NMDA receptors in the midbrain periaqueductal gray mediate hypothalamically evoked hissing behavior in the cat

BRAIN RESEARCH ELSEVIER

Brain Research 762 (1996) 80-90

Research report

NMDA receptors in the midbrain periaqueductal gray mediate hypothalamically evoked hissing behavior in the cat K. Schubert l, M.B. Shaikh, A. Siegel * Department of Neurosciences, Medical Science Building, H-512, New Jersey Medical School, 185 South Orange Al~enue, Newark, NJ 07103, USA

Accepted 13 February 1996

Abstract The present study was designed to test the hypothesis that the descending pathway from the medial hypothalamus to the dorsal periaqueductal gray (PAG) is critical for the expression of defensive rage behavior in the cat and utilizes excitatory amino acids as a neurotransmitter. In the first phase of the study, monopolar stimulating electrodes were implanted into the medial hypothalamus from which defensive rage behavior could be elicited by electrical stimulation. For the entire study, the hissing response was used as a measure of defensive rage behavior. Cannula electrodes were implanted into the PAG from which defensive rage sites could be identified and were later used for microinfusion of the NMDA receptor antagonist, DL-2-amino-7-phosphoheptanoic acid (AP-7), into behaviorally identified sites within the PAG. Initially, intracerbral microinjections of the NMDA receptor antagonist, AP-7 (0.2, 2.0 nmol), which were placed directly into sites within the PAG from which defensive rage had been elicited, blocked the occurrence of hypothalamic hissing. Microinjections of similar doses of AP-7 into the PAG also blocked the facilitatory effects of medial hypothalamic stimulation upon hissing behavior elicited from the PAG. However, microinjections of 2 nmol into the PAG had no effect upon hissing that was also elicited from the region of the injection site. This finding indicates that AP-7 selectively blocks hissing elicited from the medial hypothalamus and that the suppressive effects of AP-7 cannot be the result of anesthetic or other nonselective properties of the drug. The next phase of the study, which employed immunohistochemical, receptor autoradiographic techniques, identified NMDA receptors to be present in highest concentrations in the dorsolateral aspect of the PAG where defensive rage is typically elicited. The final phase of the study, which employed a combination of retrograde labeling procedures following microinjections of Fluoro-Gold into defensive rage sites in the dorsal PAG and the immunocytochemical labeling of glutamatergic neurons, identified large numbers of neurons in the medial hypothalamus that were labeled positively for both Fluoro-Gold and glutamate. The overall findings of this study support the hypothesis that descending fibers of the medial hypothalamus that supply the dorsal aspect of the PAG mediate defensive rage behavior and utilize excitatory amino acids that act upon NMDA receptors within the dorsal PAG. Keywords: Defensiverage behavior; Medial hypothalamus;Midbrainperiaqueductalgray; Excitatory amino acid; Receptor binding; Immunocytochemical labeling; NMDA receptor

1. Introduction It is now well known that the medial hypothalamus constitutes a key structure for the integration of defensive rage behavior in the cat [13,14,16,19,22,25,28]. It is also well established that major descending projections of the medial hypothalamus include the midbrain periaqueductal gray (PAG), lateral, central and ventral tegmental fields of the midbrain and lower brainstem [2,11-14,19,20]. Although any one or more of these pathways may serve as

* Corresponding author. Fax: (I) (201) 982-5059. Present address: Department of Biology, Kean College, Union, NJ 07083, USA. (1006-8993/96/$15.00 Publishedby Elsevier Science B.V. PII S0006-8993(96)0026 1-2

descending substrates for defensive rage behavior, an increasing body of literature has suggested that the projection to the PAG may be the critical one for the expression of this form of aggressive behavior. Support for this view is derived mainly from the observations that: (1) defensive rage behavior is elicited readily by electrical or chemical stimulation of both the medial hypothalamus and PAG [2,10,19,21,24,31] and (2) the largest component of descending fibers from defensive rage sites in the medial hypothalamus project to the dorsal aspect of the PAG [13,14]. Recently, our laboratory has provided initial evidence that stimulation of the medial hypothalamus can facilitate the occurrence of defensive rage behavior elicited from the

K. Schubert et al. / Brain Research 726 (1996) 80-90

PAG and that the anatomical substrate for such facilitation is the projection from the medial hypothalamus to the PAG [19,23]. In addition, this study provided preliminary data suggesting that hypothalamic modulation of PAG elicited defensive rage is mediated by excitatory amino acids (EAA) that act upon NMDA receptors. That functional relationships between the medial hypothalamus and PAG may be mediated by EAA is also suggested by other studies conducted in rodents [5-8]. In view of the importance of the descending pathways from the medial hypothalamus in eliciting defensive rage, the present study was undertaken in order to provide further support for the hypothesis that NMDA receptors are essential for this response and that the pathway mediating its expression involves fibers that project from the medial hypothalamus directly to the PAG.

