Effects of lordosis-relevant neuropeptides on midbrain periaqueductal gray neuronal activity in vitro

Peptides.Vol. 13, pp. 965-975, 1992

0196-9781/92 $5.00 + .00 Copyright ~ 1992 Pergamon Press Ltd.

Printed in the USA.

Effects of Lordosis-Relevant Neuropeptides on Midbrain Periaqueductal Gray Neuronal Activity In Vitro SONOKO

OGAWA, j LEE-MING

KOW

AND

DONALD

W. P F A F F

Laboratory of Neurobiology and Behavior, The Rockefeller University, 1230 York Avenue, New York, N Y 10021 R e c e i v e d 7 F e b r u a r y 1992 OGAWA, S., L.-M. KOW AND D. W. PFAFF. Effects of lordosis-relevant neuropeptides on midbrain periaqueductal gray neuronal activity in vitro. PEPTIDES 13(5) 965-975, 1992.--Certain neuropeptides can facilitate lordosis by acting on midbrain periaqueductal gray (PAG) in estrogen-primed female rats. Here, we investigated responses of individual PAG neurons in vitro, to five neuropeptides: substance P (SP), luteinizing hormone-releasing hormone (LHRH), prolactin (PRL), oxytocin (OT), and thyrotropin-releasing hormone (TRH). Substance P, OT, and TRH excited spontaneous activity of PAG neurons through neurotransmitter-like actions in a dose-dependent manner, whereas LHRH and PRL virtually never affected PAG neurons this way. Oxytocin acted through oxytocin receptors located on the recorded PAG neurons, since excitatory actions of OT were 1) not abolished by synaptic blockade, 2) mimicked by the OT-specific agonist [Thr4,GlyT]OT but not by arginine vasopressin, and 3) blocked by the OT-specific antagonist [d(CHz)5,Tyr(Me)2,OrnS]vasotocin. Although LHRH had no neurotransmitter-like action on spontaneous activity of PAG neurons, it, as well as SP, could modulate responses of some dorsal PAG neurons to GABAA and GABAB agonists or norepinephrine. Neuromodulatory actions of LHRH and SP could help facilitate lordosis through PAG neurons. In vitro Electrophysiology Reproductive behavior Neuromodulation Estrogen Substance P Luteinizing hormone-releasing hormone Prolactin Oxytocin Thyrotropin-releasing hormone GABAA GABAB Norepinephrine

IT has been known that midbrain periaqueductal gray (PAG) neurons participate as essential neural components in the neural circuit for lordosis in female rats (35). Electrical stimulation of dorsal and dorsolateral PAG can facilitate lordosis (41), and lesions of this brain area reduced lordosis in estrogen-primed rats (42). The most important feature of the PAG control of lordosis is that it serves as a relay station between the hypothalamus, which provides tonic hormone-related facilitation to the PAG to prime it for the induction of lordosis (34), and the lower brainstem, where electrical stimulation excites the deep lumbar axial muscles (8,9,39), which are activated during the lordosis posture (47). Although a number of substances may affect PAG neuronal activity, some neuropeptides are assumed to be especially important for activation of lordosis-relevant PAG neural circuity, since microinfusions of them into the PAG facilitate this behavior in estrogen-primed females. Thus, microinfusions of substance P (SP) (6), luteinizing hormone-releasing hormone ( L H R H ) (37,38,43,50), or prolactin (PRL) (10) are known to facilitate lordosis in estrogen-primed female rats. Immunocytochemical studies have shown that there are SP (23,28)-, L H R H (2,22,48)-, or PRL ( 10,11)-immunoreactive nerve terminals and

SP (25,54) or L H R H (13,14) binding sites in the PAG. Cell bodies containing SP (1) in the ventromedial nucleus of the hypothalamus (VMN) or those containing PRL (49) in the arcuate nucleus also accumulate estrogen. Moreover, for SP, projections from its synthesis sites in the V M N to behaviorally effective PAG sites have been defined in rats (5) and guinea pigs (30). All these findings support the idea that hormone-dependent activation of projecting neurons from the mediobasal hypothalamus to the PAG and subsequent release of certain neuropeptides from the nerve terminals in the PAG could alter the neuronal activity of behaviorally relevant PAG neurons. Thus, definition of their electrophysiological actions on PAG neurons was required to build on previous studies which indicated that some of these neuropeptides indeed alter neuronal responsiveness through either direct neurotransmitter-like action and/or indirect neuromodulatory action in hypothalamic ( 17,18), hippocampal (55), or PAG neurons (4,44) of female rats. Thus, in the present in vitro extracellular recording study, we aimed to characterize the electrophysiological actions of these three neuropeptides in the PAG. In addition to SP, L H R H , and PRL, we studied oxytocin (OT) and thyrotropin releasing horm o n e (TRH). The importance of O T in the PAG may have

J Requests for reprints should be addressed to S. Ogawa.

