medical Research, Mill Hill, London NW7 IAA, UK 2Department of Functional Neuroanatorny, Institute of Experimental Medicine, Budapest, P.O.B. 67, H-1450, Hungary 3Department of Neurobiology, The Weizmann Institute, Rehovot 761 00, Israel

Key words: spatial learning, interneurons, calbindin, calretinin, parvalbumin

Abstract

Combined with a partial cholinergic deficiency, serotonergic lesions induce severe spatial learning deficits. Serotonergic lesions, however, have additional effects, such as reduced body weight and disruption of thermoregulation, which may be the cause of the observed learning deficits. Restoration of the serotonergic innervation of the hippocampus by raphe grafts reduces these learning deficits. The effects of the grafts may result from a direct support of spatial learning but may also be an indirect result of preventing some of the other effects of serotonergic lesions. In the present study we used raphe grafts to examine the selectivity and specificity of the effects of serotonergic lesions in the rat, and used the behavioural effects as an indication of successful transplantation in order to examine the fine details of such grafts. Raphe grafts in the hippocampus did not prevent the effects of the lesions on body weight, thermoregulation and exploratory behaviour but did minimize the effects of the lesions on spatial learning. In contrast, raphe grafts in the hypothalamus reduced the effects of the lesions on thermoregulation but failed to support learning. The grafted fibres showed termination specificity with the interneurons, which is typical of the serotonergic innervation of the normal hippocampus. The results indicate that the serotonergic innervation of the hippocampus functions locally to support spatial learning. This role of serotonin is independent of its involvement in modulation of body weight, thermoregulation or exploratory behaviour. The results confirm that the modes of serotonergic action in the hippocampus include the selective innervation of specific interneuron subpopulations.

Introduction

The importance of the serotonergic system in learning and memory is widely accepted but the nature of its involvement is debatable (Altman and Normile, 1988; Hunter, 1989; Dickson and Vanderwolf, 1990; McEntee and Crook, 1991). Since the hippocampus is a major target of the serotonergic system (Azmitia and Segal, 1978; Steinbusch, 1981) and is crucial for spatial learning (O’Keefe and Nadel, 1978) i t was assumed that serotonergic lesions should affect this type of learning. Contrary to this expectation, destruction of serotonergic neurons does not have a clear effect on spatial learning (Asin et al., 1985; Altman et al., 1990; Carli et al., 1992; Volpe et al., 1992). However, when serotonergic lesions are combined with cholinergic damage, spatial learning is severely impaired (Nilsson et al., 1988; Dickson and vdnderwolf, 1990; Richer-Levin and Segal, 1991a; Riekkinen et al., 1991). Raphe grafts in the hippocampus minimize these deficits (Richter-Levin and Segal, 199 la,b, 1992). These effects of the raphe grafts depend on the serotonergic nature of the grafts (Richter-Levin and Segal, 1991 b) and correlate positively with effects of the grafts on hippocampal commissural functions (Richter-Levin and Segal, 1991a). The ability of raphe grafts in the

hippocampus to ameliorate the behavioural deficits suggests that the serotonergic innervation of the hippocampus has a unique role in spatial learning (Richter-Levin ef a/., 1993). However. forebrain serotonin depletion affects other, non-cognitive functions like body weight and thermoregulation (Hillegant, 199 I ), which may indirectly influence the ability of rats to perform spatial learning tasks. Thus the selectivity and specificity of the serotonergic innervation in relation to learning and memory has yet to be examined.

A key assumption underlying the utilization of raphe grafts is that they reinnervate the target site in a pattern similar to the native serotonergic innervation of that site. Raphe grafts in the hippocampus were shown to survive (Azmitia er al., 1981; Anderson et a/ . , 1986), develop normal-like electrophysiological properties (Segal and Azmitia, 1986), synthesize serotonin (Auerbach et d.. 1985), hyperinnervate the host hippocampus (Azmitia et a/ . , I981 ; Zhou et a/., 1987; Tsubokawa et a/., 1988) and autoregulate serotonin release to levels comparable to those in the intact brain (Daszuta et al., 1989). Upon stimulation these fibres induce serotonergic-like effects on hippocampal cells, in the in vitro slice preparation (Segal,

Correspondence to: G. Richter-Levin, as above

Received 25 January 1994, revised 18 March 1994, accepted 26 May 1994

Differential effects of serotonin I72 1

1987) and in the anaesthetized rat hippocampus (Richter-Levin and Segal, 1990).

