input from the amygdala to the rat nucleus accumbens: its...

Pergamon 0306_4522(94)E0142-Q

.%uros&nce Sol. 61, No. 4, pp. 851-865. I994 Eisevier Science Ltd

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INPUT FROM THE AMYGDALA TO THE RAT NUCLEUS ACCUMBENS: ITS RELATIONSHIP WITH TYROSINE

HYDROXYLASE IMMUNOREACTIVITY AND IDENTIFIED NEURONS

L. R. JOHNSON, R. L. M. AYLWARD, Z. HUSSAIN and S. TOTTERDELL

University Department of Pharmacology, Mansfield Road, Oxford OXI 3QT, U.K.

A~aet-Both tyrosine hydroxyla~-positive libres from the m~olimbic dopamine system and amygdala projection fibres from the basolateral nucleus are known to terminate heavily in the nucleus accumbens. Caudal amygdala fibres travelling dorsally via the stria terminalis project densely to the nucleus accumbens shell, especially in the dopamine rich septal hook. The amygdala has been associated with the recognition of emotionally relevant stimuli while the mesolimbic dopamine system is implicated with reward m~hanisms. There is ~havioural and electrophysiolo~~l evidence that the amygdala input to the nucleus accumbens is modulated hy the mesolimbic dopamine input. but it is not known how these pathways interact anatomically within the nucleus accumbens. Using a variety of neuroanatomical techniques including anterograde and retrograde tracing, immunocytochemistry and intracellular filling, we have demonstrated convergence of these inputs on to medium-sized spiny neurons. The terminals of the basolateral amygdala projection make ~ymrnet~cal synapses p~dominantly on the heads of spines which also receive on their necks or adjacent dendrites, symmetrical synaptic input From the mesolimbic dopamine system. Some of these neurons have also been identified as projection neurons, possibly to the ventral pallidum.

We have shown a synaptic level how dopamine is positioned to modulate excitatory limbic input in the nucleus accumbens.

The amyg~ia is a key limbic structure believed in animals to be involved in the direction of behaviour to biological goals.” Together possibly with the septum, it is the element of the limbic system (a term applied to structures of the limbic lobe thought to be important in controlling affective behaviour) that is most closely associated with emotional responses, although neither area features in the classical Papez circuit. Since. the major inputs to the amygdala come from the ass~iat~onal cortex the incoming infor- mation probably includes not just what is happening now. but also responses to a similar situation in the past, i.e. the memory elements of its function.19 A major output from the basolateral~basomedial nuclei (BLa) is via the stria terminalis to the nucleus accumbens (ventral striatum) and the medial parts of the dorsal striatum.23~24~33 However, there is also evidence that the projection to the rostrai part of the

Abbrealntions: BDHC, benzidine dihydrochloride; BLa, basolateral/basomedial amygdaloid complex; DAB, di- amino~n~dine tetmhydr~hlo~de; HRP-WGA, horseradish ~roxi~as~whcatge~ agglutinin complex; LY, biotinylated Lucifer Yellow; NA, nucleus accumbens; NMDA, N-methyl, o-aspartate; PB, phosphate buffer, pH 7.4; PBS, phosphate-buffered saline; TH, tyrosine hydroxyla~; TH-IR tyrosine hydroxylase immunoreactive; VP. ventral pallidum; VTA ventral tegmental area.

nucleus a~umbens may also arrive via the ~ongitudi- nal association bundIe. The BLa to accumbens projection arises from “pyramidal” type neurons239 which are thought to be glutamatergic.‘0,39

In the nucleus accumbens (NA), the amygdalar input is influenced by dopaminergic input from the ventral tegmental area (VTA). It has been shown that NA units are excited by BLa stimulation and that this is attenuated by concurrent stimulation of the ventral tegmental area.“s2 Mogenson and Yim went on to show that this attenuation was due to the release of endogenous dopamine, possibly as a consequence of presynaptic dopamine D2 receptor action. which they argue may result in decreased transmitter release from the amygdalar terminals.53 A decrease in loco- motion caused by bilateral injection of the glutamate agonist NMDA (N-methyl, D-aspartate) into the basolateral amygdala was reversed by bilateral injection of dopamine into the NA. Alternatively, the increase in dopamine levels could be brought about from endogenous sources, such as seen with picro- toxin injections into the VTA, which reduce intrinsic inhibition of the dopamine neurons projecting to the NA.29 However, the central, basolateral and cortico- medial nuclei of the amygdala receive a dopaminergic input from the VTA2’ while the central nucleus projects back to the substantia ni~a~VTA.~~ Thus the interaction between the amygdala and the dopamine

851

852 L. R. Johnson ef al.

ceils of the midbrain can occur via a direct re- ciprocal connection as well as through interactions between the terminals of both ceil groups within the NA.

The functions of the amygdala are complex. In humans, electrical stimulation of the amygdaia, carried out in patients with epileptic foci in the temporal lobes can produce complex hallucinations, feelings of ditjji vu, fear, pleasure, anger or anxiety.‘* In animals, the structural integrity of the amygdaia in infancy would seem to be essential in the development of normal social (play, mutual grooming, hostility etc) and non-social behaviours (self grooming, manipulat- ing objects, feeding), although the effects of lesions may develop over periods of several years.45 Social deficits, in primates at least, may involve impairment of specific groups of “face recognition” neurons.4” The amygdaia seems to play a crucial role in the way in which previously neutral stimuli acquire incentive properties.’ However, it is interesting to note that in humansi and monkeyP lesions of the amygdaia do not seem to result in appreciable memory deficits. Recent evidence would suggest that the amygdaials and especially the amygdaia-accumbe~s pathway” is specifically involved in the association of stimuli with reward value.

