Exp Brain Res (1997) 115:369–374
© Springer-Verlag 1997
R E S E A R C H N OT E
&roles:Zsolt Borhegyi · Csaba Leranth
Distinct substance P- and calretinin-containing projections from the supramammillary area to the hippocampus in rats; a species difference between rats and monkeys &misc:Received: 18 October 1996 / Accepted: 21 January 1997
&p.1:Abstract Our recent studies showed the co-existence of substance P and calretinin in the supramammillo-hippocampal pathway of monkeys, as well as species differences in the synaptic targets of extrinsic substance P fibers in the hippocampi of monkeys and rats. Experiments used: (1) single and multiple stereotaxic injection of wheat germ agglutinin-conjugated HRP into the hippocampus and immunostaining for substance P in the supramammillary area; (2) colocalization of substance P and calretinin in supramammillary area cells; and (3) colocalization of these two neurochemicals in retrogradely labeled supramammillary projective cells of both male and female rats. These demonstrated: (a) many calretinin- and fewer substance P-immunoreactive neurons retrogradely labeled in the ipsilateral supramammillary area; (b) approximately 74% of all substance P cells contain calretinin and 9% of the calretinin neurons co-contain substance P; and, most importantly (c) none of the retrogradely labeled supramammillary cells colocalize calretinin and substance P. These results indicate the presence of two distinct supramammillo-hippocampal projections in the rat, one that contains substance P and the other calretinin. The latter innervates the same areas as those in the monkey, and the former terminates only in the CA2 hippocampal subfield. &kwd:Key words Species differences · CA2 · Retrograde tracing · Colocalization · Theta rhythm · Rat&bdy: Z. Borhegyi Department of Functional Neuroanatomy, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Z. Borhegyi · C. Leranth (✉) Yale University, School of Medicine, Department of Obstetrics and Gynecology, P.O. Box 208063, New Haven, CT 06520–8063, USA; Tel: +1-203-785-4748, Fax: +1-203-785-7684, e-mail:
[email protected] C. Leranth Section of Neurobiology, Yale University, School of Medicine, New Haven, CT 06520–8063, USA&/fn-block:
Introduction Our previous studies have demonstrated that, in the nonhuman primate brain, all of the parent neurons of the supramammillo-hippocampal system, which can very effectively control the electrical activity of dentate granule cells, CA2 area pyramidal neurons (Nitsch and Leranth 1993; Seress et al. 1993), and specific populations of interneurons (Leranth and Nitsch 1994), colocalize calretinin (CR) and substance P (SP; Nitsch and Leranth 1993). On the other hand, our recent study has shown that in rats, supramammillary area (SUM) SP fibers terminate only in the CA2 region, but not in the dentate gyrus (Borhegyi and Leranth 1997). However, CR fibers seem to occupy the same hippocampal areas in the rat (Maglóczky et al. 1994; Nitsch and Leranth 1996) as both CR- and SP-containing SUM afferents do in monkeys (Nitsch and Leranth 1993, 1994; Seress et al. 1993; Seress and Leranth 1996). These findings prompted this study to determine whether in rats, like monkeys: (1) the extrinsic hippocampal SP-containing fibers originate in the SUM; and, if so (2) whether all of these projective neurons cocontain CR.
Materials and methods Adult Sprague-Dawley (300–320 g) male and female rats (n = 16) were used in this study. Animals were kept under standard laboratory conditions, with tap water and regular rat chow ad libitum, in a 12-h light, 12-h dark cycle. Eight animals received a large (2 µl) wheat germ agglutinin-conjugated HRP (WGA-HRP; 2.5% diluted in saline) injection into the temporal hippocampus (AP –5.6 mm, L 5.4 mm, V 3.6–6.6 mm), and four rats (two male and two female) multiple WGA-HRP injections (0.3 µl each) along the septotemporal axis of the hippocampal formation (AP1 –5.8 mm, L1 4.8 mm, V1 4.0–7.5 mm; AP2 –5.2 mm, L2 4.8 mm, V2 5.2–6.4 mm; AP3–4.5 mm, L3 4.6 mm, V3 4.4 mm; AP4 –3.8 mm, L4 2.5 mm, V4 3.2 mm, and AP5 –2.8 mm, L5 1.4 mm, V5 3.4 mm) (Fig. 1) via a glass micropipette (40–50 µm outer diameter connected to a microsyringe). Forty-eight hours later, colchicine (25 µl of 3 mg/600 µl) was injected into the lateral ventricle.
