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Distribution of thyrotropin-releasing hormone (TRH) in the hippocampal formation as determined by radioimmunoassay
314
Neuroscience Letters, 103 (1989) 3 l 4 31 ~) Elsevier Scientific Publishers Ireland Ltd.
NSL 06272
Distribution of thyrotropin-releasing hormone (TRH) in the hippocampal fo ation as determined by radioimmunoassay Walter C. L o w 1,4, Janet Roepke l, Shereen D. Farber 1, Timothy G, Hitl 5, Albert Sattin 3-5 and Michael J. Kubek 2-5 Departments of 1Physiology and Biophysics, 2Anatomy and JPsychiatry, the ¢Program in Medical Neurobiology, Indiana University School of Medicine and the 5R.L. Roudebush Veterans, Administration Medical Center, Indianapolis, IN 46223 (U. S.A. ) (Received 25 November 1988; Revised version received 18 April 1989; Accepted 18 April 1989)
Key words: Thyrotropin-releasing hormone; Hippocampal formation; Fornix pathway The distribution of thyrotropin-releasing hormone (TRH) in the hippocampal formation was determined using a radioimmunoassay (RIA) specific for TRH. RIA of hippocampal subregions revealed that the CA3 region of the hippocampal formation contained the highest amount of TRH, followed by intermediate levels in region CA1 and the dentate gyrus. The hilus and subiculum contained the lowest levels. The issue of whether hippocampal TRH is derived from extrinsic and/or intrinsic sources was evaluated by making lesions of the major subcortical afferent to the hippocampus, the fornix pathway. Analysis of the hippocampal formation by RIA revealed that the ventral hippocampus contains higher levels of TRH than the dorsal hippocampus (6.01 +_0.62 pg/mg tissue weight vs 1.11 + 0.19 pg/mg tissue weight). Lesions of the fornix produced significant decreases in ventral TRH to 52.9% of its control level and in dorsal TRH to 28.8% of its control level. The results from these studies suggest that (1) there is a differential distribution of TRH in the hippocampal formation, (2) the hippocampal formation might be composed of extrinsic and intrinsic sources of TRH, and (3) extrinsic sources of TRH might enter the hippocampus via the fornix pathway. In addition (4) the greater post-lesion decrement in ventral vs dorsal hippocampal TRH suggests that TRH fibers traversing the fornix innervate the ventral hippocampal formation in preference to its dorsal counterpart.
Thyrotropin-releasing hormone (TRH) is a tripeptide (pyroglutamyl-histidyl-prolyl-amide) that was originally shown to evoke the release of thyroid-stimulating hormone from the anterior pituitary [2]. More recent studies have demonstrated that TRH and its receptor are also localized within discrete regions of the limbic system and other areas of brain [3, 9, 18]. Among the limbic system structures, TRH has been found in differing amounts in the hippocampal formation, amygdala, piriform cortex, septum, cingulate gyrus, entorhinal cortex and hypothalamus [10]. Many of these areas of the limbic system have long been known to be involved in seizure activCorrespondence." W.C. Low, Dept. of Physiology and Biophysics, 635 Barnhill Dr., Indiana University School of Medicine, Indianapolis, IN 46223, U.S.A. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
315
ity [7]. Previous studies have documented sustained elevations of hippocampal TRH as a result of electroconvulsive shock [21], chemically induced seizures [20], chemical kindling [14] and electrical kindling [11]. A quantitative distribution of TRH within the hippocampal formation, however, has yet to be determined. In addition, whether TRH found in the hippocampus is derived from extrinsic and/or intrinsic sources remains to be resolved. This study addresses these issues by examining the distribution of TRH within the hippocampal formation and by evaluating the possibility that a portion of TRH found within the hippocampal formation is derived from extrinsic sources. Ten adult male Sprague-Dawley rats (175-250 