The principal pathologies of the amygdaloid region are associated with epilepsy, although the amygdaia has also been implicated in schizophrenia. Reynolds3’ found elevated levels of dopamine in the left amygdala of schizophrenic patients. A study by Roberts et al.j’ has shown that levels of the peptide choie- cystokinin were reduced in the amygdaia of type 2 schizophrenic patients, post mortem, while vasoactive intestinal protein was increased in type 1 patients. The same study showed changes in somatostatjn levels in other limbic brain areas.

We have previously examined the connections between peptide interneurons in limbic brain areas and projection neurons from those areas to the nucleus accumbens, a common output target.z.4’ We have demonstrated that hippocampai input converges on to NA neurons which are postsynaptic to tyrosine hydroxyiase-immunoreactive (TH-IR) ter- minais,“8 presumably representing the dopaminergic input from the VTA. In this study we set out to examine the input from the basoiaterai nucleus of the amygdaia to the NA, comparing three different methods for iabeiiing the boutons in the NA. The identity of some of the postsynaptic target neurons was revealed using in vitro intracellular filling with biotinylated Lucifer Yellow (LY), and in some experiments the output of these filled neurons was established by retrograde transport. In order to estabiish the synaptic relationship between the amygdalar input and that from the dopaminergic neurons of the VTA, we examined tissue in the electron microscope for examples of convergence of amygdaiar and dopaminergic input on to single targets.

Some of these results have already been communi- cated in preliminary form at the Fourth IBAGS Meeting, 1 992.“2

EXPERIMENTAL PROCEDURES

This study involved the combination of anterograde tracing and immunocytochemistry with intracellular filling in fixed slices. The tissue was prepared for light and electron microscopy.

Female Wistar rats (160-240 g) from Banting and King man were anaesthetized with equithesin (0.3 ml/100 g) and placed in a stereotaxic frame. In two rats pressure injections of 500 nl of biocytin were made bilaterally into the caudal basolateral amygdala, using the coordinates A4.6, L4.6. V7.2 and 7.4, modified from Paxinos and Watsons2 and Kiinig and Klippel. 2b The injections were made using air pressure via micropipettes with glass tips pulled to a diam- eter of 30pm. Two other rats received unilateral injections of biocytin into more rostra1 regions of the amygdala, represented by coordinates P2.0 (from bregma), L3.6 and V7.5. In four rats unilateral lesions were made by passing a current of 3 mA for 4 s via a microelectrode consisting of d fine glass pipette encasing a silver wire with 1-2 mm of wire exposed at the tip. The coordinates used were A4.8-5. I, L4.0-4.7, V7.4. In a ninth rat 40 nl of horseradish peroxidase-wheatgerm agglutinin (HRP-WGA) was injected into the basolateral amygdala. The survival time for animals with biocytin inject&s was one day, whereas two days were used for animals with lesions and HRP-WGA iniections.

In one of the rats an amygdaloid lesion was c&bined with an injection of rhodamine-la~ll~ latex beads (Lumafluor. NY) made into the ventral pallidum (A7.1. L0.8, V7.0) during the same surgery session.

Per&ion

Rats were deeply anaesthetized and perfused through the aorta with 150 ml 0.9% NaCl solution followed by 200 ml of 0.1% gluta~ldehyde and 3% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB) followed by 200 ml of 3% paraformaldehyde in 0.1 M PB. The brains were cut coronally at 100 pm in 0. I M PB on a vibrating microtome. Sections through the amygdala were either collected in vials and processed where appropriate to reveal the biocytin or HRP-WGA or, for the lesion sites, they were collected on gelatine-coated slides and Nissl stained.

In four of the rats, two with lesions and two with injections of biocytin, cells in the NA were filled in vitro with an aqueous 4% solution of LY conjugated to biotin (Molecular Probes, Oregon) using the method of Biihl.” In a further one rat that had an injection of rhodamine beads into the ventral pallidurn (VP) the retrogradely labelled neurons were identified by their tluorescence and were also filled with LY.

Fluorescence was visualized with a Zeiss fixed stage microscope using an ultra-violet light filter (excitation wave length 390-420 nm). Sections were stored on ice in order to maintain ultrastructural integrity and visualized at room temperature in 0.1 M PB. Glass electrodes of 2pm tip diamerer were filled with LY. Using micromanip~ators ~agashiki, Japan) the electrode was guided either into rhodamine Auorescing retrogradely fabelled cells or directly into the tissue until clean soma and dendritic filling commenced. The cells were allowed up to 15 min to be completely filled while passing a posit&e current of 2 nA iontophoretically (World Precision Instruments, U.S.A.). One to six cells were filled per section.