Earlier studies showed that serotonergic axons of the midbrain raphe form multiple contacts with only certain subpopulations of hippocampal non-principal cells (containing the calcium-binding protein calbindin or calretinin) but avoid others (parvalbumin-con- taining cells) (Freund et al., 1990; Halasy et al., 1992; Acsfidy el al., 1993). Although the grafts restore the serotonergic innervation of the hippocampus, the fine detailed pattern of reinnervation has not been established.

In the present study we examined the effects of lesions and grafts on a variety of parameters, including body weight, thermoregulation, exploratory behaviour and learning of non-spatial (active avoidance) and spatial (Moms water-maze) tasks. We also compared the effects of raphe grafts in the hippocampus and hypothalamus to assess the importance and specificity of the contribution of the serotonergic innervation in each of these regions. Following the behavioural tests, we examined the detailed morphology of the serotonergic lesions and grafts. Double immunocytochemistry for serotonin and calcium- binding proteins was performed on hippocampal sections containing the grafts. In addition, we injected gold-conjugated Phaseolus vulgaris leucoagglutinin (gPHAL) into the dorsal hippocampus to study the origin of serotonergic and non-serotonergic fibres.

Materials and methods Subjects were male Wistar rats from a local breeding colony. The rats were housed in a temperature controlled room, 3 4 per cage, 12 h/12 h light/dark cycle, with free access to food and water. Lesions and transplantation were camed out at the age of 6-8 weeks. Following surgery rats were returned to their cages for 5 months until the behavioural experiments commenced. Behavioural experi- ments were conducted over a period of 3 months. Immunocyto- chemistry and gPHAL labelling were performed following the behavioural experiments.

A bilateral intraventricular injection of 5,7-dihydroxytryptamine (5,7-DHT) (200 pg (free base) in 20 pI saline containing 0.2 mg/ml ascorbic acid) was administered to anaesthetized rats (chloral hydrate 3.5% solution, 10 ml/kg, i.p.) pretreated with 30 mgkg desipramine (in I ml saline, i.p.).

For transplantation, the midline tegmentum (-1 mm3) of embryonic day 14 E l 4 embryos was dissected out. Tissue was mechanically dissociated and a total volume of 1 1.11 was injected bilaterally into each host brain (coordinates: hippocampus: -4.5 mm from bregma, 5.2 mm lateral, depth from -6 mm to -4 mm; hypothalamus: 4 mm from bregma, 1 mm lateral, depth from -9.5 to -7 mm) as described before (Segal and Azmitia, 1986).

The following groups were tested: control (n = 8). serotonin- depleted (DHT group, n = 8), and DHT rats with raphe grafts in the hippocampus (HG group, n = 7) or hypothalamus (HTG group, n = 9).

The water-maze was a circular pool of water ( 1 30 cm diameter, 50 cm high rim and water level of 15 cm, water temperature -24 2 I'C), to which milk powder had been added. A hidden platform (top surface 10 cm in diameter, 3 cm below water level) was located in the middle of one quadrant. All rats were injected daily with atropine (40 mgkg, in I ml saline, i.p.) 20 rnin before the first daily trial. Rats were placed in the maze near the rim and facing it, and allowed to swim freely for a maximum of 1 rnin or until they reached the hidden platform. At the end of each trial the rat was either led to or left on the platform for 15 s. Rats were given each

day three blocks of two trials, for 2 consecutive days. During the second day every two trials started from a different starting point. On the third day rats were given two retention trials. The platform was then removed from the maze and the rat was kept in the pool for 30 s. Their swimming path was analysed as follows. The water- maze area was divided into four quadrants. The number of times a rat crossed into or out of a circle centred in each quadrant was used as an estimate of the time the rat had spent in that quadrant.

