Lateral Hypothalamic Stimulation Inhibits Dentate Granule Cell LTP: Direct Connections

Brain Research Bulletin, Vol. 43, No. 1, pp. 5–15, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00

PII S0361-9230(96)00425-X

Lateral Hypothalamic Stimulation Inhibits Dentate Granule Cell LTP: Direct Connections MATTHEW J. WAYNER, 1 CLYDE F. PHELIX AND DEBORAH L. ARMSTRONG Division of Life Sciences, The University of Texas at San Antonio, San Antonio, TX 78249-0662

ABSTRACT: We discovered that angiotensin II (Ang II) applied directly to the dentate gyrus inhibited LTP induction in medial perforant path–dentate granule cell synapses and that the inhibition can be blocked by losartan, an Ang II AT1 receptor specific antagonist. In the first part of this study we found that electrical stimulation of the lateral hypothalamus (LH) inhibits LTP in these synapses and the inhibition can be blocked by pretreating the animals with losartan, indicating that LH angiotensin-containing neurons project to the dentate gyrus. Results of the second part of the study demonstrate clearly that some angiotensin-containing LH neurons project directly to dentate granule cells. LH neurons were identified by retrograde tracers applied to the granule cell layer. Double-labeled neurons containing angiotensin and HRP were sparsely distributed and both fusiform and multipolar LH neurons appeared in a small cluster lateral and ventral to the fornix at the level of the paraventricular nucleus. Large numbers of angiotensin staining neurons were observed in the hypothalamus. Results support our hypothesis that some angiotensin containing LH neurons project directly to the dentate gyrus. Q 1997 Elsevier Science Inc.

tions related to angiotensin receptors, relatively high levels of Ang II in the hippocampus [25], and hippocampal pyramidal cell sensitivity to extracellular Ang II [7,8,18] remained obscure, even though the hippocampus is clearly involved in the memory process. In 1991, we discovered that Ang II inhibited long-term potentiation (LTP) in hippocampal medial perforant path–dentate granule cell synapses [5]. Following this observation, we found that ethanol and diazepam, well known for their anterograde amnesia effects, inhibited hippocampal dentate granule cell LTP, in a dose-dependent way, and that the inhibition could be blocked by losartan, an Ang II AT 1 specific receptor antagonist [1,31,32]. Furthermore, Ang II administered directly and bilaterally into the dorsal hippocampal dentate gyrus was found to impair the 24-h retention of an inhibitory shock avoidance response, and the inhibition could be blocked by pretreatment with losartan [12]. Impairment of the aerial righting reflex by ethanol can also be blocked, but not completely, by losartan. The intoxicating effect of ethanol on the aerial righting reflex can be completely blocked, however, by pretreatment with both losartan and PD123319, an Ang II AT 2 specific receptor antagonist [28]. These results provide strong support for an important role for Ang II containing neurons that project to the hippocampus dentate gyrus in certain types of anterograde amnesia associated with ethanol, diazepam, and possibly age-related dementias. When we discovered that some dentate gyrus granule cells were differentially sensitive to Ang II [ 35 ] , our major interest at that time was to study the sensitivity of lateral hypothalamic ( LH ) neurons to Ang II and ethanol, because some of these LH neurons were osmosensitive and involved in drinking [17 ] . Results demonstrated a clear interaction between Ang II and ethanol in the enhancement of spontaneous discharge frequencies in some LH neurons [ 36 ] . Therefore, it seemed reasonable to assume that some of the Ang II neurons projecting to the dentate gyrus might originate in the LH and project directly to the dentate gyrus. In 1993, we reported that electrical stimulation of the LH inhibited LTP in medial perforant path – granule cell synapses and that the inhibition could be blocked by pretreatment with saralasin, a nonspecific Ang II antagonist [ 32 ] . More recently, low-dose ethanol perfusion of the LH inhibited dentate granule cell LTP, and the inhibition was blocked with losartan pretreatment as expected; however, the same doses applied directly to the dentate were without

KEY WORDS: Angiotensin II, Long-term potentiation, Hippocampus, Memory, Losartan, Hypothalamus, Electrical stimulation, Dementia.