g) were used to determine the subregional distribution of TRH by radioimmunoassay (RIA). Animals were decapitated, brains were quickly dissected from the skull and immersed in a beaker containing chilled saline. Hippocampi were then individually placed on an ice-chilled plate and slices approximately 1 mm thick were cut transverse to the longitudinal axis. Each slice was then dissected under low power magnification into separate subregions consisting of CA1, CA3, hilus, dentate, and subiculum. Individual subregions were immediately placed on aluminum foil and frozen on dry ice until all of the slices from an animal's hippocampi were completely dissected and then stored at -90°C until extracted for assay. Rats were anesthetized with Equithesin (0.33 ml/100 g b.wt.) and placed in a stereotaxic apparatus. Bilateral lesions were utilized to study the effect of fornix lesions on TRH in the dorsal and ventral hippocampus. In these experiments, 6 animals received bilateral aspirated lesions of the fornix pathway while another 4 received bilateral control lesions. Two to 3 weeks after the lesion, animals were killed by decapitation, and the brains were removed and dissected. The fornix pathway was examined in each animal to verify the completeness of the aspirated lesion. In animals that received unilateral lesions of the fornix or control lesions on the right side of the brain, only the ipsilatTABLE I T R H C O N T E N T IN H I P P O C A M P A L S U B R E G I O N S Comparisons of T R H content: CAI vs CA3, P<0.001; CA1 vs dentate gyrus, P < 0 . 0 2 ; CA1 vs hilus, P > 0 . 0 5 ; CAI vs subiculum, P<0.001; CA3 vs dentate gyrus, P<0.001; CA3 vs hilus, P < 0 . 0 1 ; CA3 vs subiculum, P<0.001; dentate gyrus vs hilus, P > 0 . 0 5 ; dentate gyrus vs subiculum, P < 0 . 0 5 ; subiculum vs hilus, P < 0.02. Hippocampal region
Total T R H (pg + S.E.M.)
Tissue weight (nag + S.E.M.)
T R H content (pg/mg tissue weight +S.E.M.)
CA 1 (n = 9) CA3 ( n = 8 ) Dentate gyrus (n = 7) Hilus (n = 7) Subiculum (n = 9)
116.4 + 8.84 152.0+5.80 76.5+__7.69 26.6 ___3.90 38.4 __+5.62
33.8 + 2.74 33.1 + 1.58 31.8__+2.14 10.1 + 0.74 27.9 __-2.7
3.40 __+0.21 4.63+0.20 2.34+0.31 2.76 + 0.62 1.39 __+0.23
316
eral hippocampal formation was removed for the TRH assay. In animals with bilateral fornix lesions or bilateral control operations, hippocampi from both sides of the brain were removed to be assayed. In the evaluation of TRH in the dorsal and ventral hippocampus, the hippocampi were bisected in a plane transverse to the longitudinal axis. Dissected hippocampi were immediately weighed, frozen on foil with dry ice and stored at -90°C until they were extracted. In all, a total of 20 animals were used in this study. Tissue samples were extracted with HAc and TRH was quantified by a specific radioimmunoassay as previously described [12]. TRH content was expressed in pg (mean + S.E.M.) per mg tissue weight. Statistical comparisons were performed on log transformed data using analysis of variance and t-tests. The determination of TRH by RIA in various subregions of the hippocampal formation is shown in Table I. The CA3 region contained the highest concentration of TRH, followed by intermediate levels in the CA I region and the hilus. Lowest levels were observed in the dentate gyrus and subiculum. TRH levels were significantly (P < 0.05) different among all regions except between the CA1 and hilar regions, and between the hilus and dentate gyrus. The determination of TRH levels within the dorsal and ventral regions of the hippocampal formation revealed that the ventral hippocampus contained significantly
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Fig. 1. Dorsal and ventral distribution of h i p p o e a n g ~ l T R H a n d e f f e c t s of fimbria-fornix (Fx) lesions. Ventral hippoeampus: sham vs Fx lesion, P < 0.005. Dorsal hipppocampus: sham vs Fx lesion, P < 0.005. Sham ventral hippocampus vs sham dorsal hippocampus, P < 0.001.
