Extra-hippocampal projections of CCK neurons of the hippocampus and subiculum

Peptides, Vol. 4, pp. 331-334, 1983. ~ AnkhoInternationalInc. Printed in the U.S.A.

Extra-Hippocampal Projections of CCK Neurons of the Hippocampus and Subiculum G A I L E. H A N D E L M A N N , * M A R G E R Y C. B E I N F E L D , ? T H O M A S L. O ' D O N O H U E , I : J E N N I F E R B. N E L S O N * A N D D O U G L A S E. B R E N N E M A N §

*Lab. Clinical Science, N1MH, Bethesda, MD 20205 ?Department o f Pharmacology, St. Louis University, St. Louis, MO 63104 SUnit on Neuroendocrinology, NINCDS-ETB, Bethesda, MD 20205 §Lab. Developmental Neurobiology, NICHD, Bethesda, MD 20205 R e c e i v e d 21 M a r c h 1983 HANDELMANN, G. E., M. C. BEINFELD, T. L. O'DONOHUE, J. B. NELSON AND D. E. BRENNEMAN. Extrahippocampal projections of ('('K neurons of the hippocampus and subiculum. PEPTIDES 4(3) 331-334, 1983.--A population of neurons in the hippocampus and subiculum contains cholecystokinin (CCK), Following transection of the dorsal fornix, a major afferent pathway of the hippocampus and associated structures, CCK levels were reduced in the septum and hypothalamus. A microdissection analysis indicated that the loss of CCK occurred in nuclei receiving direct projections from the hippocampus and subiculum, suggesting that CCK-containing neurons in the hippocampus and subiculum project to extrahippocampal regions. Cholecystokinin

Fornix

Hippocampus

Subiculum

C H O L E C Y S T O K I N I N (CCK), originally detected and sequenced as a peptide in gastrointestinal tissue [14, 16, 21], has been demonstrated to be a biologically active peptide in central and peripheral nervous tissue [8]. In brain tissue, CCK~, the C-terminal octapeptide of CCK, is synthesized [26], released from nerves [25], and has postsynaptic actions on neurons [9]. CCK is present in high concentrations in rat brain [3,29], where it has an interesting regional distribution. Unlike other neuropeptides, CCK is more abundant in cortical than in noncortical areas, and is especially concentrated in cortical areas associated with the limbic system, such as entorhinal and cingulate cortex. Substantial amounts are also found in limbic structures, particularly hippocampus and septum. Other regions containing large amounts are hypothalamus, thalamus, striatum, olfactory bulb, and olfactory tubercle [3, 15, 18]. The fact that CCK is abundant in limbic structures that are highly interrelated suggests that the peptide might act as a neurotransmitter or neuromodulator among these regions. CCK is present in both neuronal cell bodies and terminals in the hippocampus and subiculum, and also in the areas with which they most communicate: the septum and entorhinal cortex. Within the hippocampus, CCK-containing neurons are located in all cell layers [I 1,12], but are most prevalent in the stratum radiatum, the layer subjacent to the pyramidal cell layer [12]. The cells appear polymorphic, with long axons extending into the pyramidal cell layer where large numbers of CCK-positive terminals are located [ 11,12]. CCK has been shown to be excitatory to these pyramidal cells [9]. The CCK neurons might therefore be expected to act as interneurons modulating the activity of pyramidal cells. In support of this hypothesis, isolation of the hippocampus from its afferents by destruction of the fornix and entorhinal

cortex has no effect on the CCK content of the hippocampus, indicating that the CCK-positive terminals are derived from the intrinsic neurons and not the afferent sources [12]. True interneurons would not project to extrahippocampal regions. However, destruction of the neurons in the hippocampal region inferior with kainic acid caused a decrease in CCK content of the septum [13]. In addition, Greenwood and colleagues observed CCK-positive fibers in the dorsal fornix [11]. These findings suggest that CCK-containing neurons in the hippocampus project through the fornix to the septum. The present experiment was designed to investigate this possibility, and in addition, the possibility that CCKcontaining neurons in the subiculum contribute afferents via the fornix to subicular projection areas: the septum, mammillary bodies of the hypothalamus, and anterior nuclei of the thalamus [6, 20, 28, 31]. In these experiments, the fornix was transected to interrupt the passage of fibers from the hippocampus and subiculure which enter the pre- and post-commissural columns of the fornix. The CCK content of regions receiving these projections was then measured, either in macro- or microdissected samples. Hippocampal neurons send projections through the pre-commissural fornix which terminate in the lateral septum [31]. The neurons of the subiculum sending projections via the fornix have a wider distribution. Some fibers enter the pre-commissural fornix and terminate in lateral septum and the bed nucleus of the stria terminalis (NIST) [31,32]. Others project through the post-commissural fornix to the anterior nuclei of the thalamus and the mammillary bodies of the hypothalamus [6, 20, 28, 30, 31]. A decrease in CCK content in these projection regions would support the hypothesis that hippocampal and/or subicular CCK neurons project to extra-hippocampal regions.