INTRODUCTION In 1973, we discovered that some hippocampal dentate granule cells were sensitive to the extracellular microiontophoretic administration of angiotensin II (Ang II) [34]. The significance of this observation remained unknown until 1991 [5]. During the intervening 18 years, considerable evidence accumulated on the distribution of Ang II in different brain regions [25] and the characterization and differential development of Ang II receptors [16,29]. Most of these data confirmed the physiological importance of the well-established role of brain Ang II in drinking and the regulation of body fluids and in the control of blood pressure [37]. In addition, significant observations were made on the possible role of Ang II, Ang III, and Ang IV in cognitive processes and mood elevation [30,38]. Some of the data were obtained in elderly individuals who were being treated for hypertension with angiotensin converting enzyme (ACE) inhibitors [4,27]. These individuals demonstrated improved memory and reported mood elevation while on captopril, an ACE inhibitor. However, func1

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WAYNER, PHELIX AND ARMSTRONG substances and several hippocampal injection sites revealed that anterior regions of the hypothalamus do have significant input to the dorsal hippocampus. In 1984, Ko¨hler and coworkers had reported a similar finding that only a small population of LH Ang II neurons project to the entorhinal area. Several other peptides were found in LH neurons that project directly into the dorsal hippocampus [11] . It is also important to emphasize that many of these hypothalamic afferents contain acetylcholinesterase [10 ] . Results of our preliminary immunocytochemical and retrograde tract tracing experiments [ 3 ] confirmed our LH electrical stimulation data, which showed that the level of the hypothalamic paraventricular nucleus would be the most likely site of Ang II producing neurons projecting to the dentate gyrus. More recent preliminary anterograde tracing data [ 22 ] supports this hypothesis that these more anterior LH neurons also innervate the dorsal hippocampus. The purpose of the present study was ( a ) to replicate the earlier experiment on LH electrical stimulation and LTP inhibition, utilizing losartan to block the inhibition; and ( b ) to provide additional supporting neuroanatomical data for direct projections from Ang II containing LH neurons to hippocampal dentate granule cells. Results confirm that losartan blocks inhibition of dentate granule cell LTP by LH electrical stimulation and that direct projections exist from angiotensin containing LH neurons to the dentate gyrus. LH ELECTRICAL STIMULATION INHIBITS HIPPOCAMPAL DENTATE GRANULE CELL LTP Methods

FIG. 1. Fourteen pEPSPs traces used in determining an input–output curve. (A) Before tetanization; (B) at the end of the experiment illustrating LTP in both the enhanced slope of the pEPSPs and the increased amplitudes of the PSs. Single vertical deflections are the stimulus artifacts. Calibration: 2 ms and 2 mV.

effect [ 33 ] . These data not only confirm a differential sensitivity of some LH neurons to ethanol but indicate that the effects are presynaptic and cannot be attributed to a direct postsynaptic action of the ethanol. Hypothalamic – hippocampal connections have not been studied extensively, particularly those involving the LH. Previously, several retrograde tract tracing investigations of hypothalamic innervation of the hippocampus suggested a direct connection originating in the posterior parts of the LH and supramammillary nucleus [ 9,19,20 ] . Anterograde tract tracing studies of these hypothalamic regions have shown a direct monosynaptic innervation of the dorsal dentate gyrus with hypothalamic axons forming axodendritic synapses with proximal dendrites of granule cells [ 6 ] . Electrical stimulation of these posterior hypothalamic regions monosynaptically inhibits firing of dorsal hippocampal dentate granule cells [ 24 ] . Our early experiments involving numerous retrograde tracing