Two arenas were used to assess exploratory behaviour. An open field and an elevated plus-maze. The open field consisted of a white ( 1 X 1 m) square arena which was divided into 16 squares by thin black lines. Rats were put in for 5 rnin and the number of squares visited was used to estimate exploratory behaviour. The plus-maze consisted of four arms (75 cm each) on a SO cm high stand. Two of the arms had no railing (open arms) and the other two were protected by a 10 cm high opaque railing (closed arms). The ratio between the number of visits to the open and closed arms was used to estimate exploratory behaviour.

For estimation of thermoregulation, baseline body temperature was measured twice over 5 min and then rats were put into a jar containing ice-cold water for 3 min. Body temperature was then measured every minute for 3 min. The reduction in body temperature over those 3 min was used to estimate thermoregulation.

Four HG, four HTG and three DHT rats were used for the anatomical investigation, of which one HG animal, two HTG animals and one DHT animal received a unilateral gPHAL injection into the dorsal hippocampus for retrograde tracing. This tracer, when pressure- injected into the hippocampus, was found to retrogradely label cells within all major input areas of the hippocampus, i.e. entorhinal cortex, medial septumdiagonal band of Broca complex, supramammillary region and median raphe. The animals received a volume of 250 nl at each of the four injection sites under Equithesin (chlomembutal 0.3 mlkg) anaesthesia. Coordinates for the injections were: antero- posterior, bregma -3.6; lateral, 1.5 and 3; dorsoventral, -2.5 and 3.8.

Three days later the operated animals together with the non- operated ones were deeply anaesthetized and perfused first with saline for 1-2 min, then with a fixative containing 4% paraformaldehyde, 0.05% glutaraldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB). Vibratome sections (60 pm thick) were cut at the coronal plane from the hippocampi, from the brainstem at the level of the posterior hypothalamus and from the median raphe of each animal, and were washed in PB several times. (The sections for electron microscopy were washed in 0.1 M PB containing 10% sucrose for 10 min and in 0.1 M PB containing 30% sucrose overnight, then freeze-thawed three times by lowering them to the surface of liquid nitrogen repeatedly in a small aluminium foil boat. For serotonin immuno- staining the sections were incubated with monoclonal rat anti- serotonin antibody (Consolazione er al., 1981) (150) then with biotinylated sheep anti-rat IgG (Fab fragment, Boehringer, 1: 100) for 6 h, followed by avidin-biotinylated horseradish peroxidase complex for 2 h, (Vector ABC, 1:lOO). The immunoperoxidase reaction to visualize serotonin-positive elements was developed by ammonium- nickel sulphate-intensified 3,3'-diaminobenzidine (DAB-Ni) as a chromogen .

For double immunostaining for serotonin and calcium-binding proteins, the sections were incubated with a mixture of monoclonal rat anti-serotonin ( I :SO) and rabbit anti-calbindin D28K antibody (R202, 1: 1500; Bairnbridge et al., 1982). rabbit anti-calretinin antibody (1:SOOO; Rogers, 1989) or rabbit anti-parvalbumin antibody (R 301, 1: 1000; Baimbridge et al., 1982) for 2 days, followed by biotinylated sheep anti-rat IgG together with goat anti-rabbit IgG (ICN, 150)

1722 Differential effects of serotonin

for 6 h. After incubation with ABC, as above, the serotonin- immunopositive elements were developed by DAB-Ni (dark blue reaction product). The first immunoperoxidase reaction was followed by treatment with rabbit peroxidase-antiperoxidase complex (PAP, Dakopatts, 1:100) and the second reaction to visualize the different calcium binding protein-containing cells was developed with DAB alone, which gives a brown reaction product. All the washing and dilution of the antisera were in 50 mM Tns-buffered saline. To enhance penetration of the antisera the sections were either treated with 0.5% Triton X-100 or freeze-thawed as described above.

Following double immunostaining for serotonin and the calcium- binding proteins, the sections were osmicated ( 1 % Os04, 1 h), adding 7% glucose to the solution to preserve colour differences. The sections were dehydrated, embedded in Durcupan (ACM, Fluka) and examined under the light microscope. The areas containing contacts were re-embedded for further ultrathin sectioning.