Amygdala-TH convergence in nucleus accumbens 853

Immunoc.vtochemistry and peroxidase reactions

Immediately following cell filling, sections were placed in a cryopro&ant solution (25 g sucrose, 10 ml glycerol, 25 ml 0.2 M PB in 100 ml) and allowed to equilibrate for at least 30 min, but often o&might at 4°C. Thk next morning sections were flattened on to small mesh nets; thorough drying by blotting on tissue paper before immersion in isopentane seemed to reduce section cracking. Sections were frozen first in slushy isopentane and then liquid nitrogen.” Finally they were allowed to thaw in the cryoprotectant. before being washed thoroughly in phosphate-buffered saline (PBS). After washing, the sections were placed in a solution containing the primary antibody (rabbit anti- tyrosine hydroxylase; Eugene Tech, New Je&ey) at 1: 1500 m PBS, to which was added Vectastain ABC Reagent (Vector. Laboratories, U.K.). The sections were gently shaken at 4°C for 48 h.

Following incubation, sections were washed thoroughly in PBS and then incubated in a biotinylated secondary antibody (Vector Laboratories) for 2 h. After washing they were incubated for an additional 3 h in Vectastain ABC reagent, transferred to 50 mM Tris-HCl buffer (PH 7.6) and then incubated for 7.5 min in 0.005% 3,3’ diaminobenzidine tetrahydrochloride (DAB, Sigma) in the same buffer. Hydrogen peroxide was added to give a final concentration of 0.01% for 5 min at room temperature until the peroxidase reaction product in the LY-filled cells could be seen with the aid of a binocular microscope. It was usually also possible at this stage to detect the delineation by the anti-tyrosine hydroxylase reacted fibres of the striatal borders. Biocytin fibres were also revealed by this reaction. The reaction was stopped by washing in fresh Tris-HCl buffer and then the sections were transferred to 0.1 M PB.

The sections containing anterogradely transported HRP-WGA were reacted with the chromogen o-tolidine. Briefly, they were equilibrated in a citrate-acetate buffer, pH 5 and then transferred to a reaction mixture of 30 mg o-tolidine in lOOm1 0.1 M citrate-acetate buffer to which was added immediately before use I ml 5% sodium nitro- prusside anQf” 1 1 1% H,O,. The incubation was carried out at O”C, m darkness for 6&9Omin. Sections were then washed briefiy in fresh buffer and transferred to a stabilization solution consisting of 50mg DAB in lOOmI PB plus 1 ml I% cobalt acetate and 15 pl 30% H,Oz for 5 min at 0°C. Finally thev were washed in 0.1 M PB. These sections were thin incubated with TH antibody, which was revealed using the peroxidase-antiperoxidase system and the chromogen benzidine dihydrochloride (BDHC).

All sections were then flattened on watch glasses and immersed in 1% osmium tetroxide in 0.1 M PB for 30 min. After washing in H,O, the tissue was dehydrated through a series of alcohols, including 40 min in 70% alcohol containing 1% uranyl acetate, to propylene oxide and placed in Durcupan resin (Fluka, U.K.) overnight. The following day sections were carefully transferred to slides, a coverslip put on and the resin allowed to polymerize for 48 h at 60°C.

Microscopy

Slides were examined in the light microscope. The distribution of the amygdalar input to the NA was assessed using the sections with biocytin filled fibres. These were compared to a series of sections stained for calbindin in order to mark the core and shell compartments. Neurons filled with LY were identified, drawn with the aid of a camera lucida and photographed.

Blocks of NA tissue were prepared for electron microscopy. Areas likely to receive amygdalar input which contained good TH-immunoreactivity, were also selected, cut out and mounted on a preformed resin block with cyanoacrylate glue. Where possible these blocks included a LY-filled neuron. Serial ultrathin sections were cut,

collected on single slot, Pioloform coated, copper grids, stained with lead citrateIs and examined in a Philips 201C electron microscope.

To evaluate the postsynaptic targets of the amygdalar input, blocks were prepaied ?rom core and shell regions of the NA for each of the three labellinn methods. Ultrathin sections were collected from within 5LlOgm of the tissue surface. One section per grid, chosen at random, was scanned and all anterogradely labelled structures were recorded, whether or not they were synaptic. Boutons which were synaptic were classified by their target structures in single sections and for small profiles the default category was “spine”. The target number of degenerating or biocytin labelled boutons to be counted in each of the core and shell was 100. Since the counts for the boutons labelled anterogradely with HRP-WGA were from a single animal, the numbers were smaller.

ControIs

In our preliminary experiments the first incubation with ABC, to reveal the LY-filled neurons, was carried out in the absence of the TH antibody, and the bound peroxidase was ,reacted with DAB before the immunocytochemical incu- bations were started. In each experiment at least one section containing LY-filled neurons was processed without TH- antibody, but with all the other steps. The majority of sections in an experiment were not used for cell filling and contained only the anterograde tracer and TH immunoreactivity. Blocks for electron microscopic examination were prepared from the NA of unlesioned tissue or from animals where the lesions failed to involve the basolateral] basomedial nuclei of the amygdala and examined for the presence of degenerating boutons.