317
greater amounts of TRH (Fig. 1). In control tissue, TRH content in the ventral hippocampus averaged 6.01 ___0.62 pg/mg tissue weight while the dorsal hippocampus averaged 1. l 1 + 0.19 pg/mg tissue weight. After lesions of the fornix, the magnitude of the decrease in TRH was greatest for the ventral in comparison to the dorsal hippocampus: 3.18 _ 0.41 pg of TRH per mg tissue remained in the ventral hippocampal formation after fornix lesions as opposed to 0.32 +0.06 pg/mg tissue in the dorsal hippocampus. The results of the radioimmunoassay from microdissected subregions indicate that there is a differential concentration of TRH within the hippocampal formation. The greatest amount of TRH is found in the CA3 region followed by region CA1, hilus, dentate, and subiculum. This distribution for TRH in the hippocampus appears to differ from that of its receptor where the highest apparent binding sites are found in the ventral subiculum and the dentate gyrus while lower levels are found in the CA fields [18]. These differences in the localization of TRH and its receptor might be explained by possible TRH efferents from the CA fields that project to the dentate and subiculum. In support of this notion, studies by Hjorth-Simonsen [8] have shown that the CAl pyramidal cells innervate the subiculum. Furthermore, immunocytochemical studies by Kubek et al. [15] have shown that these pyramidal cells are immunoreactive to TRH. The loss of hippocampal TRH after fornix lesions demonstrates for the first time that a significant portion of hippocampal TRH may be derived from an extrinsic source(s) which projects to the hippocampus via the fornix pathway. The cells of origitr for such fornical TRH have yet to be identified. A preliminary study of the other major afferent pathway to the hippocampal formation, the perforant path, indicates that perforant path lesions do not affect basal hippocampal TRH [17]. Residual amounts of TRH remaining after fornix lesions can thus be interpreted as that which is derived from sources intrinsic to the hippocampal formation; possibly CA3 pyramidal cells that have been shown to contain TRH pro-hormone [16] and those which are immunoreactive to TRH [15]. In support of this notion, preliminary studies have shown the presence of a TRH mRNA-derived cDNA fragment from the hippocampal formation using methods to amplify small amounts of specific mRNA [l 3]. The greater reduction of TRH in ventral as opposed to dorsal hippocampus after fornix lesions suggests that TRH fibers innervate the ventral hippocampus more densely than its dorsal counterpart. This finding correlates well with receptor densities being higher in the ventral vs. dorsal regions of the hippocampus. This differential dorsal and ventral distribution has also been noted for the cholinergic, noradrenergic and serotonei-gic afferents [5, 19] and might indicate that the ventral hippocampal formation is receptive to afferent innervation earlier in development than the dorsal hippocampus. We cannot discount, however, the possibility that TRH is entirely intrinsic to the hippocampal formation; and that lesions of the fornix result in either (1) a retrograde degeneration of hippocampal TRH neurons, or (2) a deafferentation-induced transsynaptic degeneration. If the decrease in hippocampal TRH is a result of the latter, then non-TRH fibers from the fornix might exert atrophic effect on intrinsic TRH.
318 The functional significance o f T R H in the h i p p o c a m p u s remains to be determined. Some studies have suggested that T R H might play a role in arousal. Injections o f T R H into the dorsal h i p p o c a m p u s in sleeping squirrels produces an increase in body temperature, E E G desynchronization, and increase in E M G activity [23]. Furthermore a reduction in sleep duration is also seen in rats anesthetized with pentobarbital [4] when T R H is injected into the septal nucleus. This structure is a target site o f CA3 efferents [22, 24] and thus m a y be modulated by CA3 pyramidal cells that contain T R H p r o - h o r m o n e [16]. In addition to its arousal effects, e