For silver intensification in combination with serotonin immuno- reaction, the sections of gPHAL-injected animals were washed several times in PB followed by rinses in distilled water, then incubated in IntenSE M Silver Enhancer (Amersham) for 30 min. The reaction was stopped with distilled water and fixed with 1% sodium-thiosulphate dissolved in water. The reaction product appeared as black granules dispersed in the cytoplasm. The intensification was followed by serotonin immunoreaction (as described above) using DAB as a chromogen (brown reaction product).

Results are presented as mean 5 SEM. ANOVA (followed, where required by Student-Newman-Keuls post hnc tests, with a significance level of 0.05) or the paired t-test were used for statistical comparison.

Results Behaviour and physiology Body weight Rats were weighed prior to being tested in the Morris water-maze (5 months after lesions and transplantation) and again at the end of behavioural testing (3 months later). All groups gained weight during this period (8 versus 5 months: control, 37658 versus 42027 g, T = 4.55, P < 0.004; DHT, 33251 1 versus 353k13 g, T = 4.25, P < 0.04; HG, 33054 versus 358k 11 g, T = 3.36, P < 0.01; HTG, 349210 versus 36359 g, T = 3.89, P < 0.0046, paired t-test). However, there was a significant difference in body weight between control rats and each of the lesioned groups, which weighed less than normal (F = 5.08, P < 0.006, with significant differences between control and each of the lesioned groups in post hoc tests).

Exploratory behaviour For estimation of exploratory behaviour, rats were first put in an open field for 5 min and immediately afterwards in the elevated plus-maze for an additional 5 min. All serotonin-depleted rats showed a similar reduced tendency to explore the open field, and although there was no significant difference between any of the lesioned groups and control rats in open field test there was a significant effect of the serotonergic lesion [control versus (DHT + HG + HTG); T = 2.47, < 0.01]. Control rats tended to spend more time in the closed arms

of the elevated plus-maze but visited the open arms as well. All serotonin-depleted groups, including grafted ones (DHT, HG, HTG), avoided the open arms of maze (F = 3.07, P < 0.04, ANOVA, with significant differences between control and each of the lesioned groups in post hoc tests).

Thermoregulation There was no significant difference in basal body temperature among the groups. Exposure to ice-cold water for 3 min led, in control rats, to a reduction of 0.83 + 0.3"C. It led to a significantly greater reduction of body temperature in the DHT and HG groups (DHT versus control, F = 9.1, P < 0.004, HG versus control, F = 12.1, P < 0.001, ANOVA) (Fig. I ) . In the HTG group the reduction in body temperature was smaller (although not significantly different) compared to the other lesioned groups, and was not significantly different from the control group.

Spatial learning task The water-maze task has two components representing non-spatial and spatial learning. The non-spatial aspect of the task (i.e. learning that there is no escape from the maze other than getting on the platform) was acquired by all groups, as was evident by a significant reduction in the escape latency between the first and last blocks of trials (controls, T = 19.23, P < 0.0001; DHT, T = 3.19, P < 0.007; HG, T = 14.9, P < 0.0001; HTG, T = 8.08, P < 0.OOOl). There was a significant effect of groups on the performance of the spatial

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c 5 x U 0 m

FIG. 1 . The effects of serotonin depletion and raphe grafts on thermoregulation. Reduction of core temperature over 3 min following a 3 min exposure to ice- cold water was significantly greater in the DHT and HG rats compared to control (DHT, F = 9.1, P < 0.004; HG, F = 12.1. P < 0.001, ANOVA). The HTG group was not significantly different from either the control or the other lesioned groups.

0 1 2 3 4 5 6 7 8

Trial number

FIG. 2. The Morris water-maze spatial learning task. There was a significant effect of groups on the performance of the spatial learning part of the task ( F = 4.3, P < 0.006, ANOVA for repeated measurements). Serotonin depletion reduced the ability of atropine-treated rats to locate and escape to the invisible platform (control versus DHT, F = 10.3, P < 0.003). While HTG rats performed as poorly as the DHT rats (control versus HTG. F = 12.6, P < 0.001). HG rats were similar to controls and significantly better than the DHT ( F = 4.53, P < 0.04) and the HTG ( F = 5.96. P < 0.02) rats.

