A1 Noradrenergic action on medial preoptic-medial septal neurons: A neuropharmacological study

SYNAPSE 2:494-507 (1988)

A1 Noradrenergic Action on Medial Preoptic- Medial Septa1 Neurons: A

Neuropharmacological Study YANG I. KIM, CAROL A. DUDLEY, mi) ROBERT L. MOSS

Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235

KEY WORDS Norepinephrine, Medial preoptic area, Septum, 6-Hydroxydopamine Alpha receptor, Beta receptor, A1 noradrenergic region

ABSTRACT In an attempt to determine whether the excitatory and inhibitory orthodromic responses of single medial preoptic-medial septal (MPO-S) neurons to discrete electrical stimulation of the A1 noradrenergic region were mediated specifically by norepinephrine WE) and involved different types of adrenoreceptors, a series of electrophysiological and neuropharmacological experiments was conducted. Extracellrrlar single unit recording and local drug application techniques were used in female rats under urethane anesthesia. Chemical lesion of the catecholaminergic nerve terminal plexus in the medial preoptic area with 6-hydroxydopamine abolished both excitatory and inhibitory orthodromic effects of A1 region stimulation on MPO-S neurons, suggesting the noradrenergic nature of the effects. This conclusion was corroborated by the observation that the orthodromic effects were mimicked by locally applied exogenous NE. The excitatory effects were reliably mimicked by a low concentration of NE (0.5 mM, in-barrel concentration) and methoxamine (1.0 mM, an alpha-1 agonist), but not by either low or high concentrations (1 and 100 mM) of clonidine (an alpha2 agonist) and isoproterenol (a beta agonist). The inhibitory orthodromic effects of A1 region stimulation were reliably mimicked by a high concentration of NE (50 mM), clonidine (100 mM) and isoproterenol (100 mM), but not by a low concentration of NE (0.5 mM), methoxamine (1 mM), clonidine (1 mM) or isoproterenol (1 mM). A high concentration (100 mM) of methoxamine mimicked the inhibitory effects less than 40% of the time. The low concentration (0.5 mM) NE-induced excitation that matched the excitatory orthodromic effect of A1 region stimulation was blocked by phentolamine (100 mM), an alpha blocker, but not by timolol(100 mM), a beta blocker. On the other hand, the high concentration (50 mM) NE-induced inhibition that matched the inhibitory orthodromic effect of A1 region stimulation was blocked by timolol, but not by phentolamine.

Taken together, the present results are consistent with the hypotheses that the ascend- ing noradrenergic projections from the A1 region affect the excitability of MPO-S neurons directly through NE and that the excitatory and inhibitory orthodromic effects involve different types of adrenoreceptors, i.e., alpha-1 and beta receptors, respectively.

INTRODUCTION The medial preoptic-medial septal (MPO-S) area is con-

sidered to be an important site of noradrenergic (NE; norepinephrine) action for the control of luteinizing hormone (LH) secretion (Kalra and Kalra, 1983). Support for this notion is provided by data from different lines of research. From a neuroanatomical perspective, the MPO-S area has been shown to contain LH-releasing hormone (LHRH) neurons (Krey and Silverman, 1983) and to be heavily innervated by noradrenergic fibers (Fuxe and Hokfelt, 1969; Lindvall and Bjorklund, 1974; Ungerstedt, 1971). In addition, close anatomical apposi- tions between noradrenergic fibers and LHRH peri- karya in the preoptic-septa1 complex have been demonstrated to exist (Jennes et al., 1982).

Data from endocrinological experiments also support the idea that the medial preoptic (MPO) area is an important site for noradrenergic control of LH secretion. NE turnover rates in this site have been demonstrated to parallel serum LH levels (Homma and Wuttke, 1980; Wise et al., 1981). Furthermore, application of exogenous NE to the MPO area or chemical depletion of endogenous NE in this site was shown to affect LH secretion (Ferris et al., 1984; Hanke and Wuttke, 1979; Leipheimer and Gallo, 1985; Simpkins et al., 1979; Simpkins and Kalra, 1979).

Received August 31, 1987; accepted April 5,1988

Address for Correspondence: Dr. Robert L. Moss, Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235.

0 1988 ALAN R. LISS, INC.

A1 NORADRENERGIC ACTION ON MPO-S NEURONS 495

Recently, we reported (Kim et al., 1987) that electrical stimulation of the A1 noradrenergic region evokes an orthodromic response (either excitatory, inhibitory or complex) from a subpopulation of MPO-S neurons, including some neurons projecting to the arcuate nucleus/ median eminence (ARCME). Although in the aforemen- tioned report some indirect indications were provided for the idea that the orthodromic responses resulting from electrical stimulation of A1 noradrenergic region were NE-mediated events, conclusive evidence was lack- ing. Therefore, in the present study we sought to provide such evidence through a series of neuropharmacological experiments.

The present report consists of four experiments. In the first experiment, we examined the effects of destroying the noradrenergic nerve terminal plexus of the MPO area with 6-hydroxydopamine on the orthodromic responses of MPO neurons to A1 region stimulation. In the second experiment, the ability of locally applied exogenous NE to mimic the excitatory or inhibitory orthodromic effect of A1 region stimulation on MPO-S neurons was tested. In the third experiment, the effects of locally applied selective adrenergic agonists, methoxamine, clonidine and isoproterenol, on the single unit activity of MPO-S neurons were assessed and compared to the orthodromic effects of A1 region stimulation. In the last experiment, the ability of selective adrenergic blockers, phentolamine and timolol, to antagonize the NE-induced excitation or inhibition of MPO-S neurons was tested. "his testing was conducted only on neurons which were excited or inhibited by both A1 region stimulation and NE.