Animals. Harlan male Sprague–Dawley rats weighing between 290 and 375g were used. Animal rooms were maintained at 21 { 17C with a 12 L:12 D cycle, lights on at 0600 h. The rats were housed in plastic cages containing corn cob chips and covered with ventilator tops. Commercial Tekland rat chow and water were available ad lib. All rats were allowed a minimum of 2 weeks acclimation before being used. The rats were food deprived 24 h before surgery. Surgery. Following a brief period of mild metofane inhalation, animals were anesthetized using a dose of 1.4 g / kg 25% urethane ( Sigma ) , administered IP. The rats were then placed in a Narishige stereotaxic instrument with the head fixed such that the Paxinos and Watson’s [ 21] coordinate system could be used. The surface of the skull was exposed, two holes were made with a dental drill, and the dura matter was incised. Surgical procedures were completed within 1 h. To prevent drying, 0.9% saline was periodically applied to the exposed skull and brain. Throughout the surgery and experiment, core body temperatures were monitored and maintained at 35 { 1.07C with a feedback control system and a heated pad. Body temperature was monitored visually and recorded at 1.0-min intervals. Recording and stimulating electrodes. Micropipette recording electrodes were prepared from single barrel borosilicate glass tubing 1.2 mm o.d. 1 0.6 mm i.d. pulled on a Narishige vertical puller and filled with 3 M NaCl. Resistance ranged from 1 – 3 Mohms. At the beginning of an experiment, the recording electrodes were positioned approximately 3.5 mm posterior to bregma and 2.0 mm lateral to the midline. The dentate gyrus was identified by single unit activity characteristic of granule cells as well as by an electrode depth of 3.0 – 3.5 mm below the brain surface. The position of the recording electrode was adjusted to produce an optimal popu-

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FIG. 2. Mean { SEMs potentiation measured in terms of pEPSP slopes plotted as a function of time in minutes. Vertical deflections occurred when the four tetanii were administered. (A) Demonstrates that LH electrical stimulation inhibits dentate granule cell LTP. (B) Shows that pretreatment with 10 mg/kg IP losartan blocks the inhibition. Five animals in each group.

lation excitatory postsynaptic potential ( pEPSP ) in the granule cells. Frederick Haer #17-90-1 stainless steel concentric electrodes were used for stimulation. The stereotaxic coordinates were adjusted for variation in rat ages and to maximize the monosynaptic responses of the positive-going pEPSPs produced by granule cells in response to stimulation of the dorsomedial perforant path. Average stimulating coordinates were 8.5 mm posterior to bregma and 4.2 mm lateral to the midline. At the end of the experiment, placement in the dorsomedial perforant path was verified with 100 Hz tetanic stimulation [15 ] . Electrophysiological recording. Stimulation consisted of 50 ms duration monophasic constant current pulses delivered once per minute. Input – output curves were obtained over the range of 25 – 460 mA before baseline and at the end of each experiment. Fourteen increasing stimuli were used. In these experiments, actual baseline intensities selected ranged from 50 to 251 mA. These stimulus intensities produced pEPSP amplitudes of 5 – 7 mV that were at or slightly below threshold for evoking a negative-going waveform, population spike ( PS ) , on the declining phase of the pEPSP. Representative pEPSPs before and after tetanization, input – output curve data, are illustrated in Fig. 1. The top traces in A were obtained in a control animal. In Panel B, the 14 traces and slope enhancement are clearly discernible in the same animal following tetanization and completion of the experimental protocol. Also, the threshold for the PS and increased PS amplitude following tetanization can also be seen. The slope of an individual pEPSP was measured over the first ms of the approximately linear rising phase. The slope of each trace was measured by adjusting the cursors in the on-line display and calculating the difference in mV. These differences constitute the raw data and are referred to as the slopes. Once determined, stimulus current remained constant throughout the experiment. After recording 30 min of baseline responses, four sets of tetanic stimulation were administered to induce LTP, separated by intervals of 10 min at 30, 40, 50, and 60 min as illustrated in Fig. 2 in the Results section. Each set contained five trains, 10 pulses per train at 400 Hz, delivered at a rate of one train per second for 5 s. The pulse width in the trains were 50, 100, 150, and 200 ms, respectively. The posttetanic potentiation due