Differential effects of serotonin I723

learning part of the task (GR, F = 4.3, P < 0.006). Post hoc tests revealed the following. The DHT rats were impaired in their ability to locate and escape to the hidden platform (DHT versus control, F = 10.3, P < 0.003) (Fig. 2). HG rats were significantly better than the DHT rats (HG versus DHT, F = 4.53, P < 0.04). confirming

previous observations (Richter-Levin and Segal, 1991a, b). In contrast, the HTG group was as impaired as the DHT group and significantly worse than both control and HG groups (HTG versus control, F = 12.6, P < 0.001; HTG versus HG, F = 5.96, P < 0.02).

This difference between raphe grafts in the hippocampus and

FIG. 3. Light micrographs of serotonin-immunostained embryonic raphe transplants in the hippocampus following 5,7-DHT lesion. (A) The graft, containing several serotonin-immunoreactive cells (arrows), is located within the fissure of the ventral hippocampus. (B) In another animal the graft survived in the ventricle attached to the white matter of the overlying neocortex. A moderate number of serotonin-immunopositive neurons (arrows) were present in this graft, which were shown to innervate the hippocampus (see F). (C) Serotonin-immunopositive neurons and axons within the transplanted tissue. (D, E) Serotonin- immunoreactive axons originating from the graft restore the original pattern of innervation. Dense fibre labelling is present in the hilus of the dentate g y m (D), in stratum radiatum of the CA3 region (E), and in stratum oriens of the CAI region (F). The principal cell layers are almost devoid of serotonin-positive fibres. D and E are from the same animal as A, and F is from the same animal as B. Scales: A. B, 200 pm; C-F, 30 pm.

1724 Differential effects of serotonin

hypothalamus was also evident in the quadrant analysis test ( F = 7.5, P < 0.0008). Control rats spent most of the test trial time in the third quadrant. in which the platform was previously located. DHT rats and HTG rats spent significantly more time in the other quadrants while the HG rats performed similar to controls and significantly better than the DHT and HTG groups.

Anatomy Effect of5.7-DHT lesion The effects of the lesion were evaluated in 5.7-DHT-treated rats, in the hippocampus of the HTG animals and in the hypothalamus of the HTG rats.

In the hippocampus, the serotonergic innervation was very sparse

FIG. 4. Light micrographs of serotonin-immunostained embryonic raphe transplants in the hypothalamic region following 5.7-DHT lesion. (A, B ) Grafts containing several serotonin-immunopositive neurons (arrows) are present in both hemispheres, in the ventral premammillary region on the left side and just above the mammillothalamic tract on the other side. B shows the graft situated on the right side some sections away caudally. at its greatest cross-sectional area. (C) Serotonin-immunoreactive fibres densely innervating the supramammillary region of the hypothalamus. (D) High-power light micrographs of serotonin- containing neurons and axons within a graft placed in the hypothalamus. Scales: A, B. 500 pm: C. D. 30 pm.

Differential effects of serotonin 1725

following the lesion. In one of the HTG rats the innervation of the hippocampus was normal unilaterally. This occasional failure of lesion has been reported previously (Van Luijetelaar et al., 1991). The neocortex overlying the hippocampus was also almost completely devoid of serotonergic fibres. In hypothalamic areas, the supramam-

millary region, known to have a dense serotonergic innervation, was compared among different animals. Thus, while the density of fibres was found to be slightly higher than in the cortical areas, it was still well under the normal level. The parent cells o f these fibres, the serotonergic neurons of the dorsal and median raphe nucleus, were