370 ted goat anti-rabbit IgG) followed by ABC. Visualization of tissue-bound peroxidase was accomplished by a final DAB reaction. Consecutive sections were mounted on gelatin-coated slides (one pair of sections per slide; one section immunostained for CR and the other for SP) so that the posterior side of the first section and the anterior side of the second section were face up. Sections were dehydrated and mounted in Permount (Sigma). Light-microscopic examination and camera lucida drawings from the surface of both sections were then made, and photographs were taken from the same identified perikarya to investigate the extent of colocalization of the two substances. Antisera According to a previous characterization, the SP antiserum binds 40% of 125I-labeled substance P trace at a 1:250 000 final dilution. Half-maximal displacement by SP is 30 pM. No measurable displacement is observed at 100 pM with substance K, physalaemin, metenkephalin, or eledoisin (Carraway and Leeman 1979). This SP antiserum has been used in other immunohistochemical experiments in the rat septum (Szeidemann et al. 1995) and monkey hippocampus (Leranth and Nitsch 1994; Nitsch and Leranth 1994). To further test the specificity of the antiserum on rat supramammillary tissue, two controls were performed: (1) sections were incubated with an SP antiserum (diluted to 1:5000) preadsorbed (24 h at 4°C) with a 23.5 µM solution of SP (S6883; Sigma, St. Louis, Mo.); and (2) the SP antiserum was replaced with normal rabbit serum. In both cases, no immunoreaction could be observed. The CR antiserum is a widely used and well characterized (Rogers 1989) immunoreagent. Fig. 1 Low-power light micrograph (captured with a stereo-zoom microscope) demonstrates a vibratome section of the hippocampal formation dissected from a rat that received multiple injections of WGA-HRP and cut parallel to its longitudinal axis. Note that all hippocampal subregions are filled with WGA-HRP. Scale bar 2 mm (S septal pole, T temporal pole)&ig.c:/f
Results
In both male and female rats, the results were identical. CR-immunoreactive cells were homogeneously distributed through the entire posterior hypothalamic area, including the SUM (Fig. 2), supramammillary decussation, After a 24-h survival period, animals were killed under ether and lateral hypothalamic and periventricular areas. Two anesthesia by transcardial perfusion of 50 ml saline followed by a different cell types could be distinguished. The first fixative containing 5% acrolein in 250 ml phosphate buffer (PB; 0.1 M; pH 7.35). Sixty-micron vibratome sections cut from the in- group of neurons was small (10–15 µm) and homogejection site and SUM were rinsed (3 × 15 min) in PB, incubated neously populated all of the aforementioned areas. The for 20 min in 1% sodium borohydride, and the WGA-HRP was vi- second type was large (nearly 30 µm) and could be obsualized by a glucose oxidase reaction (15 mg DAB, 12 mg am- served only around the mammillary peduncle in the monium chloride, 0.12 mg glucose oxidase, and 60 mg β-D-gluSUM and in the medial part of the periventricular area. cose in 30 ml PB for 30–45 min). Thereafter, SUM sections were incubated first in 10% normal These two morphologically distinct cell types were goat serum for 45 min, followed by a 60-h incubation at 4°C in a found among the SP-containing cells, as well. SP-immupolyclonal rabbit anti-SP antibody (a generous gift of Dr. S. Leeman; noreactive cells were found mostly in the midline area of 1:5000 dilution in 1% normal goat serum in PB containing 0.04% the posterior hypothalamus with the highest density in Triton X-100 and 0.1% sodium azide). After this, the sections were incubated in the secondary antibody (biotinylated goat anti- the middle portion, along the longitudinal axis of this rabbit IgG, 1:250 in PB, Vector, Burlingame, Calif.) and, then, in structure (Fig. 2). The density of the CR-immunoreactive ABC Elite (1:50 in PB, Vector), each