MATERIALS AND METHODS Animal Preparation

Female Sprague-Dawley rats (n = 73; 220-350 g) were housed in a temperature-controlled room with a stan- dard light: dark cycle (14 h light: 10 h dark; the light period began at 500 a.m.) with a minimum 4 weeks of adaptation. Animals were allowed access to Purina chow and water ad libitum. All animals were ovariectomized (OVX) at least 3 weeks prior to electrophysiological recording and were subcutaneously injected with 10 pg estradiol benzoate a t 8:OO a.m., two days before the day of the experiment and with 2.5 mg progesterone, 48 hours after the estradiol injection. This steroid injection schedule was shown to result in an increase in the proportion of MPO-S neurons responding orthodromically to A1 region stimulation (Kim et al., 1987). In addition, a similar steroid injection schedule in unanesthetized, OVX rats has been shown to induce an LH surge in the afternoon of the day of progesterone injection (Caligaris et al., 1968; 1971; Kalra et al., 1972).

On the day of the experiment, each animal was anes- thetized with urethane (25% w/v solution injected i.p. at 1.2-1.4 g/kg body weight) and placed in a stereotaxic instrument with the incisor bar set at 5 mm above the horizontal plane. A rectangular area of skull overlying the MPO-S area was removed using a high-speed drill. The dura was dissected away with a fine ophthalmic knife and the exposed brain surface was covered with a small cotton ball soaked with saline to prevent it from drying. Concentric bipolar stainless steel electrodes were then placed in the ARCME (in Experiments 2 , 3 and 4) and in the A1 noradrenergic region. Electrode (base

diameter: 0.2 mm; tip separation: 0.5 mm) placement in the ARCME was through a dorsal approach in which the electrode was lowered into brain tissue at a 30" angle from the vertical plane using the following anterior-posterior (A-PI, lateral (L) and ventral (V) coordinates: A-P: -5.0 to -5.5 mm from bregma; L: 0.0 mm from the mid-sagittal suture; V: 9.7 to 10.3 mm from the surface of brain. Electrode (base diameter: 0.1 mm; tip separation: 0.5 mm) placement in the A1 region was accomplished after removing some muscle tissue from the neck and tearing the occipito-atlantic membrane to expose the dorsal part of the medulla. The electrode was angled 45" from the vertical plane and positioned using the following coordinates: A-P: - 1.0 to - 1.5mm from the obex; L: 1.7 to 1.8 mm from the midline; V: 2.3 to 2.4 mm from the brain surface. Electrode placement in the A1 region was ipsilateral to the electrophysiological recording site. The body temperature was maintained at 37 + 1°C with a hydraulic heating pad throughout the experiment.

Preparation of Single-Barrel Electrode and Multi- Barrel Micropipette Assembly

For extracellular single unit recordings from MPO-S neurons without testing of any drug effect on the neuronal activity, single-barrel micropipettes were pulled from glass capillary tubing (A-M Systems, Everett, WA) and were filled with 0.5 M sodium acetate blfier containing 2% Pontamine Sky Blue (PSB; pH 7.3). The impedance of the single-barrel electrodes was brought to the range of 5.0 to 11.0 megohms by breaking the tips of the electrodes. The final tip diameter was less than 1.0 pm.

For both single unit recordings and testings of the effects of drugs, which were locally applied by iontophoresis or by micropressure, 7-barrel micropipette assem- blies were prepared from glass capillary tubing according to the method developed by Moss et al. (1978). The tip of the micropipette assembly was broken to a diameter of 1.5-3.5 pm. Single unit recordings were made through the center barrel filled with 4 M NaC1. The impedance of the center barrel ranged from 4.0 to 11.0 megohms. Each of outer barrels was filled with one of the following solutions: monosodium glutamate (GLUT0.5 M, dissolved in distilled water; pH KO), PSB, 2 M NaCl (pH 7.4), artificial cerebrospinal fluid (ACSF; 126.0 mM NaC1, 5.0 mM KCl, 1.3 mM MgS04, 1.24 mM NaH2P04.H20, 26.0 mM NaHC03, 2.0 mM CaC12, 10.0 mM glucose; pH 7.41, HEPES buffer (10 mM; pH 7.45), NE-HC1 (NE; 0.5 or 50 mM, in ACSF or HEPES buffer; pH 7.2-7.41, methoxamine-HC1 (MOX; 1 or 100 mM, in HEPES buffer; pH 7.2-7.41, clonidine-HC1 (CLD; 1 or 100 mM, in HEPES buffer; pH 7.2-7.4), phentolamine-mesy- late (PT; 100 mM, in distilled water; pH 5.0-6.0) and timolol-maleate (TM; 100 mM, in distilled water; pH 5.0- 6.0). The impedance of the outer barrels ranged from 9 to 40 megohms. Selection of drugs for the outer barrels depended on the design of a specific experiment (see below).

Extracellular Single Unit Recordings and Antidromic and Orthodromic Identification

The MPO-S area was approached from the dorsal aspect of the brain according to the stereotaxic atlas of Pellegrino et al. (1981). Using a micromanipulator, the

496 Y.I. KIM ET AL

Fig. 1. Photomicrographs showing representative examples of A1 region (A) and ARCME (B) stimulation sites (arrow heads). The A1 noradrenergic region is encircled.


Fig. 2. (A) Photomicrograph of a coronal brain section a t the level of the medial preoptic area, which illustrates the 6-hydroxydopamine injection cannula tract (small arrows) and the first and the last electrophysiological recording sites (940 um apart according to the stereotaxic readings) marked with blue spots (arrow heads). The end of the injection cannula tract, i.e., the actual injection site is indicated by a large arrow. (B) Catecholamine histofluorescence micrograph obtained from

the medial preoptic area in the intact side of the brain which corre- sponds approximately to the 6-hydroxydopamine-injected site. Note the two fluorescent nerve fibers (arrows) crossing the blood vessel (BV). Also, note the strong fluorescence along the vascular walls. Abbrevia- tions: AC: anterior commissure, BV: blood vessel, OC: optic chiasm, 3V: the third ventricle.