2. Materials and methods A total of 10 adult cats of either sex, weighing between 3.0 and 4.0 kg were utilized in this study. The animals were individually housed and had free access to food and water throughout the course of all experiments. A detailed description of the general surgical and pharmacological methods used in this experiment are similar to those described elsewhere [19,25-27]. During aseptic surgery, animals were deeply anesthetized with Isoflurane (1%). Sixteen stainless steel guide tubes (17 gauge, 10 mm in length) and filled with bone wax, were then stereotaxically mounted bilaterally according to the atlas of Jasper and Ajmone-Marson [17] and cemented over holes drilled through the skull overlying the medial hypothalamus and dorsal midbrain PAG. Three stainless steel stylets attached to the skull served as indifferent electrodes. Three bolts were anchored to the skull with dental acrylic and were attached to a plastic safety cap that protected the entire assembly.

2.1. Elicitation of defensive rage behauior In the present study, several different experimental paradigms were employed and are described in detail below. One set of experiments utilized single stimulation of either the medial hypothalamus or PAG from which defensive rage behavior was elicited and the effects of intracerebral infusion of drugs upon defensive rage behavior from either of these two brain regions were determined over a postinjection test period. A second set of experiments sought to determine the effects of drug infusion upon medial hypothalamic facilitation of defensive rage behavior elicited from the PAG. One week following surgery, freely moving animals were placed in a wooden observation chamber (61 X 61 × 61 cm) with a clear Plexiglas door. The guide tubes permitted vertical insertion of stimulating electrodes into the medial hypothalamus and

81

cannula electrodes into the PAG for the elicitation of defensive rage in both brain regions, as well as for microinjections of AP-7 or saline controls into the midbrain PAG. The cannula electrodes (27 gauge, 50 mm long, (Plastics One, Inc., Roanoke, VA) were calibrated and insulated with an oil-base paint, except for 0.5 mm at the tip of the electrode. In order to find a reliable defensive rage response, electrodes were lowered through guide tubes in 0.5 mm increments, and stimulation was applied to either the PAG or medial hypothalamus. Stimuli consisted of rectangular electrical pulses (0.05-0.9 mA, 62.5 Hz, 1 ms half cycle duration). Electrical stimulation was generated by Grass S-88 stimulators that were connected through constant current units (Grass SIU6) to the cat. A Tektronix 5000 series oscilloscope was used to monitor peak-to-peak current. For studies involving paired trials of dual stimulation, biphasic pulses were separated by 4 ms. The duration of stimulation was limited to 15 s. If the response could not be elicited within 15 s, a response latency score of 15 s was recorded for that trial even though stimulation was not effective; and when the response was elicited within the 15 s time frame, stimulation was terminated immediately following the response, and the response latency was then recorded. When a response was repeatedly elicited in a stable manner within 15 s following the onset of stimulation, electrodes were cemented in place with dental acrylic. The latency of the hissing response, which was used as a standard measure of defensive rage, was defined as the duration of time required for the animal to exhibit the hissing response following the onset of stimulation [19,27,28]. The rationale for using hissing as a standard measure of defensive rage is described elsewhere [19]. In brief, hissing constitutes an integral part of the defensive rage response and occurs whenever defensive rage is elicited. It should also be pointed out that other forms of vocalization can also be elicited from the PAG of the cat. These responses include 'howling' and 'growling' which can also be associated with threat displays. However, these responses are typically elicited following stimulation of more caudal aspects of the PAG [3]. In all experiments, a 2-min intertrial interval was employed.