RESULTS

Light microscopy : connectivity

The injections in this experiment were mainly targeted on the caudal part of the basolateral nucleus of the amygdala (Fig. 1C). Biocytin-labelled fibres could he followed as they passed dorsally and then caudally in the stria terminalis, before moving ros- trally and then ventrally into the nucleus accumbens, where they formed varicose fibres which were mainly in the shell, although a few were also found in the core. However, injections of biocytin that involved the basomedial nucleus (Fig. 2A) gave rise to exten- sive labelling (Fig. 2D) in both the core (Fig. 2E) and shell (Fig. 2F) and to fibres in the contralateral NA. There was also considerable labelling in the medial part of the ipsilateral dorsal striatum, especially along the edge of the lateral ventricle. Large injections of biocytin into the BLa resulted in almost Golgi-like labelling of pyramidal neurons in ipsilateral and contralateral parietal cortices.

The apparent injection site for the HRP-WGA, revealed with DAB, was large (Fig. lA), but centred within the BLa. Anterogradely labelled varicose fibres were seen in both core (Fig. 28) and shell of the NA and in the rostra1 part of the nucleus. They were also found in the dorsal striatum, as described for biocytin labelling. As with biocytin, the anterogradely labelled fibres were coarse, relative to those immunostained for TH. This was especially clear in this material, where different chromogens had

L. R. Johnson CI a/.

B

Fig. 1. A. C and D show the amygdala at approximately the same coronal section. A pressure injection of HRP~-WGA is illustrated diagrammatically in A while C shows the injection site for biocytin and D the extent of an electrolytic lesion. B shows an injection site for rhodamine-labelled latex beads in the dorsal VP, which gave rise to retrogradely labelled cells in the NA, some of which were filled with LY (see Fig. 6 AX). The pressure injection of WGA-HRP targeted in the BLa (A) gave rise to the anterograde WGA-HRP fibres in the NA, shown in Fig. 2B. Ic, internal capsule; is. injection site; vp.

ventral pallidum.

been used to reveal the anterograde marker and Retrogradely labelled neurons, identified following the TH-IR. The lesion sites were also centred on the the injection of rhodamine beads into the dorsal caudal BLa, but tended to involve more of the ventral pallidurn (Fig. I B), were found in the caudo- nucleus (Fig. ID). medial NA. notably in the septal hook.

Fig. 2.(A) Biocytin injection into the basomedial nucleus of the amygdala. This injection gave rise to the labelling in E and F. (B) Fibres of the amygdalar input to the nucleus accumbens core adjacent to the anterior commissure (AC) labelled by the anterograde transport of HRP-WGA, revealed with DAB (arrowheads), (C) A neuron in the nucleus accumbens, intracellularly filled with biotinylated LY. Note the spines emerging from the distal dendrites (arrows). (D) Low power micrograph of the nucleus accumbens showing the lateral ventricle (LV) and the anterior commissure (AC). The boxed areas appear in E and F. (E) Biocytin fibres in the core of the nucleus accumbens (arrowheads) from the injection illustrated in A. For orientation note the position of the anterior commissure (AC) and the fibre bundles. (F) Biocytin fibres in the shell (arrowheads) from the same injection. For orientation note the capillary

containing an erythrocyte (asterisk). Scale bars = 20 pm.

856 L. R. Johnson et al.

Light microscopy : immunocytochemistry

TH-IR fibres were found throughout the NA. Their distribution tended to be somewhat patchy and an intensely immunoreactive area was frequently seen in the septa1 hook of shell, just ventral to an area of relatively low levels of TH-IR. In material that had been stained for both TH-IR and biocytin it was possible to distinguish the two labels, even when both had been revealed with DAB as the TH-IR fibres were restricted to the surface layers of the sections whereas the biocytin-labelled fibres penetrated through the entire section thickness.

Light microscopy : cell filling

Intracellularly filled neurons were clearly revealed following incubation with ABC (Fig. 2C). They were all found to be of medium size and spiny, although the extent to which the spines were revealed varied from neuron to neuron. It is probable though that they all belonged to the class of medium-sized densely spiny neurons that is the most common in the NA. Although considerable leakage of LY from the soma occurred at times, it was possible to select for further study neurons where the dendrites could be clearly followed from the cell body. We saw no evidence that local axon collaterals of these neurons had also filled with LY. In total, 49 neurons were filled in five animals.

In the rat that had received the injection of rhodamine beads into the VP, cells that fluoresced with the rhodamine were selected for LY filling. Careful records were kept in order to identify those sections which contained such dual-labelled neurons. A total of 24 neurons were filled in this rat, of which 19 were retrogradely labelled. Two dual-labelled neurons from the area of NA most likely to contain amygdalar inputs were examined in the electron microscope.

Amygdalar efferents to the nucleus accumbens

In the electron microscope synaptic boutons in the core and shell regions were identified. The postsynaptic elements of the point of synaptic contact were compared for the core and shell, and for the NA as a whole. Of the 712 boutons examined, there was considerable agreement between the methods as regard targets in NA (Fig. 3) and there were no significant differences between the targets in core or shell labelled by any of the methods. Comparing methods, in rats with lesions of the BLa 82% of degenerating boutons contacted one spine (Figs

A: Amygdala targets in the NA Core & Shell Biocytin labelled

loo 1

Core (n=154) Shell (n=105)

Difference not significant (p > 0.7)

B: Amygdala targets in the NA Core & Shell Degeneration labelled

Core ( ~ 1 5 6 ) Shell ( ~ 2 2 3 )

Difference not significant (p > 0.8)

Fig. 3. The targets of amygdalar afferents to the NA. The percentages are compared for core and shell targets of one spine, two spines and dendrites, for biocytin-labelled boutons (A), and degenerating boutons (B). There was no difference between targets in the core and shell for either method (chi-squared values for biocytin: 0.51 (P > 0.7), and

for degeneration: 0.37 (P > 0.8) both with 2 d.f.).