n d o g e n o u s hippocampal T R H exhibits a longterm enhancement in response to electroconvulsive shock [21], electrical kindling [I 1], and chemical kindling [14]. The long-lasting nature o f this T R H elevation is particularly intriguing in view of its role in Ca 2+ mobilization that partially involves the inositol triphosphate p a t h w a y [1, 6]. Calcium ions have been shown to be involved in the potentiation o f synaptic transmission [25]. T h r o u g h this cascade, T R H might play a role in the long-term plasticity o f neural responses in the hippocampai formation after repetitive activation o f afferent systems. The technical assistance o f M. Zaphirou, S.H. M u r p h y , and J.L. Evan is gratefully acknowledged. This w o r k was supported by grants from the Veterans Administration Medical Research Service (A.S. and M.J.K.), the Muscular D y s t r o p h y Association (M.J.K.) and P H S G r a n t s NS-25661 (M.J.K.) and AG-5575 (W.C.L.). I Albert, P.R. and Tashjian, A.H., Thyrotropin-releasing hormone-induced spike and plateau in cytosolic free Ca-`+ concentrations in pituitary cells, J. Biol. Chem., 259 (1984) 5827-5832. 2 Boler, J., Enzmann, F., Folkers, K., Bowers~ C.Y. and SchaUy, A.V., The identity of chemical and hormonal properties of the thyrotropin releasing hormone and pyroglutamyl-histidyl-proline amide, Biochem. Biophys. Res. Commun., 37 (1969) 705-722. 3 Brownstein, M.J., Palkovits, M., Saavedra, J.M., Bassiri, R.M. and Utiger, R.D., Thyrotropin-releasing hormone in specific nuclei of rat brain, Science, t85 (1974) 267--269. 4 Brunello, N. and Cheney, D.L., The septal-hippocampal cholinergic pathway: role in antagonism of pentobarbital anesthesia and regulation by various afferents, J. Pharmacol. Exp. Ther., 219 (1981) 489- 495. 5 Gage, F.H. and Thompson, R.G., Differential distribution of norepinephrine and serotonin along the dorsal-ventral axis of the hippocampal formation, Brain Res. Bull., 5 (1980) 771 773. 6 Gershengorn, M.C., Geras, E., Purrello, V.S. and Rebecchi, M.J., Inositol trisphosphate mediates thyrotropin-releasing hormone mobilization of nonmitochondrial calcium in rat mammotropic pituitary cells, J. Biol. Chem., 259 (1984) 10675~10681. 7 Goddard, G.V., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 29~330. 8 Hjorth-Simonsen, A., Some intrinsic connections of the hippocampus in the rat: an experimental analysis, J. Comp. Neurol., 147 (1973) 145-161. 9 Jackson, I.M.D. and Reichlin, S., Thyrotropin-releasing hormone (TRH): distribution in hypothalamic and extrahypothalamic brain tissues of mammalian and submammalian chordates, Endocrinology, 95 (1974) 854-862. I0 Kubek, M.J., Thyrotropin-releasing hormone: localization of specific hypothalamic and extrahypothalamic sites of CNS modulation. In R.C.A. Frederickson, H. Hendrie, J.Hingtgen and M. Aprison (Eds.), Neuroregulation of Autonomic, Endocrine and Immune Systems, Martinus Nijhof, Boston, 1986, pp. 265 301.
319 11 Kubek, M.J., Bates, V.E. and Meyerhoff, J.L., Elevations of CNS thyrotropin-releasing hormone in an animal model of epilepsy, 64th Meeting Endocrinol. Soc., 1982, p. 233. 12 Kubek, M.J. and Hill, T.G., Methods of thyrotropin-releasing hormone measurement. In: J.N. Hingtgen, D.H. Hellhammer and G. Huppmann, (Eds.), Advanced Methods in Psychobiology, Hogrefe International, Toronto, 1987, pp. 261-279. 13 Kubek, M.J., Larsen, S.H. and Melendez, A., Thyrotropin-releasing hormone (TRH) mRNA is present in the rat hippocampus, Soc. Neurosci. Abstr., 14 (1988) 399. 14 Kubek, M.J. and Morzorati, S.L., Chemically kindled seizures increase thyrotropin-releasing hormone levels in specific areas of the rat brain, Brain Res. Bull., in press. 15 Kubek, M.J., Roepke, J., Zaphirou, M., Murphy, S.H., Hill, T.G., Sattin, A. and Low, W.C., Thyrotropin-releasing hormone (TRH) in subregions of the hippocampal formation: Immunocytochemical localization and quantitation by radioimmunoassay (RIA), Soc. Neurosci. Abstr., 13 (1986) 995. 16 Lechan, R.M., Wu, P. and Jackson, I.M.D., Immunolocalization of the thyrotropin-releasing hormone prohormone in the rat central nervous system, Endocrinology, 119 (I 986) 1210-1216. 