for 2 h at 20°C. The tissue- neurons was much higher than that of the SP-containing bound peroxidase was visualized by using a brown DAB reaction cell population. (15 mg DAB, 165 µl 0.3% H2O2 in 30 ml PB). Between each incuThe results of colocalization experiments and semibation step, sections were thoroughly rinsed in several changes of ice-cold PB. After the immunostaining procedure, sections were quantitative analyses on five rats (three after a single and placed on gelatin-coated slides, dehydrated, and mounted in Per- two after multiple WGA-HRP injection) revealed that mount (Sigma). the majority (approximately 74%) of the SP cells contain To determine the coexistence of SP and CR in the same neurons, the very specific ”mirror” colocalization technique of Kosa- CR, while only a small population (approximately 9%) ka et al. (1985) was used. Adjacent vibratome sections of the of the CR- immunoreactive neurons cocontain SP SUM (from four animals) were immunostained for either SP (see (Figs. 2, 3, Table 1). The distribution of the double-laabove) or CR (rabbit anti-CR, a generous gift of Dr. J. H. Rogers, beled neurons was correlated with the rostrocaudal disdiluted to 1:5000 in PB containing 0.1% sodium azide and 1.0% tribution pattern of the SP-immunopositive neurons. normal goat serum). In order to limit the extent of the immunostaining to the surface of the sections, no Triton X-100 was used. Thus, the majority of CR-containing SP cells (approxiSections were further processed in secondary antisera (biotinyla- mately 15%) were found in the middle portion of the
371 Fig. 2 Composite camera lucida drawings (based on the rat brain atlas of Paxinos and Watson, 1986) demonstrate the distribution patterns of calretinin(circle), substance P (SP)(filled circle), and calretinin (CR) plus SP-containing (triangle) neurons in the posterior hypothalamus at three different rostrocaudal levels. CR cells are in the entire nucleus, while the majority of SP-labeled neurons are in the middle section, in the medial part of the posterior hypothalamus. CR plus SPimmunoreactive cells are only in the latter area. (mt mammillothalamic tract, MMn medial mammillary nucleus, median, mp mammillary peduncle, MM medial mammillary nucleus (medial), f fornix)&ig.c:/f
Table 1 Results of a semiquantitative analysis on five rats. Data from the rostral, middle, and caudal sections of the posterior hypothalamic area. The first three columns show the percentage of calretinin (CR)- and substance (SP)-containing neurons in the total number of cells. The fourth and fifth columns contain the total Single labeled (%)
&/tbl.:
Rostral Middle Caudal Total
CR
SP
91%±6.0 80%±7.0 92%±6.0 88%±5.0
2%±0.6 6%±0,8 1%±0.2 3%±0.4
Double labeled (%) 7%±0.4 14%±1.0 7%±0.3 9%±0.8
number of SP and CR cells (single-plus double-labeled) counted in five animals. The last two columns present the number of SPplus CR-containing cells as a percentage of the total number of either CR- or SP-immunoreactive neurons&/tbl.c:& Total number
CR + SP in total number (%)
CR
SP
CR
SP
1240 1,165 1,495 3,905
112 275 2,028 588
7%±0.3 15%±0.7 7%±0.2 9%±0.4
77%±3.0 68%±2.0 83%±4.0 74%±3.0
372
Fig. 3 Light micrographs demonstrate the result of a colocalization experiment for CR and SP (A, B) on adjacent sections, and retrogradely labeled CR- (C) and SP-immunoreactive (D) neurons in the supramammillary area. A, B Letters with arrowheads point at neurons immunoreactive only for SP (a, l, n, o) or CR (b, h, i, j,
k), and at cells containing both SP and CR (c, d, e, f, g, m). Asterisks label landmark capillaries. Neurons shown in C and D are of the large type. Note the characteristic nuclear staining of the retrogradely labeled CR-containing cell (C). Scale bar 10 µm&ig.c:/f
373