SA, B, 6B, C), 12% contacted two spines in a single section (Figs 4E, SC, & D) and 6% were in synaptic contact with dendrites (Fig. 4C). With biocytin- labelled terminals, 84% were found on single spines (Fig. 4D) 8% on two spines (Fig. 4B) and 8% on dendrites. There was no significant difference (chi- square = 4.0) between these two distributions. For

Fig. 4. Examples of amygdalar input to the shell (A-C) and core (D-F) of the nucleus accumbens. Boutons are in asymmetrical synaptic contact (arrows) with spines (A, D) two spines in a single section (B, E) or dendrites (C, F). Boutons in A and F were labelled by anterogradely transported HRP-WGA (compare unlabelled bouton in F) in B and D with biocytin and C and E by degeneration. Note that the degenerating bouton in C is also making an asymmetrical synaptic contact with a spine (asterisk) and that the postsynaptic dendrite gives rise to a spine (sp). In D the spine can be seen to emerge from a dendrite (d).

Scale bars = 0.2 pm.


Fig. 4.

858 L. R. Johnson PI U/

Fig. 5. Examples of convergence between amygdalar input and TH-IR boutons on to unlabelled target neurons in the nucleus accumbens shell. In A and B boutons degenerating as a result of a lesion in the amygdala (db) are in asymmetrical synaptic contact (arrowheads) with spines that are also postsynaptic to TH-IR boutons (asterisks) making symmetrical synaptic contacts (arrows). Vesicles are still distinguish- able in the degenerating bouton in A, showing it to be at a relatively early stage of degeneration. C and D are sections from a series showing in C a TH-IR bouton in symmetrical synaptic contact (arrow) with a dendrite (d) from which a spine (sp) emerges. In D this spine can be seen to receive asymmetrical synaptic input (arrowheads) from a bouton degenerating as a result of a lesion in the amygdala. The bouton is

also in synaptic contact with a second spine. Scale bars = 0.2 pm.

Fig. 6. Examples of convergence between amygdalar input and TH-immunoreactive boutons on to neurons in the nucleus accumbens shell that project to the ventral pallidum. A and B are sections through different dendrites of the same LY-filled neuron. In A one dendrite (dl) gives rise to a spine, the neck of which (sn) is contacted (arrows) by the TH-IR bouton (asterisk). In B a second dendrite (d2) also gives rise to a spine (sp) which is in asymmetric synaptic contact (arrowheads) with a bouton (db) degenerating as a result of a lesion in the amygdala. In C a bouton immunoreactive for TH (asterisk) is in symmetrical synaptic contact with a LY-filled dendrite which gives rise to a spine (sp) that is postsynaptic (arrowheads) to a bouton (db) degenerating as a result of a lesion in the amygdala. These neurons both contained

rhodamine beads retrogradely transported from the ventral pallidurn. Scale bars = 0.2 pm.


Fig. 6.

860 L. R. Johnson e! (I/

anterogradely transported HRP-WGA only 74 boutons were counted (core: 28, shell: 46), but examples were found on one spine (Fig. 4A), on two spines and on dendrites (Fig. 4F).

Using the chi-square test to compare the distribution of targets, and pooling the results from all three methods, no differences were found in the core (chi-square value: 3.0). In the shell there were significant differences between the methods (chi-square value: 12.35), but these disappeared when the data were analysed without the HRP-WGA labelled boutons (chi-square value: 3.21). Combining the data for degeneration and biocytin, in the core it was found that 83.5% of contacts were with one spine, 10% on two spines and 6.5% on dendrites, while in the shell, 82% were on one spine, 10% on two spines and 8% on dendrites. Synaptic specializations were always asymmetrical.

Electron microscopic immunocytochemisty

The electron-microscopic appearance of TH-IR has been described in detail previously. We found boutons immunostained for TH in symmetrical synaptic contact with dendrites (Figs. SC; 6C) and spines (Fig. 5A. B), especially spine necks (Fig. 6A). The accepted criteria for synaptic contacts include widening of the synaptic cleft, cleft material, accumulation of vesicles presynaptically and pre- and postsynaptic membrane thickening. The synaptic specializations in this study, since they involved relatively short appositions of membrane, were sometimes quite difficult to identify. A recent study4’ found that 25% dopaminergic synaptic specializations were visible in one section only. They were always found to be symmetrical.

Intracellularly filled neurons

The somata and proximal dendrites of intra-

cellularly filled neurons were identified in the electron microscope by the intense reaction product of the ABC-peroxidase reaction (Fig. 6). It was sometimes possible to see an area of damage near the soma, which marked the point of entry of the pipette. Such areas were also found elsewhere in the tissue, indicating the location of unsuccessful attempts at penetrating the neuron. More distally the reaction product was less intense (Fig. 6C) and this facilitated the identification of synaptic specializations along the membranes. In general it was possible to see reaction product in spines, even quite distally.