965

966

OGAWA, KOW AND PFAFF TABLE 1 PROPORTION OF THE PAG NEURONS EXCITED BY DIFFERENTDOSES OF SUBSTANCEP, OXYTOCIN, OR TRH IN OVX + E AND OVX PREPARATIONS SubstanceP 1 n_M

Oxytocin

10 ~

100 ~

1~

TRH

10 rtM

100 ~

1 nM

10 n M

100 n M

OVX + E and OVX

6/51" (12°7o)

57/95 (60%)

15/19 (79%)

8/67 (12%)

32/91 (35%)

9/17 (53%)

7/25 (28%)

26/50 (52%)

16/21 (76%)

OVX + E

3/31 (10%) 3/20 (15%)

32/57 (56%) 25/38 (66%)

8/11 (73%) 7/8 (88%)

6/39 (15%) 2/28 (7%)

23/59 (39%) 8/32 (25%)

6/10 (60%) 3/7 (43%)

3/14 (21%) 4/11 (36%)

15/35 (43%) 11/15 (73%)

8/9 (89%) 8/12 (67%)

OVX

*

Excited neurons/tested neurons.

been underestimated because it was reported that there are very few OT binding sites (52) and nerve terminals (5 l) in the PAG. In contrast to its well-defined behavioral (45,46) and electrophysiological (15,19) actions in the VMN, its actions in the PAG needed to be studied. On the other hand, TRH-containing nerve terminals (12,21) and TRH binding sites (24,26) are found in the PAG. Since TRH injections to the PAG cause thermoregulatory and respiratory changes (29) without affecting lordosis (38), it was hypothesized that TRH may affect the neuronal activity of different PAG neurons. During these experiments, we examined: 1. proportions and regional distributions of neurons responsive to each of the neuropeptides SP, LHRH, PRL, OT, and TRH; 2. pharmacological basis of neuronal responses to these neuropeptides using specific agonists and antagonists; 3. possible estrogen effect on responsiveness of PAG neurons to these neuropeptides; 4. relative responsiveness to various neuropeptides in individual PAG neurons; and

100

METHOD

Preparation of Brain Slices Female Sprague-Dawley rats (175-250 g) were ovariectomized and either implanted (OVX + E; n = 61) or not implanted (OVX; n = 28) with a 5-mm Silastic capsule filled with crystalline estradiol (Sigma) for at least 7 days prior to use for the experiments. Rats were anesthetized with Metofane and decapitated. Brains were removed and blocked in ice-cold sucrose artificial cerebrospinal fluid (ACSF) gassed with 95% 02 and 5% CO2. Sagittal sections (350/~m thick) containing PAG were prepared with a Vibratome (Lancer Series 1000). The slices were incubated in gassed sucrose ACSF for 1 h and then stored in gassed regular ACSF for at least 1 h prior to use for the recording. The regular

J i !

so

5. possible neuromodulatory actions of neuropeptides. Some of the results have been reported in preliminary form (31,32).

i

Substance P

Oxytocln

T

TRH

4

60 % of Neurons Excited 40

20

o Dorsal

Ventral

Dorsal

Ventral

Dorsal

Ventral

FIG. 1. Regional distribution of SP-, OT-, and TRH-responsive (at 10 nM) neurons in the PAG. Dorsal half and ventral half were further subdivided into three parts, rostral (solid bars), middle (shaded bars), or caudal (hatched bars) thirds of the PAG. Data from OVX + E and OVX preparations were pooled since there were no statisticallysignificantdifferencesin the proportion of excited neurons betweenthe two hormone groups. Numbers above the bars are numbers of neurons tested.

NEUROPEPTIDES AND PAG NEURONAL spikes/sec 40

ACTIVITY

967

SP

3O

a

20 10 0 0

10

20

3%,n

spikes/sec 25

SP

SP

RP

20

b

15 10

5 0 0

10

spikes/$ec 15

20

30

40

50

%1.