2.2. Medial hypothalamic facilitation of hissing elicited from the PAG The following procedures were employed to identify sites in the medial hypothalamus from which modulation of PAG-elicited hissing could be obtained. Electrodes were lowered vertically through guide tubes overlying the medial hypothalamus. Then, electrical stimulation was applied as the electrode was further lowered in 0.5 mm steps concurrently with stimulation of sites within the dorsal PAG from which hissing was elicited. Stimulus parameters applied to the medial hypothalamus were identical to those applied to the PAG except for current strength, which was

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maintained at subthreshold levels (i.e. the level of current applied to the medial hypothalamus alone which could not elicit hissing). Paired trials consisted of single stimulation of the PAG from which defensive rage was elicited and dual stimulation of the medial hypothalamus and the PAG. In order to provide a counterbalanced design for administration of trials of single and dual stimulation, paired trials were administered in an A-B-B-A manner, where A represented single stimulation and B represented dual stimulation. Response latencies for single stimulation of the PAG and dual stimulation of the medial hypothalamus and the PAG were compared. For all paired trials, current intensities for the medial hypothalamus and the PAG were held constant.

2.3. Drugs and drug administration In the next phase of the experiment, the selective N M D A receptor antagonist, oL-2-amino-7-phosphoheptanoic acid (AP-7) (Research Biochemicals, Natick, MA), was microinjected into the dorsal PAG from which defensive rage could be elicited in doses 2.0, 0.2 nmol (pH = 7.4) or saline (pH = 7.4), acting as a vehicle control with the aid of an SGE (0.5 Ixl) microsyringe. These dose levels were selected on the basis of findings obtained from a previously published paper in our laboratory involving intracerebral administration of AP-7 [19]. All experiments using drug administration were only carried out following a pre-test period of 3 days over which time the defensive rage response remained stable (i.e. there was little or no variation in response latencies for three consecutive days). AP-7 was dissolved in sterile saline and was freshly prepared immediately prior to each experiment. The order of all drug doses was determined by using a table of randomized numbers.

2.4. Microinjections of AP-7 into the PAG and hypothalamically elicited hissing In the first experiment, five animals were utilized to determined the effects of intracerebral injections of AP-7 into the PAG upon hissing behavior elicited from the medial hypothalamus. Prior to drug administration, baseline response latencies following medial hypothalamic stimulation were initially determined. Then, utilizing a 0.5 txl SGE syringe, microinjections of AP-7 were administered through a cannula electrode into the dorsal PAG (from which hissing could be elicited) at two doses (2.0 and 0.2 nmol/0.25 ~zl and vehicle). Response latencies were again determined over a 180 min, postinjection time period.

2.5. Microinjections of AP-7 into the PAG and modulation of PAG elicited hissing from the medial hypothalamus The second experiment represented an alternative approach by which the mediating role of NMDA receptors

upon medial hypothalamic inputs into the PAG was assessed. This experiment differed from the previous ones in that it utilized a design which compared the effects of NMDA receptor blockade upon medial hypothalamic modulation of PAG elicited hissing. In this experiment, five animals were employed to determine the effects of intracerebral injections of AP-7 into PAG sites (where hissing could be elicited) upon medial hypothalamic modulation of hissing elicited from the PAG. Sites in the medial hypothalamus used for modulation were ones from which hissing could also be elicited when stimulation was applied at supra threshold levels. In the course of dual stimulation, modulating currents were always maintained at levels below threshold for elicitation of hissing behavior. Initially, 10 paired trials of single stimulation of the PAG and dual stimulation of the PAG and medial hypothalamus were administered. Response latencies for hissing were determined after each trial. Paired trials of single stimulation were compared with those of dual stimulation. A minimum change in response latency of 30% following dual stimulation relative to single stimulation of the PAG was used as a criteria for identification of significant modulation. AP-7 (2.0, 0.2 nmol/0.25 txl) or saline (pH = 7.4, vehicle control) was microinjected into a site within the dorsal PAG from which defensive rage could be elicited upon electrical stimulation. Response latencies for single and dual stimulation were again compared for a period up to 180 min, postinjection.

2.6. Microinjections of AP-7 into sites within the PAG from which hissing was elicited The third experiment sought to determine whether the suppressive effects of AP-7, when microinjected into the PAG were the result of non-specific (e.g. anesthetic) properties of the drug. For this experiment, five animals were employed. The paradigm was identical to that employed for the analysis of the effects of AP-7 into the PAG upon hypothalmically elicited hissing described above with the exception that the injection and stimulation sites, located within the PAG, were the same and that only the higher dose of AP-7 (2 nmol) was employed.