Convergence of amygdalar and tyrosine hydroxyluse - immunoreactive inputs

In sections cut from blocks that contained both TH-IR and degenerating boutons, we have found a total of 16 examples of convergence of the two inputs on to single postsynaptic neurons (Fig. 5), two of which were identified medium sized spiny neurons (Fig. 6). These examples, for four different rats, were all found in the shell of the NA. Figure 6A, & B

illustrates an example where the TH-IR input and the degenerating bouton are in synaptic contact with different dendrites of the same neuron, identified

because they belong to an intracellularly-filled neuron. In Fig. 6C, a TH-IR bouton is in symmetrical synaptic contact with the dendrite of a neuron intracellularly filled with LY. A bouton degenerating following a lesion in the basolateral/basomedial amygdala is in asymmetrical synaptic contact with a spine that emerges from the same intracellularly filled dendrite. Both target cells were identified as projection neurons.

Controls

In rats with TH immunoreaction no cell bodies were found in any section of the NA, nor were any cells seen when sections were processed only to reveal the biocytin. Thus the neurons which could be identified were those filled intracellularly with LY. Sections reacted with ABC inevitably revealed both filled cells and biocytin where both occurred in the same section. However, where anterograde labelling was sparse in the area of cell filling, or in lesioned material, it was clear that LY did not fill any appreciable amount of local axon collateral.

The contralateral projections from the amygdala to the accumbens meant that it was not possible to use the unlesioned side as a control, but degenerating boutons were only found in sections from rats with well placed lesions of the basolateral and basomedial nuclei.

DISCUSSION

We have shown that the input from the amygdala to the NA makes asymmetrical synaptic contacts, principally with spines, in both the core and shell of the nucleus. In addition we have shown that the amygdalar input contacts neurons which are also postsynaptic to TH-IR boutons, presumably the mesolimbic dopamine input. Some of these neurons have been identified as belonging to the class of medium sized spiny neurons, and two have been shown to project to the ventral pallidum.

AmJjgdalar inputs to the nucleus accumbens

The use of an electrolytic lesion to interrupt the amygdala-accumbens fibres is widespread in behavioural experiments. However, it could be criticized on the grounds that it may also destroy fibres of passage, a view supported by the studies reviewed by Ever& and Robbins13 who found that the behavioural consequences of excitotoxic NMDA lesions of the amygdala in conditioned taste aver- sion studies were different from those resulting from an electrolytic destruction of the area. In our studies though, comparisons between the anterograde transport of biocytin and lesions have shown that similar populations of boutons are labelled

Amygdalr-TH convergence in nucleus accumbens 861

in the NA. The data for boutons iahelied anterogradely with HRP must be interpreted with caution since the numbers observed are relatively small. However, we did find the same targets for boutons iabelled anterogradely with HRP, namely, synaptic contacts with one spine, two spines and dendrites.

The degenerating boutons appeared to be slightly smaller than those iabeiled by the other two methods, probably due to the fact that they had already begun to shrink at an early stage of the degenerative process. However, in combination with immunocytochemistry for TH it was desirable to use degeneration to identify the amygdalar input, thus avoiding any confusion in the electron microscope between the afferent amygdalar fibres and those immunoreactive for TH, both revealed with DAB. Our results for the pattern of postsynaptic targets of amygdalar input are in agreement with those of Kita and Kitai.14 who used the iectin, Phaseolus &garis-ieucoaggiutinin to label the pathway, including the observations of boutons in synaptic contact with two spines In the same ultrathin section.

Absolute numbers of boutons labelled may vary, but this could depend either on the differences in size of injection site, or the relative sensitivities of the methods. The injection site for HRP--WGA was large but the tracer seemed to be confined to the basoiat- era1 amygdaia with a little spread of the medial areas.3i The biocytin injection sites were variable in size, but always produced some labeliing in the NA, although the extent of the inputs to core or shell depended on the exact location of the injection. Previous studies have consistently shown that rostra1 BLa projects to the rostrolaterai NA while caudai BLa projects to the caudomedial NA.16.JS It could be argued that even using these tracers, rather than a lesion, there was some possibility of uptake by fibres of passage, but care was taken to minimize damage around the tip of the pipette, and the small injections of biocytin may be considered to be free of this problem.2s Larger injections of biocytin did result in Goigi-like pyramidal cell labeiiing in both ipsiia~era~ and contralateral co&es, which was not the result of direct spread and was attributed to retrograde transport of the tracer.2’szs

The projection from the BLa to the NA is thought to be giutamatergic, and, consistent with this, we found that boutons in the NA always made asymmet~cai synaptic contact. However, there is evidence that a number of peptides, including somatostatin and cholecystokinin, are present in the stria terminalis, although it is not clear whether these would be in the input or output pathways of the amygdala.3* There is also a GABAergic projection from the central nucleus that gives rise to Ebres in the stria terminalis, a part of the “extended amygdala” of Alheid and Heimer,’ but this projection probably does not reach the NA, terminating in the bed nucleus of the stria terminalis.’ A peptidergic

or GABAergic pathway would be expected to terminate in boutons making symmetrical synaptic specializations in the NA, but none were found in this

study. The similarity of projection targets in the core and

shell regions of the NA was interesting. Meredith et al.,** have shown that core neurons are spinier and more highly branched than neurons in the shell and it has also been reported that TH-IR boutons show differences in their ~stsynaptic targets between these two area?“ with more inputs to dend~tic shafts in the shell. However, it may be valid to consider that the modulation of amygdaiar input to the NA by dopamine could differ depending on whether the input reaches the core, where TH-IR synapses are likely to be on spines and spine necks, a fine control, or to the shell, where the TH-IR boutons are more likely to be on to the adjacent dendrites, perhaps providing a more generalized interaction.