30

40

50

60

30

40mi n

50

60

[DPDT-SP lOOnMJ

10

0 0

spikesv'sec 25

10

20

ZO,.,n

]DPDT-SP lOOnM1

2O

d

15 10

0 0

10

20

FIG. 2. Firing rate histograms of SP-responsive PAG neurons from OVX (a and b) or OVX + E (c and d) preparations. Firing rates (number of spikes/s) were originally recorded for every s and then mean rates for every 4 s were calculated to make these histograms. Arrows indicate the time of the injections of peptides into the infusion tube. Numbers marked with * indicate the numbers of spikes evoked, which were calculated as total number of spikes above the baseline during 10 min after injection of agents. (a) Application of a lower dose of SP (1 nM) evoked fewer spikes compared to that of a higher dose of SP (10 nM). (b) PAG neurons were not desensitized by repeated applications of SP with intervals of 15 min. (c) An SP analog, DPDT-SP, slightly attenuated excitatory action of SP in two out of six tested neurons. (d) In four out of six tested neurons, DPDT-SP failed to affect the excitatory action of SP.

ACSF consists of (in mM): NaCl 124, KC1 5, KH2PO4 1.2, MgSO4 1.3, CaC12 2.4, NaHCO3 26, a n d D-glucose 10. T h e sucrose ACSF is a modified ACSF with all NaCI replaced with sucrose to protect n e u r o n s from a possible overstimulation during preparative procedures.

Recording of Extracellular Single-Unit Activity A PAG slice was laid on a nylon net submerged in a recording c h a m b e r (approximately 1.5 ml in volume), which was contin-

uously perfused with gassed regular ACSF at a rate of 2 m l / m i n . In some preparations, we used a high Mg 2+ ACSF (9 m M MgSO4 a n d 0.3 m M CaCI2), which has been shown to block synaptic transmission in the V M N (16). The perfusing solution was prew a r m e d by passing the infusion tubing t h r o u g h a water bath kept at 36°C. T h e p H of the gas-saturated ACSF was 7.35-7.55. Extracellular single-unit activity o f P A G n e u r o n s was recorded with ACSF-filled glass micropipettes ( 4 - 1 0 Mf~). The firing rates ( n u m b e r of spikes/s) were m o n i t o r e d a n d stored on-

968

OGAWA, KOW AND PFAFF

spikes/sec 15

10

a

0 0

10

20

30mi n

spikes/sec 15

10

b

0 0

10

20

spikes/sec 6 OT lOrlM

30

TG-OT lOnM

40

50

60mi n

AVP lOnM

C 2

oJ 0

10

20

30

40

50

60mi n

FIG. 3. Firing rate histograms of OT-responsive PAG neurons from estrogen-treated (a and b) or nontreated (c) female rats. Numbers with * denote spikes evoked above baseline. (a) Application of a lower dose ofOT ( 1 nM) evoked fewer spikes compared to that of a higher dose of OT (10 nM). (b) Exposure to an experimental ACSF with high concentration of Mg2+ and low concentration of Ca 2+ (duration indicated with horizontal line) did not abolish the excitatory action of OT. (c) Responses to OT, TG-OT (an OT receptor-selective agonist), or AVP were examined in the same neuron. Note 10 nM of OT or TG-OT evoked similar numbers of spikes in this nearly silent neuron, whereas AVP at the same concentration evoked only one-tenth the number.

line with an AT compatible personal computer interfaced with a 12-Bit High Speed A / D board (ADI000: Real T i m e Devices, Inc.) for analog-to-digital conversion, and displayed both online and off-line as firing rate histograms. The data acquisition and analysis software was developed by Sonoko Ogawa.

Experimental Procedure A PAG neuron was tested with several neuropeptides and related agents after its firing rate became stable. Fifty ul of these solutions dissolved in saline was delivered into the perfusing solution through the infusion tube with a glass microsyringe. The following agents with the concentrations of 1 n M to 1 tzM (calculated peak concentrations in the chamber after dilution) were used: substance P (SP, Bachem), luteinizing hormone-releasing hormone (LHRH, Bachem), oxytocin (OT, Bachem), arginine vasopressin (AVP, Peninsula), thyrotropin-releasing hormone (TRH, Bachem), prolactin (PRL, NIH), [Thr4,GlyY]OT (TG-OT, OT-specific agonist, Peninsula), [D-Pro2,D-Trp7'9]Sp(DPDT-SP, SP antagonist, Peninsula), [d(CH2)5,Tyr(Me)2,OmS]vasotocin (ET-OV, OT antagonist, Peninsula), [Pmp~,O-Me-Tyr2]AVP (V~-receptor antagonist, Pen-