331

H A N D E L M A N N ET AL.

332 TABLE 1 PEPTIDE C O N T E N T OF W H O L E SEPTUM A N D H Y P O T H A L A M U S A F T E R FORNIX L E S I O N (pg/tzg PROTEIN, M E A N -+ S.E.M.)

CCK

VIP

Control

Lesion

Control

Lesion

Septum

2.52 + 0.31

1.04 _+ 0.14"

0.82 _+ 0.07

1.07 _+ 0.06*

Hypothal

1.26 _+ 0.08 (n= 11)

0.96 _+_0.10 (n= 15)

0.63 _+ 0.05 (n=ll)

0.72 -+ 0.05 (n= 15)

*Significantly different from control, p<0.05, Student's t-test.

EXPERIMENT 1 METHOD

Subjects Twenty-six male albino rats weighing 300-350 g were used.

Surgery The lesions of the dorsal fornix were performed stereotaxically under Chloropent anesthesia, by passing radio frequency current, as previously described [22]. Sham lesions were performed according to the same procedure except for lowering the electrodes and passing current.

Tissue Preparation and CCK Measurement Seven days after surgery, the rats were killed by decapitation and the brains rapidly removed. The septum and hypothalamus were removed from each brain with microscissors and quickly frozen on dry ice. The tissue samples were later homogenized by sonication in 0.1 N HCI, and an aliquot taken for protein determination [19]. The CCK content was extracted and measured by radioimmunoassay as previously described [4]. The CCK antiserum used in the assay cross-reacts with gastrin. The dominant CCK-like material found in the rat brain, however, coelutes with sulfated CCK8 and separates from gastrin on G-25 sephadex and high pressure liquid chromatography [2]. To control for possible non-specific effects caused by the surgical procedure, another peptide, vasoactive intestinal peptide (VIP), was also measured in the samples by radioimmunoassay [10]. RESULTS

As shown in Table 1, the fornix transections significantly reduced the CCK content of the septum and slightly reduced the levels in the hypothalamus. VIP levels were not reduced in either region, but were instead increased in septum.

EXPERIMENT 2 METHOD

Subjects Seventeen male albino rats weighing 275-340 g were used.

Surgery To prepare the rat brains for microdissection, discrete fornix transections were made. Each rat was anesthetized with Chloropent (1 ml/rat) and placed in a stereotaxic apparatus with the incisor bar set at 0. The scalp was retracted and a rectangular hole was drilled through the skull 0.5 mm caudal to bregma. The hole extended slightly more than 2 mm on each side of the sagittal suture. The transection was made by first lowering microscissors opened to a width of 4 mm to a depth of 4 mm ventral to the brain surface and making an incision. The same cut was then made stereotactically with a scalpel blade. The scissor incision was necessary to disrupt the corpus callosum, which otherwise prevented access to the fornix. For sham surgery, the scissors were inserted to a depth of 3 mm and an incision was made, but there was no scalpel incision.

Tissue Preparation and CCK Measurement Seven days after surgery, the rats were killed by decapitation and the brains rapidly removed and frozen in powdered dry ice. The frozen brains were cut into 300 izm thick sections in a cryostat. Various brain nuclei were microdissected with metal cannulae, according to the method of Palkovits [24]. The dissected tissue, except for hippocampal samples, was sonicated in 0.1 N HCI and an aliquot taken for protein determination. CCK content was extracted and measured as previously described. To verify the fornix transections, the choline acetyltransferase (CHAT) activity of the hippocampi was measured [17]. The dissected hippocampal tissue was sonicated in 10 mM EDTA, 0.2% Triton-X 100. ChAT activity was determined by measuring the difference between the activity in the presence and absence of HNP (N-hydroxyethyl-4-[1napthylvinyl] pyridium bromide; Calbiochem-Behring, La Jolla, CA [5,27]. RESULTS

As indicated by ChAT activity in the hippocampus, the fornix transections were effective in interrupting the septohippocampal pathway. The control rats had a mean of 1372+_112 pmol ChAT activity/min/mg protein (range= 1044--1783), while the rats with fornix transections had a mean of 263-+51 pmol/min/mg protein (range= 115-521). The fornix transections produced a significant decrease in CCK levels in five of the areas measured (Table 2). These were lateral septum, NIST, mammillary bodies, the anteroventral nucleus of the thalamus, and subiculum.