Intracellular filling

The use of in vitro ceil filling to identify the postsynaptic neurons had several advantages. Initially we tried a detection protocol using anti- bodies to LY, which requires rc-sectioning of the tissue after cell filling. However, we then developed a method which is simpler and still well-suited to electron microscopy, using the biotinyiated form of LY.*’ Unlike the report from Hill and Oliver:’ we avoided the use of Triton X-100 by subjecting the tissue to freezing and thawing; with lOO-pm-thick sections the detection then consisted of a simple incubation with ABC and revelation with our standard DAB reaction. Penetration of the ABC molecule into this section thickness would not seem to be a problem and we thus avoid the need to rc-section, with the probable loss of tissue. In our hands this method is more reliable than photo- oxidation,6 with the benefit that ail sections can be processed simultaneously.

Neurons could also be filled in precisely those areas of the NA where amygdalar input was expected following earlier tracing experiments. The potential for the morphological characterization of projection neurons is much greater than with the Golgi-HRP method,” since it is possible selectively to fill neurons which are fluorescing with the retrograde label, choosing those neurons which lie in the terminal area of the input of interest, AI1 of the neurons filled in this study were medium sized spiny neurons.

We encountered some problems with this method however. It was essential that the giutaraldehyde content of the fixative was kept low and this inevitably produced some compromise of the uitra- structural preservation, although this was beneficial to the quality of the TH immunostaining. We also found that lOO+m-thick sections were a good compromise for completeness of ceil filling and ABC penetration but the TH-IR never penetrated

862 L. R. Johnson et al.

the entire depth of tissue. Other difficulties of interpretation could arise from the use of DAB as the chromogen for both the filled cells and the TH-IR. However, in tissue which contained only filled cells we did not see local axon collaterals coming from the neurons, although these are known from Golgi studies to exist.” Since these collaterals almost certainly make symmetrical synaptic contacts with other medium-sized spiny neurons,44 if they were to be labelled during cell filling they could be confused with the TH immunoreactivity, also revealed with DAB. In fact the local axon collaterals of medium sized spiny neurons have been shown to be in synaptic contact principally with dendritic shafts,” whereas the TH-IR terminals in this study were frequently associated with spines or spine necks. In addition, although sometimes leakage from the filled neuron can obscure the cell body, neurons were selected for this study only if it was clear that the filled dendrites could be traced back to the soma. Boutons of amygdalar origin were only seen to make asymmetrical synaptic specializations and thus those labelled with biocytin from the amygdala, also revealed with DAB, were unlikely to be confused with either local axon collaterals or TH-IR structures.

However, a major difficulty exists in the confir- mation of the filled cell as a projection neuron. We are not aware of any method that would allow us to identify by fluorescence a retrogradely labelled neuron which we could then confirm in the electron microscope as a projection neuron. The current method involves the photography of the neuronal cell body labelled with the fluorescent retrograde marker, and then re-photographing during the cell filling. 6.28 It is not feasible to document all retrogradely labelled neurons that are subsequently filled in this way.

The choice of retrograde marker is further com- plicated by the fact that we experienced rapid fad- ing of Fast Blue, possibly due to leaching as the sections for filling are held in buffer for long periods.5 FluoroGold is much more stable, but it is harder to distinguish labelled neurons in tissue which has been fixed with glutaraldehyde and has relatively high levels of autofluorescence, especially in the dorsal and ventral striatum.’ With rhodamine beads we found that the retrogradely labelled neurons were clearly visible in the tissue

sections.

Projection neurons

Our preliminary tracing studies had shown that the principal termination area for caudal BLa was the shell of the NA and hence we selected projection neurons from this area. The projection from the NA to the ventral pallidum has been shown to originate from neurons in both the core and shell of the NA, with the core projecting densely to the lateral VP and moderately to the medial VP, while the shell projects

to the medial VP.” However, our observation of neurons in the shell areas, especially in the septal hook, following an injection into the dorsal VP, is more consistent with the diagrams of Groenewegen et al.‘(’ Retrogradely labelled, LY-filled neurons were all medium sized spiny neurons, which have been shown in previous studies to be the projection neurons of the NA.” There is however, a projection from the NA to the medial substantia nigra (SN)/VTA, the fibres of which pass through the VP.16 Thus it is possible that the retrograde marker was also taken up by a small number of damaged fibres of passage at the injection tip and the projection neurons identified in this study might in fact be NA neurons projecting to the substantia nigra, or even neurons which project to both the VP and substantia nigra.