insula), and [ 1-adamantaneacetyl I,D-Tyr(Et)2,Val4,Abu6]AVP (V2receptor antagonist, Peninsula). Neuromodulatory actions of SP and L H R H were tested with some PAG neurons in estrogen-treated preparations. After their firing rates became stable, these neurons were first tested with either GABAA agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin3-ol (THIP, Research Biochemicals), GABAB agonist, (-)-baclofen (baclofen, kindly contributed by Ciba-Geigy), and/or norepinephrine (NE, Sigma). Fifty /11 of solution dissolved in saline, whose peak concentrations in the chamber were calculated as 4-40 #M for THIP, 200-400 n M for baclofen, and 500 riM-5 ~M for NE, was applied into the infusion tube with a glass microsyringe. Only neurons that showed consistently robust responses to these neurotransmitter agents were used for further tests. Then 50/~1 of SP, with a peak concentration of 1 nM, or LHRH, with a peak concentration of 100 nM, was applied either once or twice with a 3min interval. Starting 4 min after SP or L H R H application, the neuron was tested with either THIP, baclofen, and/or NE with 520-min intervals for up to 2 h after the peptide application. From previous work, it was estimated that the concentration of agents

N E U R O P E P T I D E S A N D PAG N E U R O N A L A C T I V I T Y

969

2000

o,

,"©

1500

O T tnl. TG-OT y-43.m+O.711X r ~.QTIl, p < O.01

• "

,.

1000

(3

o

•, "

<3• +'+

," •,"

,6

~6

soo

O T w . AVP

•

/

/y.84Z~t4+O.O4x r=.064,

n.s.

.....................

! n

i

AvP: = . . . . .

I

I

I

I

500

1000

1500

2000

IO

2500

1

3000

p| Number of Spikes Evoked by O T

FIG. 4. Comparisons of neuronal responses to OT, TG-OT, or AVP. In each of 20 neurons tested with OT and TG-OT (open circles) or 14 neurons tested with OT and AVP (solid circles), numbers of spikes evoked by TG-OT or AVP were plotted against those evoked by OT. Neurons which failed to respond to either peptides tested (12 out of 20 for OT and TG-OT and 2 out of 14 for OT and AVP) were plotted at the origin (0,0). Two lines were the best fit lines based on the linear regression analysis• Note there is a significant correlation between responses to OT and to TG-OT but not between responses to OT and to AVP (t-test for the Pearson correlation coefficients).

applied reached a peak at about 60 s, then dissipated within 5 min (19).

Response Criteria For spontaneously firing units, changes of firing rates after applications of peptides or their agonists were noted as positive effects if: 1) changes at the peak were more than two standard deviations away from the mean baseline firing rate; and 2) firing rates recovered to baseline levels. Baseline firing rate was calculated as the mean firing rate during 30 s before the injection. For silent units, evocation of more than 50 spikes during 10 min after an injection of a given test agent was regarded as excitatory. If the response to a given peptide was near the criterion, the unit was often tested again with that peptide at that concentration to reduce false positive tabulation. For neuromodulatory actions of SP and L H R H , effects of G A B A agonists or NE on firing rates were compared before and after L H R H or SP applications. Neuromodulatory actions of L H R H and SP were defined as more than 25% changes (before vs. after peptide applications) in effects of G A B A agonists or N E on firing rates.

Subdivisions o f PAG The sites of the recorded PAG neurons were classified in six different regional groups: dorsal half and ventral half were further subdivided into three parts, rostral (bregma - 5 . 0 to -6.2), middle (bregma - 6 . 2 to -7.4), or caudal (bregma - 7 . 4 to - 8 . 6 ) thirds of the PAG.

Statistics The differences in the proportion of excited neurons between groups (OVX + E vs. O V X preparations) or between subdivisions of the PAG were compared using c~Z-test or Fisher's exact

probability test• The Pearson correlation coefficients were calculated to examine the correlations between responses to O T and TG-OT, or O T and AVP. The significance of the coefficients was tested with t-test. RESULTS