to the individual tentanii appear as prominent increasing vertical displacements from baseline in Fig. 2. The pEPSPs were recorded every minute for 130 min. Percent pEPSP was calculated as follows: slope at each minute minus the mean baseline slope and then divided by the mean baseline slope and then 1 100. Baseline was the mean slope of the first 30 min. The granule cell pEPSPs were amplified by a conventional amplifier at a frequency band of 1.6 Hz to 2 kHz. All potentials were monitored on an oscilloscope, digitized at a sampling interval of 20 ms, for on-line computer display (RC Electronics). The first millisecond of the pEPSP slopes were used in the analysis. All data were stored on floppy disks. Drug administration. Losartan: losartan was prepared fresh in distilled water at room temperature. One hour prior to recording baseline responses losartan was injected IP, 10.0 mg/kg body weight. Lateral hypothalamic stimulation. An ipsilateral stimulating electrode was positioned in the LH according to the following coordinates: AP 01.8 mm, L 1.5 mm, V 8.2 mm to activate possible angiotensin containing neurons projecting to the hippocampus. Stimulation consisted of 50 ms pulses applied at 2 Hz for 30 min using 250 mA current intensity. The LH was stimulated for 30 min and then the usual 30 min baseline data were obtained before the first tetanus was applied. When the effects of losartan on LH stimulation were determined, the losartan was administered 1 h prior to LH stimulation. Following LH stimulation under these conditions, an additional 30 min was required to remove the LH stimulating electrodes and position the stimulating and recording electrodes for measuring LTP, before obtaining 30 min of baseline data. Histology At the end of an experiment and following additional pentobarbital anesthesia, the animal was perfused with 10% formalin, by means of cardiac puncture, and then the brain was removed. The brain was stored in formaldehyde, an appropriate block of tissue was removed, 40 mm sections were cut in a cryostat, and serial sections were examined by means of a microscope. Sections were then stained with thionin, mounted, cover slipped, and stored for further study. The approximate locations of the electrical stimulation electrodes in seven animals were determined.

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WAYNER, PHELIX AND ARMSTRONG induction as illustrated in Fig. 2A. In Fig. 2B, pretreatment with losartan, 10 mg/kg IP, clearly prevented electrical stimulation inhibition of LTP induction. The means { SEMs for the two groups were: 6.1 { 2.7 and 45.2 { 5.3. Results of the histological examination of electrical stimulation electrode tracts and tip locations are summarized in Fig. 3. Seven animals were required to obtain the data on inhibition of LTP by electrical stimulation in Fig. 2. In two animals, inhibition did not occur because of incorrect electrode locations; 22% LTP occurred in one animal, as illustrated in Fig. 3A, and 25% LTP as show in Fig. 3C for the other animal. Both sets of electrodes were positioned incorrectly outside of the LH. The sites of the electrode tips in the five animals in which inhibition of LTP was observed were within the shaded area of Fig. 3B. Only 6% LTP was observed in this group. These data confirm our earlier results and demonstrate that LH electrical stimulation inhibits medial perforant path–granule cell LTP induction and that the inhibition is mediated by Ang II and the AT 1 receptor because the inhibition can be blocked by pretreatment with losartan, a specific AT 1 receptor antagonist. In addition, the active stimulation site is definitely in the lateral perifornical region of the LH at the level of the paraventricular nucleus. Of the two ineffective stimulating electrode placements, the one in Fig. 3A is considerably anterior to the paraventricular nucleus, and the other in Fig. 3C is far lateral and outside the LH at the level of the paraventricular nucleus. Angiotensin-containing neurons have been found in this part of the LH (Fig. 2B), and some of them project to dentate granule cells. DIRECT CONNECTIONS BETWEEN THE LH AND DENTATE GYRUS Methods

FIG. 3. Representative cross-sections through the hypothalamus illustrating the stimulating electrode placements in the perifornical region of the LH at the level of the paraventricular nucleus. (A) One animal, stimulated anterior to the LH did not significantly inhibit dentate granule cell LTP induction. (B) Mean LTP induction of five animals with placements in this region demonstrating inhibition due to the electrical stimulation. (C) One animal stimulated in the far LH also failed to inhibit LTP induction.