EXTRA-HIPPOCAMPAL PROJECTIONS OF CCK

333

TABLE 2 CCK CONTENT OF MICRODISSECTED BRAIN REGIONS AFTER FORNIX TRANSECTION (pg//xg PROTEIN, MEAN _+ S.E.M.) Brain Region

Control

Hypothalamic Areas Preoptic area Anterior Hypothalamus Periventricular n. Supraoptic n. Paraventricular n. Median eminence Arcuate n. Ventromedial n. Dorsomedial n. Posterior Hypothalamus Mammillary Bodies Septal Areas Lateral Septum Medial Septum N. Stria Terminalis Thalamic Area Anteroventral n. Subiculum

1.66 1.68 2.32 0.84 1.26 2.55 0.98 2.17 1.66 0.74 1.11

± 0.33 ± 0.34 ± 0.33 ± 0.19 ± 0.06 ± 0.67 ± 0.12 _+ 0.58 ± 0.30 ± 0.05 ± 0.15

TRANSECTION I

a % - C ~

HIPPOCAMPUS]

Lesion

1.23 1.04 1.53 0.87 1.08 2.15 1.45 1.92 1.43 0.79 0.58

± 0.18 ± 0.12 ± 0.29 ± 0.22 _+ 0.10 -+ 0.64 +_ 0.30 ± 0.33 ± 0.22 ± 0.10 ± 0.08*

3.36 ± 0.35 1.14 ± 0.19 3.83 ± 0.32

1.95 ± 0.36* 0.80 ± 0.13 1.82 ± 0.22*

0.81 _+ 0.09 1.75 + 0.28

0.58 ± 0.06* 0.87 _+ 0.12"

I..M IpT~

//

°~,X"

CCK CONTENT DECREASED AFTER FORNIX TRANSECTION CONTAINS CCK-POSITIVE CELL BODIES AND TERMINALS .'.

CONTAINS CCK POSITIVE TERMINALS

FIG. I. Schematic diagram of the pathways associating ~he hippocampus and subiculum with the septum and hypothalamus, and indicating the placement of the fornix transection. Abbreviations: AVT=anteroventral nucleus of the thalamus; MMB=mammillary bodies; NIST=bed nucleus of the stria terminalis; VMN= ventromedial nucleus of the hypothalamus; FX=fornix. The pathway from the subiculum to the arcuate nucleus and VMN is the cortiohypotbalamic tract.

*Significantly different from control, p<0.05, Student's t-test.

GENERAL DISCUSSION Interruption of the fornix reduced the C C K content of brain regions receiving direct projections from the hipp o c a m p u s or subiculum via the pre- or post-commissural fornix. The fornix transections were discrete, as they did not d e c r e a s e the septal content of another neuropeptide, VIP, nor did they influence the C C K content o f regions receiving projections from subiculum via another pathway (Fig. 1). These findings suggest that C C K - c o n t a i n i n g fibers o f hipp o c a m p a l and subicular origin travel in the fornix. S o m e of the regions affected by the fornix lesions, such as lateral septum and mammillary bodies, contain C C K - p o s i t i v e cell bodies [1 I]. The d e c r e a s e in C C K in these cases could therefore be due to a transynaptic effect on the C C K content of these cells, caused by loss o f afferent input. The anteroventral nucleus of the thalamus, on the other hand, contains only C C K - p o s i t i v e terminals [1 I], yet showed a decrease in C C K c o n t e n t after the lesions, indicating a loss of C C K afferent input. An unexpected finding was that the C C K content of the subiculum was d e c r e a s e d by the lesions. This may indicate the presence of C C K afferents in the fornix which terminate in the subiculum. The C C K - p o s i t i v e neurons in the h i p p o c a m p u s are most n u m e r o u s in the stratum radiatum subjacent to the pyrami-

dal layer [12]. Classically, this cell layer has been thought to contain interneurons, while the pyramidal layer contained the cells which projected to e x t r a h i p p o c a m p a l regions. Chronister and colleagues, h o w e v e r , o b s e r v e d a population o f polymorphic neurons located in stratum radiatum and stratum oriens which project to septum [7]. In support o f this finding, they cited observations by Cajal of hippocampal interneurons w h o s e axons acted like those of pyramidal cells. T h e r e may therefore be two classes of C C K neurons: one with terminals within the hippocampus, and a second which sends projections to terminate in extra-hippocampal regions. Alternatively, the C C K neurons might resemble pyramidal cells of the hippocampal regio inferior, neurons with bifurcating axons whose collaterals synapse in the regio superior of the hippocampus and in the septum [1]. The fornix is an important pathway in the function of the limbic system, and lesions of this pathway produce severe behavioral deficits (see [23] for review). The contribution of a possible C C K pathway to the behavioral function of this neural system will be o f interest. ACKNOWLEDGEMENTS This work was supported in part by NIH grant NS 18335 and a grant from the American Parkinson Disease Foundation.

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