498 Y.I. KIM ET AL

TABLE I . Responses of MPOS neurons to A1 region stimulation and to low and high concentrations of exogenous NE.

Response to A1 region 0.5 mM NE (low) 50 mM NE (high) Stimulation

OD + 16 (2) 1 6 23 2 11 (2) 5 18

Response to Exogenous NE

Total t 1 + Total t 1 +

OD - 6 (1) 4 4 14 0 10 (1) 2 12 NR 12 (8) 6 41 (4) 59 l(11 24 (7) 28 (1) 53

Total 34 11 51 96 3 45 35 83 1) Neuronal responses to exogenous NE were indicated by arrows T : excitation, 1 : inhibition, - : no response 2) Numbers in parentheses indicate the number of antidromically identified neurons which fall in the specific response category.

b.

0.5 m M NE 50 m M NE O.Ppsi, 256%+ 2.lpsi, 71%+

C.

- 5 0 msec

50 mM NE 2.1psi. 03%+

*-

0.5 mM NE 0.9~4, 336% +

0.5mM NE 2.Ops1, 343%+

10 sec

Fig. 3. Effects of low and high concentrations of exogenous NE on the activity of a medial preoptic neuron exhibiting an antidromic response to ARCME stimulation and an excitatory orthodromic response to A1 region stimulation. (a) Oscilloscope tracings (five sweeps superimposed) showing the antidromicity of the neuron. The antidromic spike evoked by the first electrical shock (open arrow on the left) delivered to the ARCME collided with the spontaneous spike (not shown) which immediately triggered the electrical shock. Thus, no spike was captured following the first shock. The antidromic spike evoked by the second shock (open arrow on the right) which was triggered by the spontaneous spike at an interval much longer than the antidromic latency was captured with a constant latency following the shock. (b) A peri-stimulus time histogram showing the excitatory

orthodromic response of the neuron to A1 region stimulation. Open arrows indicate the stimulus artifacts. (c) A continuous polygraph record that shows the effects of low (0.5 mM in-barrel concentration) and high 50 mM) concentrations on NE of the spontaneous activity of the neuron. The polygraph record is composed of three traces. The upper trace is the time base. The middle and lower traces are the integration of the neuronal activity over 1.25 second epoch and firing rate, respectively. The dark bars associated with the time base illustrate the period of drug application. Information concerning NE concentration, pressure level used to eject NE, and percent firing rate change associated with NE application is provided on the top of each dark bar. The configuration of polygraph records in subsequent figures is the same as the one in the current figure.


a. D+

N = l l

C. 100

m - - 8 -

50 - C 0

0 e a

0

N=13

NR

3 N = 9

MOX N = 7 N = 7

CLD ISP

N = 9 N=12

Fig. 4. Summary of the effects of a high concentration (100 mM; in- barrel concentration) of adrenergic agonists, methoxamine (MOX; alpha-l), clonidine (CLD; alpha-2) and isoproterenol (ISP; beta), on the activity of MPO-S neurons exhibiting either orthodromic excitation (OD+; a), orthodromic inhibition (OD-; b) or no response (NR; C) to A1 region stimulation. The number of neurons tested with each adrenergic agonist is indicated at the bottom of each set of graphs.Keys for the agonist effects are provided a t the upper-right corner of (a).

single-barrel electrode or 7-barrel micropipette assembly was lowered to the following coordinates: A-P: 1.8 to 2.2 mm from bregma; L: 0.3 to 0.7 mm from the mid-sagittal suture; V: 5.0 mm from the brain surface. From this point, vertical electrode movement was controlled by a hydraulic microdrive. Conventional extracellular single unit recording techniques were used to detect unit activity. When a neuron was encountered and baseline activity recorded for several minutes, possible projection of the neuron to the ARCME was examined (only in Experiments 2, 3 and 4). For this, isolated monophasic cathodal pulses (0.2-1.0 mA intensity, 0.15 msec pulse duration, 0.2 Hz frequency) were delievered to the ARC/ ME to activate antidromically the neurons projecting to this region. The neuronal response to ARCME stimulation was recorded on a Tektronix storage oscilloscope. The two criteria employed for antidromic testing were 1) constant latency of an antidromic spike, and 2) colli- sion of the antidromic spike with a spontaneous or GLUT-induced spike (Harris et al., 1971; Lipski, 1981;

Yagi and Sawaki, 1970). Following the antidromic testing, the effect of A1 region stimulation on the neuronal activity was tested using peri-stimulus time histograms. Initially, single or paired monophasic cathodal pulses (25-150 PA, 0.1-0.2 msec, 20 msec intrapair interval, 0.2-1.0 Hz) were delivered to the A1 region. When the neuron under test showed a variable, reproducible, short latency increase or decrease in firing rate following the stimulation, it was considered to be orthodromically responsive. The onset latency (measured from the center of the first and the second stimulus artifacts) and duration of the orthodromic response were determined from a peri-stimulus time histogram (100 sweeps accumu- lated) generated using a fixed set of stimulation param- eters (paired pulses; 100 pA, 0.15 msec, 20 msec intrapair interval, 0.5 Hz). Experiment 1: Effects of 6-Hydroxydopamine Lesion

To determine whether the orthodromic responses of MPO neurons to A1 region stimulation were mediated specifically by a noradrenergic projection, the conse- quences of lesioning the catecholamine nerve terminal plexus of the MPO area were assessed. The catecholamine-selective neurotoxin, 6-hydroxydopamine (8.5 pg base in 1 pl saline) (Ungerstedt, 1968) was injected at the lateral aspect of the MPO area 24-48 h prior to the beginning of the electrophysiological experiment. Con- trol animals were injected in a similar site with vehicle alone. Unit recordings were obtained with single-barrel electrodes from neurons in the medial aspect of the injection site.