2.7. Receptor immunohistochemistry In this phase of the project, immunohistochemical techniques were employed to identify the presence of NMDA receptors in the dorsal PAG. Following the completion of pharmacological experiments, four animals were perfused transcardially with 1% paraformaldehyde, followed by 4% paraformaldehyde, under deep anesthesia. Brains were removed and placed in 4% paraformaldehyde for 2 h at 4°C and then in 10% sucrose solution for 8 h. The brain at the level of the PAG was then blocked and sectioned on a vibrotome at 40 i~m. Tissue was washed with phosphate buffer (4 times at 10 min each) on a rotary shaker, and

K. Schubert et al./ Brain Research 726 (1996) 80-90

preincubated with 10% normal goat serum (Vector Labs, CA) for 30 min at room temperature. Sections were then reacted with the purified monoclonal antibody against the NMDA-R1 receptor subtype (Pharminogen, CA) at a 1:1000 dilution in phosphate buffer for 48-72 h at 4°C. This antibody was produced against a protein corresponding to NMDA-R1 residues 660-811 representing the intracellular loop between transmembrane regions III and IV and does not react with NMDA-R2-5 subunits [30]. After four washes, sections were incubated in a 0.3% H202 solution in phosphate buffered saline (for 20 min) at room temperature in order to block endogenous peroxidase. Sections were then washed (4 times for 10 min each) and processed with a species specific Vectastain ABC Elite immunoperoxidase kit (Vector Labs, CA). After four more washes, the tissue was then stained with 3,3-diaminobenzidine tetrahydrochloride reactant (DAB) in phosphate buffered saline (Vector Labs, CA) for up to 5 min. Sections from each brain were reacted with DAB in the absence of the antibody as a control. The sections were finally transferred to a buffer wash (4 times for 10 min) and mounted on gelatin coated slides. Tissue was air-dried overnight, dehydrated over graded alcohols (50-100% alcohol) for 2 min each, and put in two separate changes of xylene (2 min each). The slides were then coverslipped with Permount and photographed by an Olympus BH2 photo microscope.

83

phosphate, pH 7.4) 3 times for 5 min each. Sections were then incubated with primary rabbit anti-glutamate antibody at a 1:8000 dilution (The Arnel Products Co., NY) in Hartman"s buffer containing 0.001% thimerosal and 0.3% Triton at 4°C. After 48 h, the tissue was again washed with Hartman"s buffer (3 times for 5 min each). The tissue was then reacted with anti-rabbit IgG, Texas Red Conjugated secondary antibody made in goat (Cappel, Westchester, PA) at a 1:50 dilution. After 60 min, the sections were washed in Hartman"s buffer (3 times for 5 rain), mounted on subbed slides, dried, and coverslipped with DPX mountant. The following controls were used to demonstrate the specificity of staining: (1) the secondary antibody (antirabbit IgG Texas Red Conjugated) was reacted with the tissue while omitting the primary antibody; and (2) the primary antibody (rabbit anti-glutamate) was preabsorbed with excess amounts of unlabeled glutamate (1 mm) which, then was reacted with the tissue and processed as described above. The sections used for immunocytochemistry, retrograde labeling or double labeling were identified and photographed with the aid of an Olympus BH-2 microscope with fluorescence illumination (UV filter: excitation wavelength = 323 rim, emission wavelength = 408 nm; filter for Texas red conjugated antibody: excitation wave length = 554 nm, emission wavelength = 573 nm). 2.9. Statistical analysis o f data

2.8. Neuroanatornical studies

The purpose of this phase of the study was to determine whether neurons situated in the anterior medial hypothalamus (from which defensive rage is typically elicited) contain: (1) immunopositive cells for glutamate and (2) project to the dorsal aspect of the PAG where defensive rage can be elicited. Upon completion of the pharmacological studies, all animals were microinjected with the retrograde axonal tracer, Fluoro-Gold (Fluorochrome Inc., Englewood, CO) into defensive rage sites within the PAG. Utilizing an SGE syringe, Fluoro-Gold (8%, dissolved in sterile water) was microinjected in a volume of 0.5 txl over a period of 20 min. After a survival period of 5 - 7 days, animals were perfused transcardially under deep anesthesia (sodium pentobarbital, 45 m g / k g ) with phosphate buffered saline and 4% paraformaldehyde (4°C). Brains were postfixed with 10% sucrose solution and sectioned at 40 txm on a freezing microtome. Alternate sections were mounted on gelatin coated slides, air dried, cleared with xylene, and coverslipped with DPX mountant. Additional sections were stained with cresyl violet for histological verification of electrode and cannula-electrode tips in the medial hypothalamus and PAG, respectively. As described elsewhere [19,27], immunocytochemical techniques were employed to identify glutamate positive neurons within the medial hypothalamus. The remaining sections were washed in Hartman"s buffer (i.e. potassium