Convergence of tyrosine hydroxylase-immunoreactitie

und amygdalar input

The identification of synaptic specializations associated with TH-IR or dopamine immunoreactive boutons is always difficult, even in material fixed with high concentrations of glutaraldehyde. A recent study using an antibody to dopamine” reports that most synaptic specializations arc visible in only one to three sections and many involve only subtle membrane densification and short appositions. This was borne out in a study which used an antibody to TH, where again synaptic contacts were small and frequently only visible in one or two sections.42 Where the apposition was particularly short, for example on a spine neck, and the membrane preservation was sub-optimal. we found it particularly difficult to identify widening of the synaptic cleft. However, it was possible to identify TH-IR structures making symmetrical synaptic

contacts. We had initially hoped that it would be possible to

avoid the use of lesions to identify the afferent input of convergence studies. However, we encountered problems with poor penetration when using BDHC as an alternative chromogen to DAB for the TH staining and were unsuccessful in attempts to use it to reveal anterograde biocytin labelling. It would have been possible to use DAB as the chromogen for both the anterograde label and the TH-IR, since in the electron microscope we had good reason to predict that the afferent boutons would be those making asymmetrical synaptic contacts while the TH-IR boutons would make symmetrical synaptic contacts. However, although for the purpose of iden- tifying convergence it would have been helpful to have both types of bouton visible in the light microscope, we were reluctant to use the same chromogen to label the afferent input, the TH-IR and in some cases the LY-filled neuron too.

Despite these technical limitations, we have shown that in the NA shell TH-IR boutons make symmetrical synaptic contact with spines and dendrites that are also postsynaptic to amygdalar input. Few of the


blocks examined at the electron microscopic level in this study, taken from the core, proved to com- bine good TH-IR and degeneration. Although several filled neurons were examined from this area, they were found to be in regions with few degenerating boutons and consequently no examples of convergence in this area have been seen.

The relative positions of the inputs are similar to those reported for inputs to the NA from hippocampus and dopamine,42,48 prefrontal cortex and dopamineG2 and in the dorsal striatum, cortical and dopamine inputs.14 The dopaminergic input is thus well-placed to modulate the excitatory input to the head of the spine. Although it has been suggested that modulation of amygdalar glutamatergic input by dopamine in the NA is likely to be the consequence of dopaminergic receptors on glutamate terminals,53 we have demonstrated a mechanism to allow this to happen postsynaptically, since dendritic spines have been shown to be capable of independently isolating and modulating depolarizing inward calcium concentrations without influencing the whole cell poten- tia1.‘7’30 It is of course possible that dopamine exhibits both pre- and postsynaptic actions in its interactions with amygdalar input to the NA. There is still no consensus about the identity of dopamine receptor types involved in the control of various behaviours,4 but our work shows that it may be necessary to consider the distribution of receptor types at the level of individual spines in order to understand interactions between afferent inputs.

We are currently investigating the question of whether there is convergence onto single neurons between hippocampal and amygdalar inputs to the NA. In extracellular recording studies it has been shown that single units in the dorsomedial NA responded to both hippocampal and amygdala stimulation.*.‘* We have now shown that both inputs contact medium sized densely spiny neurons in the NA shell, making asymmetrical synaptic contacts principally with spines and in both cases the neurons also receive TH-IR, presumably dopaminergic, input4* We have preliminary evidence that neurons that receive input from the ventral subiculum of the hippocampus project to the VP’” and we have shown here that some neurons receiving input from the amygdala might also project to the VP, which is in agreement with the results of electrophysiological

studies.” However, it is still possible that the out- puts are to different parts of the VP and that the limbic inputs are to separate populations of NA neurons. Further ultrastructural studies are needed to clarify this point.

Functional implications

A number of studies have examined behaviours thought to involve the amygdala-NA pathway.13 In behavioural paradigms such as acquisition of a new response with conditioned reinforcement, conditioned place preference and a second order schedule of reinforcement, excitotoxic lesions of the BLa produced behavioural changes which were then shown to be reversed by dopamine agonists, usually amphetamine, microinjected into the NA. From their observations with conditioned reinforcement, the authors have suggested that the function of the amygdala-NA pathway is to make the association between the conditioned stimulus and the reinforcement, whereas the dopamine input to the NA is concerned with amplifying the differential respond- ing. Our results have provided a synaptic basis for their observations.

We have now shown that neurons in two limbic areas that project to the NA make synaptic contact with neurons that are also postsynaptic to dopamine. We have also demonstrated that projection neurons from these limbic areas to the NA receive input from intrinsic peptide neurons.2A7 Thus we have provided anatomical evidence to explain how pathological changes in limbic areas of the brain, which are increasingly recognized as key features in the pathol- ogy of schizophrenia,36 might directly influence the excitatory inputs to the NA. We have also shown that dopamine is ideally placed to modulate these limbic inputs and thus provide a possible explanation for at least some of the therapeutic efficacy of D2 dopamine receptor antagonists in the treatment of schizophrenia.

Acknowledgements-We would like to thank Bristol Myers-Squibb and the Wellcome Trust for their financial support. We arc grateful to Prof. David Smith for his useful discussions of the project and to Dr Eberhard Biihl for his help with the intracellular filling. The photographic expertise of Paul Jays and Lesley Annetts and the statistical advice from Matthew Eagle are also acknowledged with thanks.

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(Accepted 7 March 1994)

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