Experiment 2: Synaptic Mimicry by NE In this set of experiments, each of the outer barrels of

the micropipette assembly contained one of the following solutions: GLUT, 2 M NaC1, PSB, ACSF, and 0.5 mM and 50 mM NE (in ACSF). When the neuron under test was orthodromically excited or inhibited by A1 region stimulation, the effects of exogenous NE at two different concentrations were tested on the spontaneous or GLUT-induced (in cases where spontaneous neuronal activity was less than 0.5 impulses/sec) activity of the neuron to see which concentration of NE mimicked the effect of A1 region stimulation. Some randomly selected neurons that did not respond to A1 region stimulation were also tested with NE for control purposes. NE and vehicle (ACSF) were applied for 30 sec with micropressure of 0.2-23.0 psi (delivered by a Medical Systems PPM 2 pneumatic pump). To insure the patency of the drug barrel, the pressure level was gradually increased until the amplitude of the action potential was slightly altered (usually decreased). For subsequent tests, the pressure was kept to the minimum level needed to elicit a reliable response. When no obvious change in action potential amplitude was observed with pressure greater than 23.0 psi and no clear response was elicited, the drug barrel was considered to be blocked. GLUT was applied with a current of 4-40 nA (delivered by a Medi- cal Systems BH-2 Iontophore Unit). Between periods of iontophoretic application, a retaining current of 2-8 nA was applied to the GLUT-containing barrel to prevent leakage. Both the ejecting and retaining currents were automatically balanced through a barrel containing 2 M NaCI.

500 Y.I. KIM ET AL.

a. U b.

10 msec U

50 msec

C. ISP, 3.2 psi MOX, 5.0 psi MOX, 5.0 psi

127% 4 191% 4 , 0% - -- ISP, 5.2 psi ISP, 5.2 psi CLD, 5.4 psi

67% t 54% t 61% t P P w -

CLD, 5.4 psi MOX, 5.0 psi HEPES, 8.0 psi 88% * 150%) 20% *

10 so=

Fig. 5. Effects of a high concentration (100 m M in-barrel concentra- the excitatory orthodromic response of the neuron to A1 region stimu- tion) of alpha-1, alpha-2 and beta adrenergic agonists on the activity of lation. Open arrows indicate the stimulus artifacts. (c) A continuous a MPO-S neuron exhibiting an antidromic response to ARCME stim- polygraph record that shows the effects of methoxamine (MOX, alpha- ulation and an excitatory orthodromic response to A1 region stimula- 11, clonidine (CLD; alpha-21, isoproterenol (ISP beta) and the vehicle tion. (a) Oscilloscope tracings (five sweeps superimposed) showing the (HEPES buffer) on the glutamate (-2.5 nA)-induced activity of the antidromicity of the neuron. For detailed explanation of the antidromic neuron. testing, see Fig. 3a legend. (b) A peri-stimulus time histogram showing

Experiment 3: Synaptic Mimicry by Adrenergic Agonists

In this particular experiment, each of the outer barrels of the micropipette assembly contained one of the following solutions: GLUT, PSB, HEPES buffer, MOX (1 or 100 mM), CLD (1 or 100 mM) and ISP (1 or 100 mM). When the neuron under test was orthodromically excited or inhibited by A1 region stimulation, the effects of the adrenergic agonists above were tested on spontaneous or GLUT-induced activity of the neuron to see which of the agonists mimicked the effect of A1 region stimulation. In addition, some neurons that were not orthodromically affected by A1 region stimulation were also tested with the agonists for control purposes. The agonists and vehicle (HEPES buffer) were applied by micropressure (0.2-23.0 psi), and GLUT was applied by

iontophoresis (4-40 nA). Iontophoretic current balancing was accomplished through PSB-containing barrel.

Experiment 4: Effects of Adrenergic Blockers on NE-induced Response

In this particular experiment, each of the outer barrels contained one of the following solutions: PSB, HEPES buffer, 0.5 mM and 50 mM NE (in HEPES buffer), PT, and TM. When the neuron under test was orthodromically excited or inhibited by A1 region stimulation, the neuron was challenged with NE in two different concentrations (micropressure-ejected). Only when the response of the neuron to a given concentration of NE was reproducible and matched the response to A1 region stimulation was the NE-induced response subjected to


a.

MOX, 2.6 psl MOX, 2.6 PSI b. 34% k 17% 4

10 uc

ISP, 2.6 psi ISP, 2.6 psi CLD, 2.6 pal 87% t 80% t 39% t

CLD, 2.6 psl ISP, 2.6 pal MOX, 2.6 psi 61% $ 70% t 15% t

~*&",l"~lldil...'"....'....'