A 3 × 4 repeated measures Analysis of Variance (ANOVA) was employed to determine the level of significance of changes in response latencies for hissing following separate doses of AP-7 or saline, (variable A) over four different periods of time, postinjection (variable B). For such tests, predrug baseline response latencies were given a value of 0% change in response latencies in order for comparisons to be made of the effects of drug administration upon medial hypothalamic elicitation or modulation of defensive rage. In addition, one-way ANOVAs were employed to compare response latencies following hypothalamic stimulation or modulation prior to and following AP-7 treatment for each dose over four periods of time, postinjection. Further, t-tests were used to determine the level of significance of the changes in response latencies between paired trials of single and dual stimulation prior to drug administration as well as between different points in time following a given dose of drug or vehicle.

3. Results

Defensive rage is characterized by a number of sympathetic and behavioral signs such as arching of the back, pupillary dilatation, piloerection, retraction of the ears, growling, hissing, and paw striking at a moving object. As described previously [4,9,13,14,16,19,24,29,32], defensive

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produced significant suppression of defensive rage response over time ( F = 6.64; df = 3,12; P < 0.005). At this dose, AP-7 significantly blocked defensive rage for up to 120 min postinjection, and returned to baseline at 180 min. The maximal effect was observed at 5 rain, postinjection, where drug infusion into the PAG resulted in 54% suppression of response latency values (t = 5.14, df = 3,12; P < 0.01). However, neither the lowest dose of AP-7 (0.2 nmol) nor saline microinjections significantly altered response latencies for hissing ( F = 1,23, df = 3,16, P > 0.3 and F = 0.73, df = 3,16, P > 0.5, respectively).

3.2. Effects of microinjections of AP-7 into the PAG upon medial hypothalamic modulation of hissing elicited ,from the PAG

Fig. 1. Maps of sites in the (A) medial hypothalamus from which hissing was elicited, and (B) PAG from which hissing was elicited and where AP-7, saline and Fluoro-Gold ( O ) were microinjected. Abbreviations: CI, internal capsule; Fx, fornix; HL, lateral hypothalamus; MH, medial hypothalamus OT, optic tract; PAG, midbrain periaqueductal gray; and RE, nucleus reuniens.

rage, which can be elicited reliably by electrical stimulation of the medial hypothalamus and by electrical or chemical stimulation of the dorsal PAG, mimics closely the response pattern that occurs under natural conditions when an animal is threatened by another animal of the same or different species [18]. In the present experiment, this response was elicited from the medial hypothalamus (Fig. IA) as well as from the dorsal half of the rostral third of the PAG (Fig. 1B). As indicated above in the Methods section, the hissing component of the defensive rage response was used throughout the study as a measure of defensive rage.

This experiment attempted to replicate the findings of a previous study [19] that examined the role of N M D A receptors in medial hypothalamic modulation of hissing elicited by the PAG (n = 5). The first part of this experiment tested the hypothesis that medial hypothalamic stimulation facilitates defensive rage elicited from the PAG. Dual stimulation of the medial hypothalamus and the PAG facilitated the occurrence of hissing from the PAG (t = 8.271, df = 20, P = 0.01) (Fig. 3). Here, dual stimulation resulted in a mean decrease of 50% in response latencies relative to those obtained by single stimulation of the PAG alone ( P = 0.001). The second part of this experiment sought to determine whether intracerebral administration of AP-7 directly into defensive rage sites within the PAG could also block the facilitating effects of medial hypothalamic stimulation of this response. Following administration of AP-7 at a dose