17 Low, W.C., Hill, T.G., Farber, S.D., Murphy, S.H. and Kubek, M.J., Differences in thyrotropinreleasing hormone (TRH) between dorsal and ventral hippocampus. Soc. Neurosci. Abstr., 12 (1986) 300. 18 Manaker, S., Winokur, A., Rostene, W.H. and Rainbow, T.C., Autoradiographic localization of thyrotropin-releasing hormone receptors in the rat central nervous system, J. Neurosci., 5 (1985) 167 174. 19 Murphy, S.H., Farber, S.D., Postlewaite, N., Paine, T., Kaseda, Y., Hofstetter, J.R., Richter, J.A. and Low, W.C., Hippocampal choline acetyltransferase (CHAT) activity and maze performance in 'old' and young rats, Soc. Neurosci. Abstr., 12 (1986) 1313. 20 Ogawa, N., Hirose, Y., Mori, A., Kajita, S. and Sato, M., Involvement of thyrotropin-releasing hormone (TRH) neural systems of the brain in pentylenetetrazol-induced seizures, Regul. Pept., 12 (1985) 249-256. 21 Sattin, A., Hill, T.G., Meyerhoff, J.L., Norton, J. and Kubek, M.J., The prolonged increase in thyrotropin-releasing hormone in rat limbic forebrain regions following electroconvulsive shock, Regul. Pept., 19 (1987) 13-22. 22 Siegel, A., Edinger, H. and Ohgami, S., Topographical organization of the hippocampal projection to the septal area: a comparative neuroanatomical analysis in the gerbil, rat, rabbit and cat, J. Comp. Neurol., 157 (1974) 359-377. 23 Stanton, T.L., Beckman, A.L. and Winokur, A., Thyrotropin-releasing hormone effects in the central nervous system: Dependence on arousal state, Science, 214 (1981) 678~81. 24 Swanson, L.W. and Cowan, W.M., An autoradiographic study ofthe organization of the efferent connections of the hippocampal formation in the rat, J. Comp. Neurol., 172 (1977) 44-84. 25 Wigstrom, H., Swann, J.W. and Andersen, P., Calcium dependency of synaptic long-lasting potentiation in the hippocampal slice, Acta Physiol. Scand., 105 (1979) 126-128.
Neuroscience Letters, 103 (1989) 3 l 4 31 ~) Elsevier Scientific Publishers Ireland Ltd.
NSL 06272
Distribution of thyrotropin-releasing hormone (TRH) in the hippocampal fo ation as determined by radioimmunoassay Walter C. L o w 1,4, Janet Roepke l, Shereen D. Farber 1, Timothy G, Hitl 5, Albert Sattin 3-5 and Michael J. Kubek 2-5 Departments of 1Physiology and Biophysics, 2Anatomy and JPsychiatry, the ¢Program in Medical Neurobiology, Indiana University School of Medicine and the 5R.L. Roudebush Veterans, Administration Medical Center, Indianapolis, IN 46223 (U. S.A. ) (Received 25 November 1988; Revised version received 18 April 1989; Accepted 18 April 1989)
Key words: Thyrotropin-releasing hormone; Hippocampal formation; Fornix pathway The distribution of thyrotropin-releasing hormone (TRH) in the hippocampal formation was determined using a radioimmunoassay (RIA) specific for TRH. RIA of hippocampal subregions revealed that the CA3 region of the hippocampal formation contained the highest amount of TRH, followed by intermediate levels in region CA1 and the dentate gyrus. The hilus and subiculum contained the lowest levels. The issue of whether hippocampal TRH is derived from extrinsic and/or intrinsic sources was evaluated by making lesions of the major subcortical afferent to the hippocampus, the fornix pathway. Analysis of the hippocampal formation by RIA revealed that the ventral hippocampus contains higher levels of TRH than the dorsal hippocampus (6.01 +_0.62 pg/mg tissue weight vs 1.11 + 0.19 pg/mg tissue weight). Lesions of the fornix produced significant decreases in ventral TRH to 52.9% of its control level and in dorsal TRH to 28.8% of its control level. The results from these studies suggest that (1) there is a differential distribution of TRH in the hippocampal formation, (2) the hippocampal formation might be composed of extrinsic and intrinsic sources of TRH, and (3) extrinsic sources of TRH might enter the hippocampus via the fornix pathway. In addition (4) the greater post-lesion decrement in ventral vs dorsal hippocampal TRH suggests that TRH fibers traversing the fornix innervate the ventral hippocampal formation in preference to its dorsal counterpart.