Fig. 6. Effects of a high concentration (100 mM; in-barrel concentration) of alpha-1, alpha-2 and beta adrenergic agonists on the activity of a MPO-S neuron exhibiting an inhibitory orthodromic response to A1 region stimulation. (a) A peri-stimulus time histogram showing the inhibitory orthodromic response of the neuron to A1 region stimula-

tion. Open arrows indicate the stimulus artifacts. (b) A continuous polygraph record that shows the effects of rnethoxamine (MOX; alpha- l), clonidine (CLD; alpha-2) and isoproterenol (ISP beta) on the Ton- taneous activity of the neuron.

possible blockade by PT or TM. A chosen blocker was firing rate during the 30 second period immediately applied iontophoretically (2-15 nA) after at least two prior to drug application and 2) application of vehicle control tests of NE. Application of blocker was begun (ACSF or HEPES buffer), other agonist(s), or different about 2 minutes before the first of the subsequent NE concentration of NE was not effective in altering the tests (at least three) and was continued until the last firing rate. NE test. Iontophoretic current balancing was through The effectiveness of the adrenergic blockers to antag- the barrel containing PSB. onize the NE-induced response was judged by the follow-

ing criteria: 1) 30% or more attenuation of the mean NE-induced percentage firing rate change and 2) a sta- tistically significant (p < 0.05, one-tailed t test) atten- Criteria for Drug Effect

A given adrenergic agonist or NE was considered to uation of the mean NE-induced response magnitude be effective in altering the single unit activity if 1) (RM). The RM was defined as an absolute firing rate application of the chosen drug resulted in a reproducible difference between 30 second pre-NE application period change in firing rate of 30% or more as compared to the and 30 second during NE application period.

Y.I. KIM ET AL 502

a.

lo0l OD+

b. 100

m Q) 0 0

g 50

r n

- - - - C Q)

0

100 C.

m 0 0 0

p 50

r n

- - - c S 0

0

0

OD -

JJ N = l 1

NR

n

AlBL N=14

I I r-

N = 1 0 N = 9

N=18 N=16 N = l o

MOX CLD ISP

Fig. 7. Summary of the effects of a low concentration (1 mM; in-barrel concentration) of adrenergic agonists, methoxamine (MOX; alpha-11, clonidine (CLD; alpha-2) and isoproterenol (ISP; beta), on the activity of MPO-S neurons exhibiting either orthodromic excitation (OD+; a), orthodromic inhibition (OD-; b) or no response (NR el to A1 region stimulation. The number of neurons tested with each adrenergic agonist is indicated at the bottom of each set of graphs. Keys for the agonist effects are provided at the upper part of (a).

Histology At the end of an experiment, PSB was iontophoreti-

cally ejected for 5 minutes at -2 pA and then for 15 minutes at -10 pA to mark the recording site with a blue spot at the tip of the electrode. Each site of electrical stimulation was also marked with a small lesion made by 200 pA of direct current passed through the stimulating electrode for 10 seconds. The animals were perfused with 4% formaldehyde in 1 M sucrose solution. Hindbrain and forebrain coronal sections (50 pm) were stained with neutral red and examined by light microscopy for localization of the blue spot(s), the electrode tract, and lesions in the ARCME and in the A1 region. In Experiment 1, a few animals were perfused with a mixture of 4% paraformaldehyde and 0.75% glutaralde- hyde to fix the brain and to convert catecholamines to fluorescent derivatives (Furness et al., 1978). Forebrain sections were examined by fluorescence microscopy to

verify the 6-hydroxydopamine-induced lesion of the catecholaminergic nerve terminal plexus in the MPO area and to localize the recording sites in the 6-hydroxydopamine-affected area.

RESULTS Orthodromic Responses of MPO-S Neurons to A1

Re-gion Stimulation The data presented in this section are combined from

results obtained in Experiments 2, 3 and 4. The orthodromic effect of A1 region stimulation was assessed in 548 neurons which were histologically verified to be located in the MPO-S area. All stimulation sites were located in the A1 region or within 300 pm of the region (Fig. la). Stimulation of the A1 region with paired pulses equal to or greater than 100 pA was always effective in evoking an orthodromic response from at least one of the neurons tested in any given animal preparation. Stimulation with one pulse or with a stimulus intensity less than 50 pA was rarely effective. To A1 region stimulation, 238 MPO-S neurons (43%) exhibited an orthodromic response and the remaining 310 neurons showed no response (NR). The orthodromic responses were clas- sified into three categories as previously reported (Kim et al., 1987): orthodromic excitation (OD +), orthodromic inhibition (OD -1 and complex orthodromic response. One hundred fifty-one of the 238 neurons (63%) showed an OD+ and 82 neurons (34%) showed an OD-. The remaining five neurons showed a complex orthodromic response. This complex response was characterized by an initial inhibition followed by an oscillatory firing pattern. The mean f SEM firing rates (impulseshec) of neurons exhibiting either OD+, OD- or NR were 5.75 f 1.03, 7.56 & 1.76 and 6.96 f 1.29, respectively. No significant differences were detected between the mean firing rates (analysis of variance test). The mean & SEM onset latencies and durations (msec) of the orthodromic excitation and inhibition were as follows: onset latency: 42.65 f 3.03 and 37.44 2.75; duration: 193.27 f 26.88 and 187.62 & 32.53. No significant differences were detected between orthodromic excitation and inhibition with respect to the onset latency and duration (t test).

Of 548 MPO-S neurons studied, 38 (7%) were antidromically identified as projecting to the ARCME (AD neurons). The mean f SEM firing rate of these AD neurons and mean _+ SEM antidromic latency were 0.92 & 0.25 impulseshec and 13.85 + 1.13 msec. Nine AD neurons exhibited an OD+ to A1 region stimulation and two exhibited an OD-. The remaining 27 neurons showed NR. A representative example of the ARCME stimulation site is illustrated in Fig. lb.

Effects of 6-Hydroxydopamine Lesion An example of a 6-hydroxydopamine injection site in

the MPO area and the electrophysiological recording sites marked with blue spots is illustrated in Fig. 2a. Irjection of 6-hydroxydopamine resulted in the destruc- tion of the catecholaminergic nerve terminal plexus around the injection site as indicated by the absence of fluorescent nerve fibers. The affected area ranged from 1.2 to 1.5 mm in diameter with the end of the cannula tract as the center. In the intact MPO area on the opposite side, fluorescent nerve fibers (Fig. 2b) were observed throughout the area.