Substance P Substance P excited PAG neurons in a dose-dependent manner between 1 n M and 100 n M (Table 1). Only 10 or 15% of the tested PAG neurons were excited by 1 n M of SP in O V X + E or O V X preparations, respectively, whereas more than a half and a majority of the tested neurons were excited by 10 n M a n d 100 nM, respectively, in both groups. There was no statistically significant difference in the proportion of excited neurons between estrogen-treated and nontreated groups at any concentration of SP. Also, both spontaneously firing neurons (25 out of 45 tested O V X + E and O V X neurons) and silent neurons (7 out of 12 neurons) were affected by SP at 10 nM. Excited PAG neurons from both types of preparations were distributed throughout the PAG (Fig. 1). Responses of PAG neurons to SP had several noticeable features. Firstly, a higher dose of SP ( 10 nM) not only excited more SP neurons but also evoked more spikes in an SP-responsive neuron compared to a lower dose of SP (1 nM) (Fig. 2a). Secondly, PAG neurons were not desensitized to SP applied with 15-min intervals (Fig. 2b). This made it possible to test the effect of an SP antagonist, DPDT-SP, on SP excitatory action (Fig. 2c-d); D P D T - S P failed to antagonize the excitatory action of SP (see the Discussion section). In two of six PAG neurons, DPDT-SP, at a concentration 10 times higher than SP, merely attenuated SP excitation (Fig. 2c), while in the remaining four neurons, D P D T - S P had no effect on SP excitation (Fig. 2d).

970

OGAWA, KOW AND PFAFF splke~/sec

IET-OV, 100nM[

15 10

5

0

10

0

20

30

50

40

60

70

80

9Oraln

TG-OT 10nM~

~

spikes/sec

15 I 10

AVP 10nM~

OT 1OhM

10rim ~T

TG-OT 10nM

~ ~0nM

b

,J

111*

0

0

o"

1lug.

10

20

40

30

50

11137•

60

70

80

1456•

90

100

110

120

130rnln

spikes/sec AVP

AVP

AVP OT

AVP

AVP

J AVP

nM

1OhM ; lOnM ~

l O~n M

1OhM ~

~OnM

1 0 ~ lOnM

5 0

~1~183;,.

0

3,

10

spikes/sec 6 OT 10nM

ms42,

20

SP 10nM

30

s~o,

40

j l ~ lo2s •

50

TRH 10nM

LHRH 100nM

gO

LHRH 100nM

10

16s7•

70

PRL 1/JM

80

90

TG*OT 10nM

5

100

110mln

TRH 10nM

0

120

TRH 10nM

T6

1

4

d

0

I. . . . . . . . . . . . . . . . . . . . . . .

10

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I ..........

20

30

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40

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50

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70

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90

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100

110

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0

120min

FIG. 5. Firing rate histograms ofOT-responsive PAG neurons (a-c) or a TRH-responsive neuron (d) from estrogen-treated (a, b, and d) or nontreated (c) female rats. Numbers with * denote spikes evoked above baseline. (a) An OT receptor-specific antagonist, ET-OV, reversibly blocked the excitatory, action of OT but did not affect the excitatory action of SP at all. Note OT actions were completely blocked at 2 rain after the injection of ET-OV, partially recovered at 15 min, and fully recovered at 30 min. Also, OT and TG-OT evoked similar numbers of spikes in this neuron. (b) ET-OV severely attenuated excitatory actions of OT and TG-OT in this silent neuron. The same dose of AVP evoked no spikes at all. (c) Excitatory action of AVP was blocked by a V]-receptor antagonist but not by a V2-receptor antagonist. Note that OT evoked a substantial number of spikes while the response to AVP was still in the process of recovery. (d) An example of the neuron that responded only to TRH. This silent neuron from an estrogentreated female did not respond to any other neuropeptides tested, i.e., OT, SP, PRL, or LHRH. Note also the consistency of the responses to TRH.

Oxytocin Substantial n u m b e r s of the tested P A G n e u r o n s were excited by 1 riM to 100 n M O T as a function of dose (Table 1). Responses of the P A G n e u r o n s to O T were primarily excitatory: a few n e u r o n s (8 of 122, 6.6%) showed inhibition to OT. Although there was no statistically significant difference, more n e u r o n s tended to be excited in the estrogen-treated group, especially at the lower doses ( 1 n M a n d 10 nM). The response size of affected n e u r o n s at 10 n M was also c o m p a r e d between the two groups. Again, the size tended to be larger in O V X + E preparations (1044 + 155 spikes; m e a n + SEM) t h a n in O V X preparations (785 + 196 spikes), but the difference was not significant. The distribution of OT-responsive n e u r o n s in the P A G was different from that of SP-responsive n e u r o n s (Fig. 1). While the latter was evenly distributed, significantly more n e u r o n s were