Thyrotropin-releasing hormone (TRH) is a tripeptide (pyroglutamyl-histidyl-prolyl-amide) that was originally shown to evoke the release of thyroid-stimulating hormone from the anterior pituitary [2]. More recent studies have demonstrated that TRH and its receptor are also localized within discrete regions of the limbic system and other areas of brain [3, 9, 18]. Among the limbic system structures, TRH has been found in differing amounts in the hippocampal formation, amygdala, piriform cortex, septum, cingulate gyrus, entorhinal cortex and hypothalamus [10]. Many of these areas of the limbic system have long been known to be involved in seizure activCorrespondence." W.C. Low, Dept. of Physiology and Biophysics, 635 Barnhill Dr., Indiana University School of Medicine, Indianapolis, IN 46223, U.S.A. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
315
ity [7]. Previous studies have documented sustained elevations of hippocampal TRH as a result of electroconvulsive shock [21], chemically induced seizures [20], chemical kindling [14] and electrical kindling [11]. A quantitative distribution of TRH within the hippocampal formation, however, has yet to be determined. In addition, whether TRH found in the hippocampus is derived from extrinsic and/or intrinsic sources remains to be resolved. This study addresses these issues by examining the distribution of TRH within the hippocampal formation and by evaluating the possibility that a portion of TRH found within the hippocampal formation is derived from extrinsic sources. Ten adult male Sprague-Dawley rats (175-250 g) were used to determine the subregional distribution of TRH by radioimmunoassay (RIA). Animals were decapitated, brains were quickly dissected from the skull and immersed in a beaker containing chilled saline. Hippocampi were then individually placed on an ice-chilled plate and slices approximately 1 mm thick were cut transverse to the longitudinal axis. Each slice was then dissected under low power magnification into separate subregions consisting of CA1, CA3, hilus, dentate, and subiculum. Individual subregions were immediately placed on aluminum foil and frozen on dry ice until all of the slices from an animal's hippocampi were completely dissected and then stored at -90°C until extracted for assay. Rats were anesthetized with Equithesin (0.33 ml/100 g b.wt.) and placed in a stereotaxic apparatus. Bilateral lesions were utilized to study the effect of fornix lesions on TRH in the dorsal and ventral hippocampus. In these experiments, 6 animals received bilateral aspirated lesions of the fornix pathway while another 4 received bilateral control lesions. Two to 3 weeks after the lesion, animals were killed by decapitation, and the brains were removed and dissected. The fornix pathway was examined in each animal to verify the completeness of the aspirated lesion. In animals that received unilateral lesions of the fornix or control lesions on the right side of the brain, only the ipsilatTABLE I T R H C O N T E N T IN H I P P O C A M P A L S U B R E G I O N S Comparisons of T R H content: CAI vs CA3, P<0.001; CA1 vs dentate gyrus, P < 0 . 0 2 ; CA1 vs hilus, P > 0 . 0 5 ; CAI vs subiculum, P<0.001; CA3 vs dentate gyrus, P<0.001; CA3 vs hilus, P < 0 . 0 1 ; CA3 vs subiculum, P<0.001; dentate gyrus vs hilus, P > 0 . 0 5 ; dentate gyrus vs subiculum, P < 0 . 0 5 ; subiculum vs hilus, P < 0.02. Hippocampal region
Total T R H (pg + S.E.M.)
Tissue weight (nag + S.E.M.)
T R H content (pg/mg tissue weight +S.E.M.)