Results and Discussion Lateral hypothalamic stimulation. Mean { SEM percent potentiation measured in terms of the relative change in pEPSP slope compared to baseline is presented in Fig. 2 as a function of time in two groups of rats. There were five animals in each group. Electrical stimulation clearly inhibited granule cell LTP

Animals. Forty five male Harlan–Sprague–Dawley rats weighing between 200 and 400 g were used in the study. Surgery. For general anesthesia, 45 mg/kg IP was employed and animals were euthanized by administering 200 mg/kg of pentobarbital IP. Similar surgical methods were employed as in the LTP experiment. The same stereotaxic coordinates were used to administer the tracers as were utilized in recording dentate granule cell pEPSPs. Retrograde tract tracing. The retrograde tract tracing methods utilized in this study included the use of different tract tracing substances, large to small injections, sensitive visualization techniques, and a double-label immunohistochemical technique. First, large injections of 500 nl to 1 ml of 30% horseradish peroxidase (HRP) with 0.1% dimethylsulfoxide, 2% wheat germ agglutinin (WGA), or 15% WGA conjugated to HRP were administered to the dorsal hippocampus to examine the extent of the LH that projects to the dorsal hippocampus. Smaller injections of 50 to 300 nl of 15% HRP or 2% WGA were utilized to examine LH projections directly into the dentate gyrus at the location of our recording electrodes. Larger injections were performed using a 1 ml Hamilton syringe. Smaller injections were made with pulled glass micropipettes, tip diameter approximately 45 mm, connected to a 1 ml Hamilton syringe with plastic tubing. Light mineral oil (Sigma) was used to fill the syringe tubing micropipette injector. Forty eight hours after tracer injection rats were euthanized and perfused transcardially with a different fixative, depending on the final histochemical processing: a 1% (para)formaldehyde–1% glutaraldehyde solution for sensitive detection of HRP and WGA-HRP; a 4% (para) formaldehyde solution for immunohistochemical detection of WGA; a 3% formaldehyde–0.5% glutaraldehyde solution for double-label im-

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FIG. 4. Bright-field photomicrographs of retrogradely filled neurons in the hypothalamus after injection into the dorsal hippocampus. (A) HRP-WGA filled neurons located around the fornix are either fusiform (small arrowhead) or multipolar (large arrowheads). (B) Higher magnification of WGA filled neurons in the region lateral to the fornix are either multipolar (large arrowhead) or fusiform (small arrowhead) and the WGA vesicles can be seen within proximal and intermediate sized (arrow) dendrites. (C) A multipolar HRP filled neuron (large arrowhead) is seen within the ventrolateral LH. HRP vesicles can be seen far into a secondary dendrite (arrow). Several HRP containing axons (blank arrows) can be seen passing laterally into the ventral amygdalofugal pathway. (D) Many HRP filled dendrites (arrows) of neurons in the central LH can be seen. This cluster was located at the level of the hypothalamic paraventricular nucleus. A large injection of 30%HRP/0.1%DMSO was used in C and D. All sections were silver postintensified. In all panels, medial is left and lateral is right. Scale Bar: A, C, and D Å 80 mm; B Å 20 mm.

munohistochemistry. Each fixative solution was prepared with 0.1 M phosphate-buffered saline (PBS), and 500 ml were perfused through each animal, with the descending aorta clamped, and after flushing the vasculature with physiological saline. The brains were removed and placed in the same fixative solutions for 2 h before sectioning in the coronal plane with a Lancer Vibratome. The section thickness varied, depending on fixative; i.e., thinner with higher aldehyde concentrations, from 40 to 70 mm. The most sensitive visualization procedure used for HRP histochemistry and WGA immunohistochemistry included a silver

postintensification of the diaminobezidine endproduct from the peroxidase histochemistry [14]. For all HRP and WGA-HRP injected tissue, sections were washed in 0.1 M PBS for 1 h before histochemical development. Optimal peroxidase detection was accomplished with 15 min development in a substrate chromogen solution of 20 mg% 3-3-diaminobenzidine (DAB, Sigma), 75 mg% para-cresol (Fluka Chemical Co., Happauge, NY), 0.003% hydrogen peroxide diluted in 0.11 M ammonium acetate buffer pH 5.0 adjusted with citric acid [26]. A wash in PBS was used to stop development and, subsequently, the stained sections were

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WAYNER, PHELIX AND ARMSTRONG tomicroscope. In some cases direct prints of entire sections were produced to determine the extent of injection and LH and perforant path stimulation sites and to verify recording sites in the DG. These direct prints were also used to produce line drawings of representative sections for mapping of these data. Results and Discussion