FIG. 5. (A-C) High-power light micrographs of hippocampal sections double-immunostained for calretinin and serotonin (A, B) or calbindin and serotonin (C). (A) A serotonin-immunopositive axon forms multiple contacts on the dendrite of a calretinin-containing interneuron situated in stratum lacunosum moleculare of the CAI region in the vicinity of a raphe transplant. Four of the boutons (bl-b.,) are labelled, which helps the identification of the dendrite on the correlated electron micrograph shown in Figure 6. (B) Serotonin-immunopositive boutons establish single contacts on two calretinin-containing cell bodies (arrows) in the molecular layer of the dentate gyrus close to a raphe gradt. (C) A calbindin-immunoreactive interneuron receives multiple contacts from a serotonin-positive fibre (arrows) in the hilus of the dentate gyrus. Some of the calbindin-positive granule cells are labelled by asterisks. (D-F) Following an injection of gPHAL into the dorsal hippocampus, a serotonin-negative neuron is labelled retrogradely in the ventral portion of the median raphe nucleus (arrow in D), as shown by the accumulation of silver-intensified gold particles. In a very few cases serotonin-positive cells were also found to project to the hippocampus (arrow in E) but most of the serotonin-immunoreactive neurons in the median raphe were not labelled by the retrograde tracer (open arrows in D and E), indicating that the 5.7- DHT lesion affected the projecting serotonergic axons rather than the cell bodies. In the hippocampus, the tracer labelled a serotonin-immunoreactive neuron within the graft (arrow in F), providing direct evidence for the serotonergic reinnervation by grafted neurons as well as pyramidal cell bodies in the CA3 region (asterisks in F) not containing serotonin. Scales: A-F, 20 pm.

1726 Differential effects of serotonin

also investigated. In all animals serotonergic cell bodies, as well as serotonin-immunopositive fibres, were found in large numbers within both nuclei, indicating that the 5,7-DHT lesion had a limited effect on serotonergic cell bodies and their local axonal arborization.

Grafts in hippocampus Four HG animals which had received bilateral raphe grafts into the ventral hippocampus were investigated. In two animals the grafts survived in the lateral ventricle, attached to the white matter of the cortex or to the fimbria (Fig. 3B). In the other two cases the grafts were located within the hippocampus centred around the hippocampal fissure or the outer blade of the dentate gyrus in the ventral hippocampus (Fig. 3A). Within the grafts only a relatively small number of cells showed serotonin immunopositivity. They were located mainly at the edge of the grafts or near blood vessels. The cell bodies were spherical, round or oval-shaped and they bore few primary dendrites (Fig. 3C). The axon distribution was similar to that seen in normal animals, i.e. the density of the fibres was high in stratum lacunosum moleculare and stratum oriens of the Ammon’s horn and in the hilus of the dentate gyrus (Fig. 3D-F). Axon density was higher than normal in the vicinity of the graft and it gradually decreased further away. There was a slight difference in the innervation pattern between the animals with grafts located within the hippocam- pus and those with grafts bordering the ventricles. In the latter case, axons innervated hippocampal areas more distant from the grafts and several fibres were observed to penetrate the overlying neocortex as well. Axons were seen to climb along the fimbria and innervate mostly the stratum oriens of CAI. In the case of grafts within the hippocampus, most of the fibres innervated the stratum lacunosum moleculare or the dentate gyrus (including the hilus and the stratum moleculare), depending on the location of the graft. The innervation of the principal cell layers was very sparse in both cases.

Grafts in hypothalamus Bilateral (three cases) or unilateral (one case) serotonergic raphe grafts were found in the posterior hypothalamus of four animals examined. The grafts varied in size and shape. They were present mainly in the lateral hypothalamic area and in the rostra1 part of the ventral tegmental area, occasionally reaching the mammillary region (Fig. 4A, B). In one case a graft survived in the third ventricle. The grafts innervated heavily the surrounding hypothalamic areas (Fig. 4C), including the supramammillary nucleus, mammillary bodies and the lateral hypothalamic area. Fibres did not extend dorsally to the medial lemniscus, and they avoided the zona incerta and the substantia nigra. This fibre distribution is similar to that described earlier in normal animals (Azmitia and Segal, 1978). Similarly to the HG animals, only a small number of cells in the graft showed serotonin immunopositivity (Fig. 4B).

Serotonergic and non-serotonergic hippocampal projections from the host and grafted raphe One DHT rat, one HG rat and two HTG rats received unilateral injections of gPHAL into the dorsal hippocampus. Sections of the hippocampus and the midbrain were silver-intensified and processed for serotonin immunocytochemistry.

The injection of gPHAL into the dorsal hippocampus resulted in the labelling of numerous somata in the median raphe nucleus (Fig. 5D, E). Serotonin immunostaining revealed that the majority of them were non-serotonergic (Fig. 5D). The cells were mainly bipolar, oriented horizontally within the median and paramedian raphe nuclei. Retrogradely labelled non-serotonergic cells were found in the entire rostrocaudal extent of the ventral part of the nucleus. A very small

normal retrograde labelling) could also be seen (Fig. 5E). suggesting that few of the serotonergic fibres in the hippocampus have a host origin.