CA 1 (n = 9) CA3 ( n = 8 ) Dentate gyrus (n = 7) Hilus (n = 7) Subiculum (n = 9)
116.4 + 8.84 152.0+5.80 76.5+__7.69 26.6 ___3.90 38.4 __+5.62
33.8 + 2.74 33.1 + 1.58 31.8__+2.14 10.1 + 0.74 27.9 __-2.7
3.40 __+0.21 4.63+0.20 2.34+0.31 2.76 + 0.62 1.39 __+0.23
316
eral hippocampal formation was removed for the TRH assay. In animals with bilateral fornix lesions or bilateral control operations, hippocampi from both sides of the brain were removed to be assayed. In the evaluation of TRH in the dorsal and ventral hippocampus, the hippocampi were bisected in a plane transverse to the longitudinal axis. Dissected hippocampi were immediately weighed, frozen on foil with dry ice and stored at -90°C until they were extracted. In all, a total of 20 animals were used in this study. Tissue samples were extracted with HAc and TRH was quantified by a specific radioimmunoassay as previously described [12]. TRH content was expressed in pg (mean + S.E.M.) per mg tissue weight. Statistical comparisons were performed on log transformed data using analysis of variance and t-tests. The determination of TRH by RIA in various subregions of the hippocampal formation is shown in Table I. The CA3 region contained the highest concentration of TRH, followed by intermediate levels in the CA I region and the hilus. Lowest levels were observed in the dentate gyrus and subiculum. TRH levels were significantly (P < 0.05) different among all regions except between the CA1 and hilar regions, and between the hilus and dentate gyrus. The determination of TRH levels within the dorsal and ventral regions of the hippocampal formation revealed that the ventral hippocampus contained significantly
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Fig. 1. Dorsal and ventral distribution of h i p p o e a n g ~ l T R H a n d e f f e c t s of fimbria-fornix (Fx) lesions. Ventral hippoeampus: sham vs Fx lesion, P < 0.005. Dorsal hipppocampus: sham vs Fx lesion, P < 0.005. Sham ventral hippocampus vs sham dorsal hippocampus, P < 0.001.
317
greater amounts of TRH (Fig. 1). In control tissue, TRH content in the ventral hippocampus averaged 6.01 ___0.62 pg/mg tissue weight while the dorsal hippocampus averaged 1. l 1 + 0.19 pg/mg tissue weight. After lesions of the fornix, the magnitude of the decrease in TRH was greatest for the ventral in comparison to the dorsal hippocampus: 3.18 _ 0.41 pg of TRH per mg tissue remained in the ventral hippocampal formation after fornix lesions as opposed to 0.32 +0.06 pg/mg tissue in the dorsal hippocampus. The results of the radioimmunoassay from microdissected subregions indicate that there is a differential concentration of TRH within the hippocampal formation. The greatest amount of TRH is found in the CA3 region followed by region CA1, hilus, dentate, and subiculum. This distribution for TRH in the hippocampus appears to differ from that of its receptor where the highest apparent binding sites are found in the ventral subiculum and the dentate gyrus while lower levels are found in the CA fields [18]. These differences in the localization of TRH and its receptor might be explained by possible TRH efferents from the CA fields that project to the dentate and subiculum. In support of this notion, studies by Hjorth-Simonsen [8] have shown that the CAl pyramidal cells innervate the subiculum. Furthermore, immunocytochemical studies by Kubek et al. [15] have shown that these pyramidal cells are immunoreactive to TRH. The loss of hippocampal TRH after fornix lesions demonstrates for the first time that a significant portion of hippocampal TRH may be derived from an extrinsic source(s) which projects to the hippocampus via the fornix pathway. The cells of origitr for such fornical TRH have yet to be identified. A preliminary study of the other major afferent pathway to the hippocampal formation, the perforant path, indicates that perforant path lesions do not affect basal hippocampal TRH [17]. Residual amounts of TRH remaining after fornix lesions can thus be interpreted as that which is derived from sources intrinsic to the hippocampal formation; possibly CA3 pyramidal cells that have been shown to contain TRH pro-hormone [16] and those which are immunoreactive to TRH [15]. In support of this notion, preliminary studies have shown the presence of a TRH mRNA-derived cDNA fragment from the hippocampal formation using methods to amplify small amounts of specific mRNA [l 3]. The greater reduction of TRH in ventral as opposed to dorsal hippocampus after fornix lesions suggests that TRH fibers innervate the ventral hippocampus more densely than its dorsal counterpart. This finding correlates well with receptor densities being higher in the ventral vs. dorsal regions of the hippocampus. This differential dorsal and ventral distribution has also been noted for the cholinergic, noradrenergic and serotonei-gic afferents [5, 19] and might indicate that the ventral hippocampal formation is receptive to afferent innervation earlier in development than the dorsal hippocampus. We cannot discount, however, the possibility that TRH is entirely intrinsic to the hippocampal formation; and that lesions of the fornix result in either (1) a retrograde degeneration of hippocampal TRH neurons, or (2) a deafferentation-induced transsynaptic degeneration. If the decrease in hippocampal TRH is a result of the latter, then non-TRH fibers from the fornix might exert atrophic effect on intrinsic TRH.