In animals pretreated with 6-hydroxydoparnine, none of the spontaneously active neurons tested (n=40) in the


a.

' In n 25[ 1 100 Al-OD+ sweeps

P

NE, 1 1 . 4 psi NE, 11 .4 psi TM: NE, 11 .4 psi NE, 1 1 . 4 psi NE, 1 1 . 4 psi 291% k R M = 1 1 . 6 7 500% 4; R M = 1 8 . 3 3 + 400% 4; R M = 3 . 2 0 388% 4; R M = 2 . 0 7 328% k R M = 1 . 9 7 . * P W U , i U l d . u * , ~ , , " , ' , , , , , ~ , u l , , ~ , , , , , , , " ~ " , l , , - * , ~ ~ ~ " ~ , , , . " , , ' ~

/ / ' \ i l l I - I , / i i i /

NE, 11 .4 psi PT, NE, 11 4 psi NE, 1 1 . 4 psi 13% 4; R M = 0 . 0 3 + 325% 2; RM = 1 . 3 0 3 4 7 % I ; RM = 1 . 7 3

I I I ~ u & u f d - ~ - ~ ~

Fig. 8. Effects of alpha and beta adrenergic blockers on low concentration (0.5 m M in-barrel concentration) NE-induced excitatory response from a MPO-S neuron which exhibited an excitatory orthodromic response to A1 region stimulation. (a) A peri-stimulus time histogram showing the excitatory orthodromic response of the neuron to A1 region stimulation. Open arrows indicate the stimulus artifacts. (b) A continuous polygraph record that shows the NE-induced

neurotoxin-affected region of the MPO area were orthodromically responsive to A1 region stimulation. In saline-pretreated control animals, 11 of 28 neurons (39%) tested were responsive to A1 region stimulation. Eight of the 11 neurons exhibited an OD+ and three showed an OD-.

Synaptic Mimicry by NE The effects of locally applied NE of two different con-

centrations (0.5 and 50 mM) on the single unit activity

excitatory response and the effects of phentolamine (m, alpha blocker) and timolol (TM beta blocker) on that response. NE was applied by micropressure while PT and TM were applied iontophoretically (+2 nA). The application periods of PT and TM were designated by "ON" and "OFF'. Abbreviation: RM: response magnitude (for definition, see "MATERIALS AND METHODS').

of MPO-S neurons exhibiting either an OD + , an OD - or NR to A1 region stimulation are summarized in Table I. In 16 of 23 cases (70%), the lower concentration of NE was excitatory to neurons exhibiting an OD+ (OD+ neurons). On the other hand, the higher concentration of NE was excitatory in only 2 of 18 OD+ neurons (11%). In fact, the higher concentration of NE was inhibitory to OD+ neurons in several instances (61%) in which the lower concentration was excitatory. The peri-stimulus time histogram and polygraph record in Fig. 3b and c

504 Y.I. KIM ET AL.

u 50 mrtc

b* NE, 5 3 p s i NE. 5 3 psi

"l*ldi I" y.lyry ,".", I I ,",*W 1.1.dd.1 L L d & d . " A ~ ~ - 98% t; RM =7 00 96% t, RM = 6 33

NE, 5 3 psi 75% t, RM = 4 00

NE, 5 3 psi TY(+15nA) ON

+ +.Ja,-d*,"*4*-AMdl *

NE, 5.3 psi 93% t; RM=6.67

NE. 5.3 psi 100% t; RM=6.33

NE, 5.3 PSI NE, 5 3 psi PT(+15nA):ON +3 100% t; RM = 5.00 100% t, RM=4.33 -ldd&&UA-*-#~- ' , , ' , I .' . / , ,

Fig. 9. Effects of alpha and beta adrenergic blockers on high concentration (50 mM; in-barrel concentration) NE-induced inhibitory response from a MPO-S neuron which exhibited an inhibitory orthodromic response to A1 region stimulation. (a) A peri-stimulus time histogram showing the inhibitory orthodromic response of the neuron to A1 region stimulation. Open arrows indicate the stimulus artifacts. (b) A continuous polygraph record that shows the NE-induced

illustrate the excitatory nature of A1 region stimulation and of the lower concentration of NE. In addition, the polygraph record also shows the inhibitory nature of the higher concentration of NE. The superimposed oscilloscope tracings in Fig. 3a demonstrate the antidromicity of this neuron to ARCNE stimulation (see the figure legend for details).

In 10 of 12 cases (83%), the higher concentration of NE was inhibitory to neurons exhibiting an OD- (OD-

inhibitory response and the effects of phentolamine PI'; alpha blocker) and timolol (TM beta blocker) on that response. NE was applied by micropressure while PT and TM were applied iontophoretically ( + 15 nA). The application periods of PT and TM were designated by "ON" and "OFF". Abbreviation: RM: response magnitude (for definition, see "MATERIALS AND METHODS')).

neurons) while the lower concentration of NE was so in only 4 of 14 cases (29%). Rather, the lower concentration of NE was excitatory in 6 of 14 cases (43%) in which the higher concentration was inhibitory.

In the majority of the cases, neither low nor high concentrations of NE were effective in altering the unit activity of neurons exhibiting no response to A1 region stimulation (NR neurons). In 12 of 59 cases (20%), how- ever, the lower concentration of NE was excitatory and

A1 NORADRENERGIC ACT

in 24 of 53 cases (45%), the higher concentration was inhibitory. Of 53 neurons tested by both low and high concentrations of NE, 10 neurons (19%) were affected in an opposite manner by the low and high concentrations. Seven of these 10 neurons were AD neurons.