FIG. 5. Direct print of a coronal section of rat forebrain through the level of the hypothalamic paraventricular nucleus. A large injection site with WGA-HRP appears white in the dorsal hippocampus ( H ) ; peroxidase endproduct was postintensified with silver. Angiotensin neurons were also stained except with nonintensified DAB chromogen and appear as white images in the hypothalamic paraventricular nucleus ( arrowheads ) . These paraventricular neurons did not contain any retrograde tracer. 3 — third ventricle; f — fornix; LH — lateral hypothalamus.

processed through a series of solutions to deposit metallic silver into the polymerized DAB within the HRP sites of the sections. This silver postintensification procedure has been described in detail elsewhere [23]. Another similarly sensitive visualization procedure for peroxidase activity detection was utilized as well. The substrate chromogen solution in this case was designed for simultaneous intensification of the DAB polymer in situ by including nickel ammonium sulfate and cobalt chloride [13]. This latter intensification protocol was used for double-label staining when HRP-DAB with nickel/cobalt was stained first, because it did not interfere with the subsequent immunocytochemical detection of angiotensin peptide. In order to detect angiotensin peptide in HRP-DAB labeled tissue, the sections were permeabilized with 0.2% Triton X-100 (Fluka) diluted in 0.1 M PBS for 1 h. After a brief wash in PBS the sections were then incubated in the primary antibody (rabbit antiangiotensin 1–7; kindly donated by C. M. Ferrario; the characterization of this antibody has been reported [2]) for 48 h at 47C. Incubations in the secondary biotinylated goat antirabbit IgG and then avidin–biotin–peroxidase solutions (Rabbit ABC Kit, Vector Laboratories, Inc., Sepulveda, CA) were preceded by 1 h washes in PBS and were performed at 47C for 48 h. A 2-h wash in PBS preceded the development in the substrate chromogen solution in the ammonium sulfate diluent with only DAB, as described above. In each case, some HRP-DAB–labeled sections were incubated in the standard antibody diluent as the omission control for the primary, before incubation with the subsequent reagents. This same immunohistochemical procedure was used to detect WGA with rabbit anti-WGA as the primary antibody (Vector), except the ammonium acetate chromogen substrate solution was used with the silver postintensification method. Subsequent to a PBS wash, sections were soaked in a 0.2% polyvinyl alcohol solution (Sigma) for 1 h before mounting onto glass microscope slides. Dried, mounted sections were cleared and coverslipped before photography with a Nikon Optiphot pho-

Ten animals received large injections with either 30% HRP– 0.1% DMSO or WGA-HRP. These provided the greatest sensitivity for detecting the maximal number of retrogradely filled neurons, revealing extensive dendritic branches and axons within pathways (Fig. 4). These large injections virtually filled the entire dorsal hippocampus with even some diffusion into the contralateral side (Fig. 5). This approach allowed mapping of the LH neurons that project into the entire dorsal hippocampus and the pathways, described below. Although some contralateral connections were observed, consistent with other reports [9,11], only the data for the side ipsilateral to the injections are shown. With these injections, LH somatodendrites were so completely filled with retrograde tracer that a second stain for angiotensin peptide could not be detected within them, even though within the same sections, angiotensin staining was easily visualized within paraventricular neurons that did contain retrograde tracer (Fig. 5). An additional 15 animals received smaller injections that were either restricted to the dorsal CA1 with some diffusion into the dentate gyrus, n Å 4 with WGA, or filled most of the dentate gyrus, in the coronal plane, as well as including some of the CA1, n Å 6 with WGA and n Å 5 with 15% HRP (Fig. 6A and B). These smaller injections had limits of diffusion ranging from 100 to 300 mm. Five animals served as control injections within the overlying cortex or within the dorsal subiculum located caudal to the typical recording site. Retrogradely filled hypothalamic neurons were located in the perifornical region (Fig. 4A), lateral to the fornix (Fig. 4B), in the ventrolateral LH (Fig. 4C), within the central LH (Fig. 4D), and directly ventral to the fornix (Fig. 7A), in all cases where the injections had significant spread into the dentate gyrus. The fewest hypothalamic neurons, i.e., typically three to five per section and scattered in the same hypothalamic regions, were observed when injections were centered within the CA1 region, as shown by the darker shading in Fig. 6B. Retrogradely filled neurons were also observed in other areas known to project into the dorsal hippocampus, for example, the thalamus, entorhinal and perirhinal cortex, and dorsal and median raphe nuclei (data not shown). With control injections, no retrogradely filled neurons were observed in the hypothalamus. Hippocampal projecting neurons were located in the hypothalamus extending from the level of the caudal suprachiasmatic nucleus, anteriorly; and to the mammillary region, posteriorly. When injections were restricted primarily to the dentate gyrus, similar to recording site locations (Fig. 6A and B and Fig. 8A), retrogradely filled hypothalamic neurons were located mostly in the more anterior and middle hypothalamic levels (Fig. 6C, D, and E). A significant cluster of hypothalamic neurons projecting to the dentate gyrus was observed at the level of the hypothalamic paraventricular nucleus, (Fig. 6D and E), where significant angiotensin peptide staining neurons could be found. This location also correlated with the most effective stimulation sites in the LH (Fig. 3B). Two morphologically distinct hypothalamic neuronal types were detected by the retrograde tracers. Some fusiform neurons (Figs. 4A and 8B) were found in both perifornical regions and within the LH. Multipolar neurons were distributed extensively within each hypothalamic region projecting into the hippocam-