dorsal hypothalamus and were concentrated near the midline (Fig. 2, Table 1). Multiple WGA-HRP injections resulted in a higher density of retrogradely labeled neurons in the SUM than after a single large injection into the temporal part of the hippocampal formation. However, the distribution pattern of these cells was the same in both cases. Most of the retrogradely labeled cells could be observed in the lateral division of the posterior hypothalamus, and a large population of these cells contained CR, but none had SP. In the central areas of the posterior hypothalamus, both CR- and SP-immunoreactive, retrogradely labeled neurons were seen. The majority of these cells were of the large type described above. Even after a single sizable WGA-HRP injection, which filled up all the subdivisions of the temporal part of the hippocampus, or after filling up the entire hippocampal formation with multiple WGA-HRP injections (Fig. 1), we were unable to find retrogradely labeled cells that cocontain SP and CR.
Discussion The location of the rat supramammillo-hippocampal projection neurons does not seem to determine their targets along the septotemporal axis of the hippocampal formation, since after single or multiple WGA-HRP injections the distribution pattern of retrogradely labeled cells was the same. This observation holds true for both sexes, indicating the absence of sexual differences in this connection. The results indicate that the rat posterior hypothalamic area contains at least five and, perhaps, six major types of neurons: (1) supramammillo-hippocampal CRor (2) SP-containing neurons; (3) CR- or (4) SP-immunoreactive cells; (5) CR plus SP-containing neurons, which are interneurons or project to other brain areas; and, possibly (because not all of the SP- and CR-containing cells can be expected to be immunolabeled, even when Triton X-100 is used) (6) supramammillo-hippocampal projective cells that do not contain either SP or CR. These data, together with other published observations on the extrinsic CR and SP innervation of the rat and monkey hippocampus (Seress et al. 1993; Nitsch and Leranth 1993, 1994, 1996; Leranth and Nitsch 1994; Maglóczky et al. 1994; Borhegyi and Leranth 1997; Nitsch and Leranth 1996; Seress and Leranth 1996) reveal a striking species difference between rats and monkeys: (1) in the monkey brain, it seems that all of the supramammillo-hippocampal SP neurons contain CR, while none do in the rat (present observation); (2) in the monkey hippocampus, extrinsic CR- and SP-immunoreactive boutons have the same synaptic targets; however, in the rat, the distribution pattern of extrinsic CR fibers seems to be identical to that of monkeys, while extrinsic SP fibers terminate only in the CA2 area. Theta activity rarely occurs in primates, in contrast to rodents (Crowne and Radcliffe 1975, Stewart and Fox
1991). The posterior hypothalamic area, including the SUM plays an important role in the regulation of hippocampal theta rhythm activity (see reviews in Kocsis and Vertes 1994; Oddie et al. 1994). There are indications that, in the rat, the same supramammillary CR neurons project to both septum and hippocampus (Kiss and Szeiffert 1995). Furthermore, in a recent study, we demonstrated that a population of supramammillo-septal CR cells are aspartate/glutamatergic (Leranth and Kiss 1996). These observations, together with the present data, raise the possibility of an excitatory, aspartate/glutamatergic supramammillo-hippocampal projection. If in monkeys, like rats, this pathway is an (SP plus CR) aspartate/glutamatergic connection, but the projection in rats does not contain SP, then, it can be speculated that the aforementioned species difference in the theta rhythm activity might be the result of the discrepancy in neurochemical content and synaptic targets of the supramammillo-hippocampal connection between monkeys and rats. &p.2:Acknowledgements The authors wish to thank Marya Shanabrough for her excellent technical assistance. The calretinin and substance P antisera were kindly provided by Drs. J. H. Rogers and S. P. Leeman, respectively. This study was supported by NIH grant NS-26068.