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3.1. Effects of microinjections of AP-7 into defensiL, e rage sites within the PAG upon hissing elicited from the medial hypothalamus This phase of the study was designed to test the hypothesis that N M D A receptors in the dorsal PAG are critical for the expression of hissing elicited from the medial hypothalamus. Infusion of AP-7 into the dorsal PAG at sites which, upon electrical stimulation, produced defensive rage behavior, resulted in a highly significant overall dose ( F = 52.64, df = 2,8, P < 0.001) and time ( F = 3.48, df = 3,12, P < 0.05) dependent blockade of hypothalamically elicited hissing (n = 5). Moreover, the blocking effect of local AP-7 administration upon hissing was also manifest by the significant interaction term of the A N O V A (i.e. dose X time) ( F = 4.82, df = 6,24, P < 0.001). As indicated in Fig. 2, the higher dose of AP-7 (2 nmol)

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Fig. 2. Effects of microinjections of AP-7 into the PAG upon hissing elicited from the medial hypothalamus. AP-7 suppressed hissing elicited from the medial hypothalamus as indicated by an increase in response latency. For this figure, baseline represents the mean predrug response latency obtained by medial hypothalamic stimulation. A value of 0% was assigned to this level of response in order to determine the effects of drug administration upon hissing elicited from the medial hypothalamus. Note that the maximum dose of AP-7 (2.0 nmol) was most effective during the 40- rain test period beginning at 5 rain, postinjection. However, neither saline nor the lowest dose of AP-7 (0.2 nmol) significantly altered hissing elicited from the medial hypothalamus (n = 5).

K. Schubert et al. / Brain Research 726 (1996) 80-90

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of 2 nmol, a significant reduction in response facilitation associated with dual stimulation was noted. Here, a maximum decrease of 50% in response facilitation was observed at 5 min, postinjection ( F = 29, d f = 3,12, P < 0.02). This decrease in response facilitation approached baseline values by 180 min, postinjection (Fig. 4). Neither the lower dose of AP-7 (i.e. 0.2 nmol) nor saline had any effect upon medial hypothalamic facilitation of hissing elicited from the P A G ( F = 1.27, df = 3,12, P = 0.33; and F = 0.73, df = 3,12, P = 0.55, respectively).

3.3. Effects of microinjections of AP-7 upon PAG elicited defensive rage The question was raised whether suppression of hissing following microinjections of AP-7 into the PAG was the result of a non-selective effect of this drug such as possible anesthetic properties that it may possess. This issue was addressed by observing the effects of microinfusion of 2.0 nmol of AP-7 into the PAG upon defensive rage elicited from the PAG. The results of this experiment, shown in Fig. 5, indicate that local microinfusion of AP-7 did not alter the latency for hissing elicited from the P A G over a 60 min postinjection test period (one-way A N O V A comparing response latencies over time: ( F = 0.35, df = 2,12, P = 0.71). Although not indicated in Fig. 5, current thresholds for elicitation of defensive rage also remained constant in spite of drug infusion. This finding indicates that, when microinjected into the PAG, AP-7 selectively blocks hissing elicited from the medial hypothalamus but has no effect upon this response when elicited from the PAG.

3.4. Distribution of NMDA-R1 receptors in the PAG The data described in this section utilized immunohistochemical methods to demonstrate the presence of glutamate receptors in the rostral half of the dorsal aspect of the PAG associated with the expression of defensive rage response. NMDA-R1 immunoreactivity was present in cell

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Fig. 4. Effects of microinjections of AP-7 (2 nmol) into the PAG upon medial hypothalamic facilitation of defensive rage elicited from the dorsal PAG. AP-7 blocked the facilitating effects of medial hypothalamic stimulation upon hissing (upper panel). Here, a value of 0% change is given to the baseline level of response facilitation established prior to drug infusion in order to depict the effects of AP-7 administration upon medial hypothalamic modulation of hissing. Thus, any reductions in response facilitation following AP-7 administration are expressed as 'positive' percent changes. Note that microinjections of saline did not have a significant effect upon medial hypothalamic facilitation (lower panel,

n= 5).

bodies located in the dorsal aspect of the PAG (Fig. 6 A - C , E ) , which receives primary inputs from defensive rage sites in the medial hypothalamus [13] and from which defensive rage can be elicited upon electrical or chemical stimulation [24]. It should be noted that NMDA-R1 labeling over cells in adjoining regions of the ventral PAG was