In the gPHAL-injected HG animal the graft was located in the ventricle attached to the CA3 region at mid-septotemporal level. Even though the injection was dorsal to the grafted site, were serotonin- immunopositive fibres were very sparse, a retrogradely labelled serotonergic cell was found at the border of the graft and host tissue (Fig. 5F). Several non-serotonergic cells were also retrogradely labelled within the graft.

FIG. 6. Correlated light and electron micrographs of the multiple contact between a calretinin-positive cell and a serotonin-positive axon shown in Figure SA in colour. Asymmetrical synaptic contact between the serotonin- immunoreactive houton (bz) and the calretinin-containing interneuron was clearly visible (arrow in A), in spite of the poor preservation. The labelled houtons on the low-Dower electron microzraohs (BI helD the correlation. c .

number of retrogradely labelled serotonergic cells (compared with Scales: A. 0.5 pm: B, 2 pm: C. 20 pm.

Differential effects of serotonin I727

Postsynuptic targets of the serotonergic mom of the grafi Earlier studies showed that serotonergic axons of the median raphe form multiple contracts with certain subpopulations of hippocampal non-principal cells containing the calcium-binding protein calbindin or calretinin but avoid others containing parvalbumin (Freund et al., 1990; Halasy et a/., 1992; AcsBdy ef a/., 1993). Double immunocyto- chemistry for serotonin and calcium-binding proteins was performed on hippocampal sections containing the graft to examine whether a similar selective pattern of innervation was re-established by the raphe grafts.

Multiple contacts between serotonin immunopositive axons and calbindin- or calretinin-containing interneurons were rather rarely observed (Fig. 5A-C). Single contacts were more frequent but still far rarer than in normal cases, even at sites near the graft with abundant serotonergic fibres. Thus, it appears that complete reinnervation of the interneurons did not take place. A multiple contact between a serotonergic axon and a calretinin-immunoreactive dendrite, detected by light microscopy, was examined in the electron microscope. Correlated light and electron microscopic observation showed that serotonergic axons established asymmetrical synapses on the inter- neuron dendrite (Fig. 6A, B). No contacts were found on parvalbumin- containing interneurons, suggesting that the specificity of the sero- tonergic innervation was preserved by the fibres from the grafted tissue.

Discussion The present results confirm previous findings that a combined disruption of serotonergic and cholinergic neurotransmission impairs the ability of rats to perform a spatial learning water-maze task (Nilsson et al., 1988; Dickson and Vanderwolf, 1990; Richter-Levin and Segal, 1991a; Riekkinen et al., 1991). Raphe grafts in the hippocampus reduced the behavioural impairment. When located in the hypothalamus such grafts had no effect on spatial learning, as was found with raphe grafts in the entorhinal cortex (Richter-Levin and Segal, 1992; Richter-Levin et a/. , 1993). In addition, we now found that the 5,7-DHT lesions affected the forebrain serotonergic fibres but had a much smaller effect on the midbrain serotonergic innervation. Together, these results suggest that, with respect to spatial learning, the serotonergic system may function locally in the hippocampus. This involvement of the serotonergic system in spatial learning was specific in that the combined lesions did not cause a general learning impairment; these rats were still able to learn the non-spatial components of the water-maze task (Nilsson et al., 1988; Richter-Levin and Segal, 199 1 a). Also, the serotonergic lesion resulted in several physiological and behavioural effects (i.e. reduced explorat- ory behaviour, reduced body weight, impaired thermoregulation), none of which was prevented by the raphe grafts in the hippocampus. The HG rats were able to perform the spatial learning task despite those impairments, suggesting that these do not underlie the spatial learning deficits.