References Borhegyi Z, Leranth C (1997) Substance P innervation of the rat hippocampal formation. J Comp Neurol (in press) Carraway R, Leeman SP (1979) The amino acid sequence of bovine hypothalamic substance P. Identity to substance P from colliculi and small intestine. J Biol Chem 254:2944–2945 Crowne DP, Radcliffe DD (1975) Some characteristics of functional relations of the electrical activity of the primate hippocampus and hypotheses of hippocampal function. In: Isaacson RL, Pribram KH (eds) The hippocampus, vol 2. Plenum Press, New York, pp 185–206 Kiss J, Szeiffert G (1995) Topographical analysis of connections between the rat septal diagonal band complex and the supramammillary area (abstract). Fourth IBRO World Congress of Neuroscience) Rapid Communications, Oxford, p 389 Kocsis B, Vertes RP (1994) Characterization of neurons of the supramammillary nucleus and mammillary body that discharge rhythmically with the hippocampal theta rhythm in the rat. J Neurosci 14:7040–7052 Kosaka T, Kosaka K, Tateishi K, Hamaoka Y, Yanaihara N, Wu JY, Hama K (1985) GABAergic neurons containing CCK-8like and/or VIP-like immunoreactivities in the rat hippocampus and dentate gyrus. J Comp Neurol 239:420–430 Leranth C, Kiss J (1996) A population of supramammillary area calretinin neurons terminating on medial septal area cholinergic and lateral septal area calbindin-containing cells are aspartate/glutamatergic. J Neurosci 16:7699–7710 Leranth C, Nitsch R (1994) Morphological evidence that the hypothalamic substance P-containing afferents are capable of filtering signal flow in the monkey hippocampal formation. J Neurosci 14:4079–4094 Maglóczky Z, Acsády L, Freund TF (1994) Principal cells are the postsynaptic targets of supramammillary afferents in the hippocampus of the rat. Hippocampus 4:322–334 Nitsch R, Leranth C (1993) Calretinin immunoreactivity in the monkey hippocampal formation. II. Intrinsic GABAergic and hypothalamic non-GABAergic systems: an experimental tracing and co-existence study. Neuroscience 55:797–812
374 Nitsch R, Leranth C (1994) Substance P-containing hypothalamic afferents to the monkey hippocampus: an immunocytochemical, tracing, and coexistence study. Exp Brain Res 101: 231–240 Nitsch R, Leranth C (1996) GABAergic neurons in the rat dentate gyrus are innervated by subcortical calretinin-containing afferents. J Comp Neurol 364:425–438 Oddie SD, Bland BH, Colom LV, Vertes RP (1994) The midline posterior hypothalamic region comprises a critical part of the ascending brainstem hippocampal synchronizing pathway. Hippocampus 4:454–473 Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates. Academic Press, Sydney
Rogers JH (1989) Immunoreactivity for calretinin and other calcium-binding proteins in cerebellum. Neuroscience 31:711–721 Seress L, Leranth C (1996) Distribution of substance P-immunoreactive neurons and fibers in the monkey hippocampal formation. Neuroscience 71:633–650 Seress L, Nitsch R, Leranth C (1993) Calretinin immunoreactivity in the monkey hippocampal formation. I. Light and electron microscopic characteristics and co-localization with other calcium-binding proteins. Neuroscience 55:775–796 Stewart M, Fox SE (1991) Hippocamal theta activity in monkeys. Brain Res 538:59–63 Szeidemann Z, Jakab RL, Shanabrough M, Leranth C (1995) Extrinsic and intrinsic substance P innervation of the rat lateral septal area calbindin cells. Neuroscience 69:1205–1221