Synaptic Mimicry by Adrenergic Agonists: Effects of adrenergic agonists of high concentration

A summary of the effects of a high concentration (100 mM) of adrenergic agonists on the activity of OD+, OD - and NR neurons is presented in Fig. 4. Of 15 OD + neurons studied, all 15 neurons were tested with MOX, and 8 and 11 neurons were tested with CLD and ISP, respectively. In seven cases (including two AD neurons), testing of all three agonists was possible. As shown in Fig. 4a, MOX was excitatory about 50% of the time while CLD and ISP were inhibitory most of the time. MOX was inhibitory 20% of the time where clonidine and isoproterenol were also inhibitory. In the two AD neurons mentioned above, MOX was excitatory and CLD and ISP were inhibitory. An example of such a situation is illustrated in Fig. 5. The drug vehicle (HEPES buffer) was without effect (less than 30% change in firing rate).

Of 14 OD- neurons studied, 13, 9 and 12 neurons were tested with MOX, CLD and ISP, respectively. In 8 cases, testing of all three agonists was possible. As shown in Fig. 4b, MOX was without effect or inhibitory while CLD and ISP were inhibitory most of the time. The inhibitory CLD and ISP effects that were typically observed in OD - neurons are illustrated in Fig. 6. On this neuron, MOX was without effect.

Of 9 NR neurons studied, all 9 neurons were tested with MOX, and 7 neurons each were tested with CLD and ISP. As shown in Fig. 4c, all three agonists were without effect most of the time, although an inhibitory effect was observed in some cases, especially with CLD and ISP.

Effects of adrenergic agonists of low Concentration A summary of the effects of a low concentration (1

mM) of adrenergic agonists on the activity of OD+, OD - , and NR neurons is presented in Fig. 7. Of 15 OD + neurons studied, 14,14 and 13 neurons were tested with MOX, CLD and ISP, respectively. In twelve cases (including one AD neuron), all three agonists were tested. As shown in Fig. 7a, MOX was predominantly excitatory while CLD and ISP were predominantly without effect, although an excitatory (especially in the case of CLD) or inhibitory effect was observed in some cases. In the AD neuron, MOX and ISP were excitatory and inhibitory, respectively. CLD was without effect.

Of 11 OD- neurons studied, all 11 neurons were tested with MOX, and 10 and 9 neurons were tested CLD and ISP, respectively. In eight cases, all three agonists were tested. As shown in Fig. 7b, the agonists were without effect more than 50% of the time. In some cases, how- ever, an inhibitory (especially in the case of ISP) or excitatory effect was observed.

Of 18 NR neurons, 16 were tested with all three agonists and two (including one AD neuron) were tested with MOX alone. As shown in Fig. 7c, the agonists were predominantly without effect. On the AD neuron, MOX was without effect.

'ION ON MPO-S NEURONS 505

Effects of Adrenergic Blockers on the NE-induced Response

In 9 OD+ neurons which were excited by the low concentration of NE, the antagonistic effect of PT a n d or TM on the NE-induced excitation was successfully tested. In four cases, both PT and TM were tested. In one case, PT alone and in four cases, TM alone was tested. In the four cases where both PT and TM were tested, PT was effective in antagonizing the NE-induced excitation while TM was without effect or enhanced the excitation. The continuous polygraph record in Fig. 8 illustrates the contrasting effects of PT and TM on the NE-induced excitation. While PT was effective in antagonizing the excitation, TM was not. Rather, TM enhanced the excitation. In neurons tested with PT or TM alone, PT was effective in blocking the excitation while TM was not.

In seven of nine OD- neurons, which were inhibited by the high concentration of NE, the antagonistic effect of both PT and TM on the NE-induced inhibition was tested. In two cases, only the TM effect was assessed. In four of the seven neurons, the NE-induced inhibition was antagonized by TM, not by PT (Fig. 9). In the remaining three neurons, neither PT nor TM was effective in antagonizing the NE-induced inhibition. In two neurons tested with TM alone, the inhibition was antagonized by the blocker.

DISCUSSION The results of the first experiment provide supportive

evidence for the idea that the orthodromic responses of MPO neurons to electrical stimulation of the A1 noradrenergic region are specifically mediated via catecholaminergic projections. This conclusion is further corroborated by the results of the synaptic mimicry experiment with NE. If the orthodromic responses evoked by electrical stimulation of the A1 region were not mediated by NE but were mediated by other neurotrans- mitter(s) as a result of stimulation of nerve fibers of passage or non-noradrenergic neurons in the region, the elimination of the orthodromic responses with 6-hydroxydopamine pretreatment would not have occurred. Furthermore, synaptic mimcry of the orthodromic effects of A1 region stimulation by locally applied exogenous NE would not be likely.

The finding that the low concentration of NE reliably mimicked the excitatory orthodromic effects, whereas the high concentration reliably mimicked the inhibitory effects, suggests that different types of adrenoreceptors may mediate the opposite orthodromic effects. Sup- portive evidence for the idea that NE, depending on its concentration, may activate different types of adrenoreceptors, is provided by previous reports (Basile and Dun- widdie, 1984; Day et al., 1985; Mueller et el., 1981, 1982; Nakamura et al., 1984). According to the report of Day et al. (1985), low concentrations (0.05 to 0.15 mM) of NE excited putative vasopressin neurons of the hypothalamic supraoptic nucleus through alpha receptors while high concentrations (1 to 100 mM) of NE inhibited the neurons through beta receptors. Although the reports by Mueller et al. (1981, 1982) are quite opposite in terms of receptor type, they do provide evidence for the idea

506 Y.I. KIM ET AL

that the excitatory and inhibitory effects of NE are dose- dependent and are mediated through different types of adrenoreceptors. According to their reports, the amplitude of the population spike recorded in the CA1 pyramidal cell layer of the hippocampus and the unit activity of CA1 pyramidal cells were increased by lower doses of NE through beta receptors and were decreased by higher doses of NE through alpha receptors.