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FIG. 6. Summary of retrograde tract tracing results for angiotensin neurons in the lateral hypothalamus (LH). (A, B) Examples of size and locations of injections in the dorsal hippocampus corresponding to recording sites in LTP experiments. (C, D, E) Representative cross-sections through the forebrain showing the distribution of retrogradely labeled neurons, solid dots, and those which also contained angiotensin, encircled solid dots. Note that the hypothalamic neurons with projections into the dorsal hippocampus are located predominantly in the LH and perifornical regions.

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FIG. 7. Examples of double-labeled, HRP – angiotensin LH neurons in the region ventral to the fornix ( A ) , lateral to the fornix ( B ) , and in the central LH ( C ) . ( A, B ) Fusiform angiotensin neurons have an unstained nucleus ( n ) and contain HRP vesicles in the cytoplasm of both soma ( arrow ) and dendrite ( arrowhead ) . ( C ) A multipolar angiotensin neuron has HRP vesicles in its soma ( small arrows ) and proximal dendrite ( arrowhead ) . Note that an angiotensin axon appears to contact the dendrite of a neighboring HRP filled neuron ( large arrow ) , that also projects to the hippocampus. Angiotensin neurons that do not project to the hippocampus surround the asterisk in B. Small HRP injections were performed in each case. Dorsal is to the top and lateral is to the right in each panel. Scale Bar: 20 mm.

pus (Fig. 4). With the more sensitive tracer detection methods, extensive dendritic trees were observed with multiple branches (Fig. 4), and in many cases these dendrites extended far into the neuropil away from the soma. Also, with the sensitive detection methods axonal pathways were found within the ventral amygdalofugal pathway (Fig. 4C), and in the medial forebrain bundle (Fig. 4D). This latter pathway passed rostrally within the ventral forebrain and ultimately into the septum where it continued dorsally, at rostral septal levels, into the fornix and transcallosal pathways terminating within the hippocampal injection sites. The tracer filled axons within the ventral amygdalofugal pathway continued caudally through the amygdala, where axons were also

observed within the fimbria fornix at the junction of dorsal and ventral hippocampus. An additional 15 animals, receiving injections of WGA-HRP, WGA, or HRP, were used to assess the double stain immunohistochemical procedure. Optimal codetection of retrograde tracer and angiotensin peptide was accomplished with utilization of the 3% (para)formaldehyde–0.5% glutaraldehyde fixative and small injections of 15% HRP. When testing the parameters and WGA-HRP injected tissue was fixed with 4% (para)formaldehyde, areas of highest concentration of WGAHRP, i.e., injection site, and of angiotensin peptide, i.e., hypothalamic paraventricular nucleus, stained intensely (Fig. 5).