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Fig. 6. Photomicrographs shown in panels A, B, C and E indicate the presence of NMDA-RI immunopositivereceptors which are distributed within the dorsal PAG. Arrows shown in panel A indicate the region of the dorsal PAG where immunopositive receptors were identified. Panel D demonstrates absence of staining in the dorsal PAG following treatment with DAB alone. Also note that few NMDA-RI immunopositivereceptors were present within the ventral PAG (panel F).

considerably lower than that observed in the dorsal PAG (Fig. 6F). As a control procedure, D A B staining in the absence of the N M D A - R 1 antibody did not reveal the presence of any immunoreactive neurons in the dorsal PA G (Fig. 6D). 3.5. N e u r o a n a t o m i c a l

observations

As noted above, microinjections of Fluoro-Gold were placed through cannula electrodes situated within the dot-

sal P A G from which defensive rage could be elicited by electrical stimulation. A photomicrographic montage of a typical Fluoro-Gold injection site placed into the P A G is shown in Fig. 7. A line drawing depicting such an injection site is also presented in Fig. 7. This injection site, as well as nine others, were confined to the dorsal aspect of the rostral half of the PAG and did not extend to other regions of the tegmentum beyond the PAG. The majority of retrogradely labeled neurons within the hypothalamus were identified at the level of the anterior half of the

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100 um Fig. 8. Photomicrographs indicate: neurons double labeled for (A) Fluoro-Gold and (B) glutamate within the medial hypothalamus following microinjections of Fluoro-Gold into the PAG; and (C) cells retrogradely labeled with Fluoro-Gold within the medial hypothalamus that (D) failed to react positively for glutamate.

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K. Schubert et al. / Brain Research 726 (1996) 80-90

Fig. 9. Maps illustrating the distribution of glutamate immunoreactive cells (closed circles), retrogradely labeled neurons with Fluoro-Gold ( © ) and neurons double labeled for both glutamate and Fluoro-Gold (stars). Note that double labeled neurons were identified within the medial hypothalamus (n = 5). Abbreviations: Ch, optic chiasm; CI internal capsule; Fx, fornix; HL, lateral hypothalamus, HP, posterior hypothalamus; HVM, ventromedial nucleus of hypothalamus; RE, nucleus reuniens.

double-labeled for both Fluoro-Gold and glutamate were present within the medial hypothalamus are shown in Fig. 8A,B. The greater majority of these cells was situated rostral and slightly dorsal to the ventromedial nucleus, but some double-labeled neurons were also noted in the ventromedial nucleus as well. Control studies omitting or preabsorbing the primary antibody (anti-glutamate) did not reveal the presence of any immunoreactive cells. Maps indicating the distribution of Fluoro-Gold and glutamate positive cells are depicted in Fig. 9.

4. Discussion The results of the present study point to the importance of the pathway from the medial hypothalamus to the midbrain PAG as one that is critical for the expression of defensive rage [12-14]. In essence, both the medial hypothalamus and dorsal half of the rostral PAG may be viewed as regions that serve to integrate defensive rage behavior. It is likely that the PAG also constitutes the most caudal region of the neuraxis of the brainstem from which integration of defensive rage is organized [24]. Recently, it was shown that the PAG is functionally organized into a series of columns extending along its longitudinal axis [3]. One such column, situated in the dorsal aspect of the PAG, constitutes a primary region from which defensive rage and threat displays can be elicited. Medial hypothalamic efferent fibers which mediate defensive rage behavior project approximately to the rostral half of this column [13,14]. Accordingly, it would appear that the rostral half of this column is critical for the integration and transmission of descending medial hypothalamic efferent fibers associated

with the expression of defensive rage behavior. However, it remains to be determined if the caudal aspect of this column may nevertheless be involved in the integration of defensive rage behavior. In particular, we do not know whether neurons situated in the caudal aspect of this column are activated indirectly from the medial hypothalamus, or whether they play little or no role in mediating defensive rage behavior elicited from this region of hypothalamus. Further studies are obviously needed to clarify this issue. In a recent study, evidence was presented that one function of the pathway linking the medial hypothalamus with the PAG is that it forms the substrate by which the medial hypothalamus can facilitate the occurrence of defensive rage organized at the level of the PAG [19]. That study further provided preliminary data, based upon pharmacological and immunocytochemical-anatomical observations, suggesting that such functions are mediated by EAA that act upon NMDA but not upon AMPA receptors within the PAG. The results of the present study thus both replicate and extend those initial findings. The present findings provide a body of new information indicating that the descending pathway from the medial hypothalamus to the PAG functions, not only to facilitate defensive rage at the level of the brainstem PAG, but that this pathway, indeed, serves as the substrate for the expression of this form of aggression. Moreover, the findings indicate that defensive rage is mediated by EAA that act upon NMDA receptors within the PAG. Several lines of evidence in support of this conclusion are based upon the following observations derived from the present study as well as related preliminary experiments from our laboratory.