The observation that in a significant proportion of MPO-S neurons exhibiting an orthodromic response to A1 region stimulation, a dual effect of NE could be induced by varying the concentration, suggests a possi- bility of the coexsistence of different types of adrenoreceptors on the same postsynaptic membrane of those MPO-S neurons. Under this condition, those exogenous NE effects that did not match the effects of A1 region stimulation may correspond to the effects exerted by noradrenergic inputs other than A1 input. Consistent with this notion, the MPO-S area has also been shown to be innervated by noradrenergic systems other than the A1 system (Day et al., 1980; Sakumoto et al., 1978; Vertes, 1985). On the other hand, it is conceivable that the mismatching exogenous NE effects might be due to the presynaptic action of NE Ganger, 1981; Starke, 1977) or due to the modulatory NE action on the non-adrenergic synaptic input(s) to the neuron studied (Rogawski and Aghajanian, 1980; Waterhouse and Woodward, 1980; Waterhouse et al., 1981). Finally, the mismatching NE effects, especially the high concentration NE-induced inhibitory effects, might be due to a non-specific action of NE on membrane sites other than adrenoreceptors (e.g., perturbations of membrane structure) (Dunwiddie et al., 1983).

The utilization of selective adrenergic agonists of two different concentrations in mimicking the orthodromic effects of A1 region stimulation proved to be useful in characterizing the types of adrenoreceptors involved. The results of this synaptic mimicry experiment; i.e., the low concentration of methoxamine reliably mimicked the excitatory effects of A1 stimulation while the high concentration of clonidine and isoproterenol mimicked the inhibitory effects, suggest that the excitatory effect of A1 region stimulation on MPO-S neurons involves alpha-1 receptors while the inhibitory effect involves alpha-2 andor beta receptors. In addition, the results suggest that the dose of a selective agonist may be a critical factor for “selective” activation of a given type of adrenoreceptor. One of the most relevant find- ings in support of this argument is the following observation: at the low concentration, methoxamine mimicked the excitatory effect of A1 region stimulation more than 80% of the time whereas, at two orders higher concentration, this agonist mimicked the excitatory effect in a much smaller percentage of the cases and was rather inhibitory in some cases. Taking into account that clonidine and isoproterenol were inhibitory to the same neurons, the inhibitory effect of the high concentration of methoxamine might be due to its non-selective action on alpha-2 andfor beta receptors.

The use of alpha and beta adrenergic blockers in the last experiment proved to be helpful in corroborating the conclusion of the synaptic mimicry experiments. With regard to the excitatory effect of A1 region stimulation, the results demonstrate that the NE-induced excitation of MPO-S neurons that matched the effect of A1

region stimulation was antagonized selectively by an alpha blocker, not by a beta blocker. In addition, the results demonstrate that, in a subpopulation of the MPO- S neurons that were excited by A1 region stimulation, the NE-induced excitation was enhanced by the beta blocker. Taken together, the results are very consistent with our current hypothesis that the excitatory orthodromic effect of A1 region stimulation on MPO-S neurons involves alpha, not beta receptors. Furthermore, the results suggest that there may exist a beta receptor- mediated inhibitory input to some of the MPO-S neurons receiving an excitatory A1 noradrenergic input.

With regard to the inhibitory effect of A1 region stimulation, the results of the last experiment demonstrate that the NE-induced inhibition of MPO-S neurons that matched the effect of A1 region stimulation was antagonized by a beta blocker in a majority of the cases, but not by a non-selective alpha blocker, phentolamine. This observation supports the hypothesis that the inhibitory effect of A1 region stimulation involves beta receptors, not alpha-1 or alpha-2 receptors. It is unclear why synaptic mimicry of the inhibitory orthodromic effect was possible with clonidine, an alpha-2 agonist as well as with isoproterenol in the synaptic mimicry experiment. Although further experimentation must be done, the inhibitory effect of clonidine could be ascribed to its presynaptic action Ganger, 1981; Starke, 1977), which would have an indirect effect by modulating the release of NE. Alternatively, the effect of clonidine might be due to a non-selective, i.e., beta receptor-mediated action.

In the present study, an attempt was made to identify the neurons recorded using antidromic testing and to compare these antidromically identified neurons (possi- bly LHRH neurons) to the unidentified adjacent neurons with respect to their response to A1 region stimulation as well as to the types of adrenoreceptors involved in the process. Although the number of antidromically identified neurons studied was small, these neurons did not appear to be much different from the adjacent neurons. One exception is that, unlike the adjacent neurons, a significant proportion of the antidromically identified neurons were affected by the low and high concentrations of NE in an opposite manner, even if they were not affected by A1 region stimulation. Although specula- tive, it may be that (nor)adrenergic systems other than A1 play a role in controlling the excitability of these antidromically identified neurons.

In summary, the results of the present study are consistent with the notion that the excitatory and inhibitory orthodromic effects of A1 noradrenergic region stimulation on MPO-S neurons are mediated directly by NE and may involve different types of adrenoreceptors, alpha-1 receptors for excitatory and beta receptors for inhibitory effect.

ACKNOWLEDGMENTS The authors wish to thank A. Sherrin for technical

assistance and J. Long for preparation of the manu- script. Research presented in this paper was supported by NM grants NS 10434 and HD 09988 V and I1 awarded to R.L.M.

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A1 Noradrenergic action on medial preoptic-medial septal neurons: A neuropharmacological study