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FIG. 8. Color bright-field photomicrographs of sections that were nickel/cobalt intensified for HRP and double stained with angiotensin antibody using nonintensified DAB chromogen. (A) Small injection in dorsal dentate gyrus (DG) of 15% HRP, 100 nl, demonstrates filling of DG with intense HRP uptake at the granule cell layer (open arrows) of the suprapyramidal blade, surrounding the tip placement of the micropipette injector. Note that all three layers including the infrapyramidal blade and the

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Areas of lower concentrations, for example, the LH, did not show staining that could be detected with other fixatives. Lower formaldehyde concentrations with some glutaraldehyde were essential to maintain a balance of HRP enzyme activity in situ and angiotensin peptide immunoreactivity. However, neither were optimal when compared with other single staining procedures. With the smaller WGA injections, the glutaraldehyde minimized in situ WGA immunoreactivity and precluded its use with these double label investigations. In five animals that received small injections of 15% HRP that were centered in the dentate gyrus (Fig. 8A), both retrogradely filled neurons and angiotensin containing neurons (Fig. 8B) were detected in the anterior and middle hypothalamic levels throughout the regions described above. However, due to the methodological compromise made with the fixative, apparently less staining was observed overall. The HRP stain was a purple/ black color due to the nickel/cobalt intensification, and the angiotensin immunoreactivity was brown due to the nonintensified DAB chromogen. Some of the hypothalamic neurons that projected to the dentate gyrus were observed to contain angiotensin peptide (Fig. 7). In these cases, the HRP stain appeared as punctate dark vesicles within the cytoplasm of soma and dendrites that also had brown diffuse cytoplasmic staining for angiotensin (Fig. 7). The distribution of these double labeled neurons is shown diagrammatically in Fig. 6. Typically, there were less than 10 double-labeled neurons per section and they tended to cluster at the level of the hypothalamic paraventricular nucleus (Fig. 6D and E). Angiotensin neurons that projected to the dentate gyrus also were either fusiform or multipolar in shape (Fig. 7). Angiotensin staining was also observed in axons within the hypothalamus, but due to subdued in situ enzyme detection, HRP containing axons were not observed. CONCLUSIONS These results demonstrate clearly (a) that electrical stimulation of the LH inhibits LTP induction in medial perforant path– granule cell synapses; (b) that the inhibition is mediated by Ang II and the AT 1 receptor; (c) the AT 1 receptor can be blocked and the inhibition prevented by pretreatment with losartan, a specific AT 1 antagonist; (d) some of the LH neurons that stain for angiotensin project to the hippocampus and they can be back filled with retrograde tracers applied to the granule cell layer; (e) the double-labeled neurons containing angiotensin and HRP are sparsely distributed, less than 10 per section, and tend to cluster laterally and ventrally to the fornix at the level of the paraventricular nucleus; (f ) angiotensin neurons that projected to the dentate gyrus were either fusiform or multipolar in shape; and (g) large numbers of angiotensin staining neurons were observed in the hypothalamus. ACKNOWLEDGEMENTS

This research was supported by a grant from The Council for Tobacco Research–U.S.A., Inc., #4038, and a Slick Fellowship in Molecular Biology and HL02914 to C. F. Phelix. The losartan was generously provided by Dr. Ronald D. Smith, the Du Pont Merck Pharmaceutical Co., Wilmington, DE. Technical assistance was provided by Barbara Smith,

M. E. Burton, J. DeLeon, L. Gutierrez, D. Gutierrez, R. Rizo, and J. C. Turner, who were supported by The University of Texas Division of Life Sciences, Howard Hughes Medical Institute Support for Undergraduate Research Training, and a Minority High School Summer Research Apprentice Program. We appreciate the help of Marianne Van Wagner in typing the manuscript.

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flexure of the DG show significant HRP uptake. Very little HRP uptake occurred in the CA1 at the injector tract and diffusion of HRP within the DG was limited dorsally at the stratum lacunosum molecular where asterisks mark blood vessels. (B) HRP–angiotensin double-labeled section of LH from same animal as in A. In bottom right, purple/black-colored HRP vesicles can be seen in the soma (small arrows) and dendrites (small arrowheads) of retrogradely filled LH neurons. In top left, brown-colored angiotensin immunoreactivity is seen diffusely distributed throughout the soma (large arrows) and dendrites (arrowheads) of neurons that do not project to the hippocampus. Note that the nucleus (n) does not stain in either case. Angiotensin axons are also seen within this field. Scale bars: A Å 500 mm; B Å 20 mm.

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