K. Schubert et al. / Brain Research 726 (1996) 80-90

One piece of evidence was obtained from a preliminary study which showed that defensive rage behavior elicited from the medial hypothalamus could be blocked by peripheral administration of a selective N M D A receptor antagonist in a dose- and time-dependent manner [23]. While peripheral administration of a drug does not permit one to draw conclusions concerning functional localization within the brain, it is consistent with the view that the circuit required for the expression of the hissing component of the defensive rage behavior requires the activation of N M D A receptors. Moreover, previous studies conducted in our laboratory and elsewhere further demonstrate that it is the downstream projections of the anterior medial hypothalamus that are critical for the expression of this response [12-14]. In fact, it was previously shown that lesions placed at different levels along the rostro-caudal axis of the medial hypothalamus effectively block defensive rage [14]. Therefore, it is most likely that peripheral administration of the N M D A receptor antagonist blocked the hissing component of defensive rage behavior as a result of its actions at the level of the midbrain PAG. The second line of evidence constituted a direct test of the hypothesis that N M D A receptor blockade observed from peripheral administration of AP-7 takes place at the level of the dorsal PAG in association with the descending pathway that mediates defensive rage from the medial hypothalamus. The results indicate that microinfusion of the N M D A receptor antagonist into sites within the dorsal PAG (from which defensive rage can be elicited) also blocks medial hypothalamically elicited defensive rage and thus mimics the findings obtained from peripheral administration of this antagonist. The third line of evidence obtained from the present study is that the facilitating effects of medial hypothalamic stimulation upon PAG elicited hissing are eliminated by N M D A receptor blockade within the PAG. This finding thus replicates a previous observation from our laboratory [19] that demonstrated that medial hypothalamic facilitation of hissing elicited from the PAG is suppressed by infusion of drugs that selectively block N M D A but not A M P A receptors. Accordingly, the results of this experiment provide further support for the existence of a functional relationship between the medial hypothalamus and dorsal PAG in the expression of defensive rage behavior and the role manifest by N M D A receptors. The fourth line of evidence stems from the receptor histochemical data. This aspect of the study demonstrated that cells which immunoreacted positively with the NMDA-R1 antibody were largely distributed within the dorsal P A G - the region that receives major inputs from those parts of the medial hypothalamus from which defensive rage can be elicited. It was also noted that immunoreactivity was present to a much lesser extent in the ventral PAG, where defensive rage cannot be elicited. These findings are also consistent with other data obtained from rodents that demonstrated the presence of significant densi-

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ties of N M D A receptor sites with a high affinity for glutamate within the rostral P A G [1,5-7,15]. The fifth line of evidence is the immunocytochemicalanatomical data that basically replicates the findings originally described in our earlier study [19]. These findings demonstrated the presence of populations of immunopositive cells for glutamate, as well as double-labeled neurons for both Fluoro-Gold and glutamate mainly in the anterior medial and dorsomedial hypothalamus. It should be noted that these cells were present in those parts of the medial hypothalamus that include the ventromedial, dorsomedial, and perifornical regions - regions from which defensive rage is typically elicited [28,29]. The discovery of a glutamatergic pathway from the medial hypothalamus to the midbrain PAG in the cat is also consistent with previous observations described in the literature [1,5,7]. In summary, the results of the behavioral-pharmacological experiments, coupled with the immunocytochemicalanatomical, receptor immunohistochemical and autoradiographic observations, clearly demonstrate that defensive rage behavior elicited from the medial hypothalamus is subserved by a pathway to the midbrain PAG whose functions are mediated by EAA that act upon N M D A receptors.

Acknowledgements This research was supported by NIH Grant NS07941-25 and by a grant from the Harry Frank Guggenheim Foundation. The authors wish to thank Drs. Hreday N. Sapru for his helpful suggestions with the behavioral pharmacological experiments and Nihal DeLanerolle for his insights concerning the receptor binding studies.

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