The raphe-originated innervation of the hippocampus contains both serotonergic and non-serotonergic fibres. Lesioned rats exhibited impaired performance in the maze even though the non-serotonergic raphe-hippocampal pathway survived the 5,7-DHT lesion (as shown by retrogradely labelled non-5-HT cells in the raphe of these rats). Thus, although the raphe grafts innervated the hippocampus with both serotonergic and non-serotonergic fibres, we suggest that it is the serotonergic component that underlies the ameliorating effects of the grafts. This view is supported by our earlier studies in which the behavioural effects of raphe grafts in the hippocampus disappeared

following a treatment with p-chlorophenylalanine (which reduces serotonin synthesis but does not affect the non-serotonergic neurons in the graft) (Richter-Levin and Segal, 1991b).

Reinnervation of the hypothalamic areas by the raphe grafts was comparable to that in the intact brain (Fig. 4C). The lack of behavioural effects of these grafts suggests that the serotonergic innervation of these area has no role in spatial learning. In contrast, raphe grafts in the hypothalamus present serotonin depletion-induced abnormal sexual behaviour (Luine et al., 1984) and seemed to reduce the effects of the lesion on thermoregulation (Fig. 2). These results further support the view that the serotonergic system is involved in specific aspects of physiology and behaviour, by local effects at defined target sites.

The majority of the serotonergic fibres in the hippocampus of the HG rats originated from the grafted tissue rather than from the host itself. This was evident since the distribution of the serotonergic fibres in the grafted hippocampi expressed a gradient of density, and was highest near the graft (Fig. 3A, B). Also there was very little retrograde labelling of serotonin immunopositive cells in the raphe nuclei of the host (Fig. 5D, E) even though the density of serotonergic fibres in the hippocampus was high (Fig. 3D-F). Post-lesion recovery could not account for this since in the DHT group the density of the serotonergic fibres remained very low.

Multiple contacts, which normally characterize the serotonergic innervation of interneurons, were rarely observed. Instead, the grafted serotonergic fibres formed single contacts and these were less frequent than in the intact hippocampus. Nevertheless, the selectivity of termination (i.e. avoiding parvalbumin-containing cells) was main- tained. Furthermore, asymmetrical serotonergic synapses were found on a calretinin-immunoreactive dendrite, indicating that the grafted serotonergic cells do form synapses with the interneurons they reinnervate.

Electrophysiological observations suggest that interneurons mediate some serotonergic functions in the hippocampus; the interneurons are more sensitive to application of serotonin-than are the principal cells (Segal, 1990). Hippocampal commissure-induced inhibition (assumed to be mediated by interneurons) is reduced in rats with combined serotonergickholinergic lesions (Richter-Levin and Segal, 1991 a). Raphe grafts, which prevent the spatial learning deficits in such double-lesioned rats, also restore the commissure-induced inhibition (Richter-Levin and Segal, 1991 a). The present anatomical observations concur with this notion that the serotonergic innervation of inter- neurons is likely to be essential for the functional recovery of the hippocampus.

In summary, a detailed analysis of the pattern of reinnervation by serotonergic raphe grafts indicated that, although this reinnervation only partially compensates for that of the lesioned fibres, it maintains selectivity with regard to parvalbumin-containing interneurons. The graft effects were region-specific, indicating that such grafts can be used to study local functions of the serotonergic and other neuromodulatory systems.

The present results indicate that the serotonergic innervation of the hippocampus has a local function which is associated with spatial learning. This role of the serotonergic system is suggested to be independent of its role in modulating body weight, thermoregulation and exploratory behaviour.

Acknowledgements We are grateful to Drs K. G. Bairnbridge, A. C. Cuello and J. H. Rogers for gifts of antisera against parvalbumin, calbindin, serotonin and calretinin, and to Dr R. A. J. Mcllhinney for the gift of gPHAL. The excellent technical

1728 Differential effects of serotonin

assistance of Mrs E. Borok, Mrs I. Weisz and Mr Terstyanszky is also acknowledged. This work was supported by the Human Frontier Science Program Organization (to T. F. F.), OTKA (no. 2920) and E7T (no. 551 91) Hungary.

Abbreviations

ABC DAB DHT

gPHAL HG HTG PB

5.7-DHT

avidin-biotin complex diaminobenzidine 5.7-DHT-treated rats 5.7-dihydroxytryptamine gold-conjugated Phaseolus vulgaris leucoagglutinin 5,7-DHT-treated rats with a raphe graft in the hippocampus 5.7-DHT-treated rats with a raphe graft in the hypothalamus phosphate buffer

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