318 The functional significance o f T R H in the h i p p o c a m p u s remains to be determined. Some studies have suggested that T R H might play a role in arousal. Injections o f T R H into the dorsal h i p p o c a m p u s in sleeping squirrels produces an increase in body temperature, E E G desynchronization, and increase in E M G activity [23]. Furthermore a reduction in sleep duration is also seen in rats anesthetized with pentobarbital [4] when T R H is injected into the septal nucleus. This structure is a target site o f CA3 efferents [22, 24] and thus m a y be modulated by CA3 pyramidal cells that contain T R H p r o - h o r m o n e [16]. In addition to its arousal effects, e n d o g e n o u s hippocampal T R H exhibits a longterm enhancement in response to electroconvulsive shock [21], electrical kindling [I 1], and chemical kindling [14]. The long-lasting nature o f this T R H elevation is particularly intriguing in view of its role in Ca 2+ mobilization that partially involves the inositol triphosphate p a t h w a y [1, 6]. Calcium ions have been shown to be involved in the potentiation o f synaptic transmission [25]. T h r o u g h this cascade, T R H might play a role in the long-term plasticity o f neural responses in the hippocampai formation after repetitive activation o f afferent systems. The technical assistance o f M. Zaphirou, S.H. M u r p h y , and J.L. Evan is gratefully acknowledged. This w o r k was supported by grants from the Veterans Administration Medical Research Service (A.S. and M.J.K.), the Muscular D y s t r o p h y Association (M.J.K.) and P H S G r a n t s NS-25661 (M.J.K.) and AG-5575 (W.C.L.). I Albert, P.R. and Tashjian, A.H., Thyrotropin-releasing hormone-induced spike and plateau in cytosolic free Ca-`+ concentrations in pituitary cells, J. Biol. Chem., 259 (1984) 5827-5832. 2 Boler, J., Enzmann, F., Folkers, K., Bowers~ C.Y. and SchaUy, A.V., The identity of chemical and hormonal properties of the thyrotropin releasing hormone and pyroglutamyl-histidyl-proline amide, Biochem. Biophys. Res. Commun., 37 (1969) 705-722. 3 Brownstein, M.J., Palkovits, M., Saavedra, J.M., Bassiri, R.M. and Utiger, R.D., Thyrotropin-releasing hormone in specific nuclei of rat brain, Science, t85 (1974) 267--269. 4 Brunello, N. and Cheney, D.L., The septal-hippocampal cholinergic pathway: role in antagonism of pentobarbital anesthesia and regulation by various afferents, J. Pharmacol. Exp. Ther., 219 (1981) 489- 495. 5 Gage, F.H. and Thompson, R.G., Differential distribution of norepinephrine and serotonin along the dorsal-ventral axis of the hippocampal formation, Brain Res. Bull., 5 (1980) 771 773. 6 Gershengorn, M.C., Geras, E., Purrello, V.S. and Rebecchi, M.J., Inositol trisphosphate mediates thyrotropin-releasing hormone mobilization of nonmitochondrial calcium in rat mammotropic pituitary cells, J. Biol. Chem., 259 (1984) 10675~10681. 7 Goddard, G.V., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 29~330. 8 Hjorth-Simonsen, A., Some intrinsic connections of the hippocampus in the rat: an experimental analysis, J. Comp. Neurol., 147 (1973) 145-161. 9 Jackson, I.M.D. and Reichlin, S., Thyrotropin-releasing hormone (TRH): distribution in hypothalamic and extrahypothalamic brain tissues of mammalian and submammalian chordates, Endocrinology, 95 (1974) 854-862. I0 Kubek, M.J., Thyrotropin-releasing hormone: localization of specific hypothalamic and extrahypothalamic sites of CNS modulation. In R.C.A. Frederickson, H. Hendrie, J.Hingtgen and M. Aprison (Eds.), Neuroregulation of Autonomic, Endocrine and Immune Systems, Martinus Nijhof, Boston, 1986, pp. 265 301.
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