responsive to O T in the rostral third c o m p a r e d to the caudal third of the P A G (p < 0.05) or its dorsal half (p < 0.05) (Fig. l). N e u r o n s in the ventral half tended to be more responsive to O T t h a n those in the dorsal half, although this difference was not significant (Fig. 1). Regularly, the lower dose of O T (1 nM) evoked fewer spikes t h a n the higher (10 riM) (Fig. 3a). In some OT-responsive neurons, characteristics of excitatory actions of O T were further e x a m i n e d pharmacologically. As shown in Fig. 3b, after an excitatory response to O T was detected, the perfusion m e d i u m was switched from regular ACSF to high Mg 2+ a n d low Ca 2+ ACSF to block synaptic transmission. This experimental ACSF did not abolish the response to O T (three out of three tested neurons). We also tested some OT-responsive n e u r o n s with TG-OT, a specific O T agonist, and A V P (e.g., Fig. 3c) to e x a m i n e specificity of O T action. The n u m b e r of spikes evoked by T G - O T or A V P is plotted in Fig. 4 against that evoked

N E U R O P E P T I D E S A N D PAG N E U R O N A L A C T I V I T Y

E+OVX

NONE

OVX

[]

NONE

and TRH

0T

E÷OVX

IoTI

NONE

INONE

OVX

E+OVX

,NE

NONE1

OVX

l--

0%

I

20% %

4

,

j

,

I

40% 60% 80% of Tested Neurons

,

(from E + O V X or O V X preparations) tested with all three peptides, 10 neurons (21%) responded to all three, nine neurons (19%) responded to only one of them, and eight neurons (17%) responded to none of them. The remaining 20 neurons (43%) responded to either two ofSP, OT, or T R H . Neuronal responses of PAG neurons tested with any two of them were further analyzed for E + O V X and O V X groups separately (Fig. 6). This revealed that: 1) all of OT-responsive neurons were SP or T R H responsive in the O V X group, whereas in the E + O V X group, there were neurons responsive only to OT; and 2) more T R H responsive neurons were O T nonresponsive in the O V X group than in the OVX + E group.

Luteinizing Hormone-Releasing Hormone and Prolactin

SP and TRH

!

971

I

100%

FIG. 6. Classification of PAG neurons based on their ability to respond to each member of a pair of peptides. For example, under OT and SP, the tabulation OT and SP shows the percent of neurons responding to both, whereas OT tabulates neurons responding only to OT. Numbers on the right side are numbers of neurons tested with the two peptides indicated above the corresponding bar graphs. by O T in the same neuron, showing that the response to O T correlated well with the response to T G - O T (r - 0.976, p < 0.01 ) but not with that to AVP (r - 0.064, NS). Excitatory actions of OT, TG-OT, and AVP were challenged with OT-, V~-, or V~receptor-specific antagonists. As shown in Fig. 5a-b, excitatory actions of O T or T G - O T were reversibly blocked by the specific O T antagonist, ET-OV. Out of six neurons, ET-OV completely blocked O T excitatory action in four neurons and attenuated it in two neurons. In contrast, vasopressin receptor antagonists, specifically the V~-, but not V2-, receptor antagonist, blocked or attenuated AVP action but not O T action in all four neurons tested (Fig. 5c).

Thyrotropin-Reh, asing Hormone Thyrotropin-releasing hormone excited PAG neurons in both OVX + E and O V X preparations, as a function of dose (Table 1). Although it was not significant, T R H tended to excite more neurons in O V X preparations. The distribution pattern of T R H responsive neurons was also different from that of SP-responsive neurons (Fig. 1). Significantly more neurons responded to T R H in the dorsal half of the PAG than in the ventral half of the PAG (p < 0.05) (Fig. 1). Importantly, T R H did not always excite the same neurons as other neuropeptides (i.e., SP or OT) did. For example, some neurons were responsive only to T R H , as shown in Fig. 5d.

Responses to More Than One Neuropeptide Responses to three neuropeptides, SP, OT, and T R H , at 10 n M were compared in individual neurons. Out of 47 neurons

Luteinizing hormone-releassing hormone or PRL, which are known to facilitate lordosis when applied in the PAG, had no direct excitatory or inhibitory actions on spontaneous activity of PAG neurons under these conditions. None of 27 tested neurons from O V X + E preparations and only one out of 18 tested neurons from O V X preparations responded to 10 or 100 n M of L H R H . Likewise, only one out of 12 tested neurons from O V X + E preparations was excited and only one out of six tested neurons from O V X preparations was inhibited by 1 ttM of PRL. For comparison, 37 out of 44 LHRH-nonresponsive and 11 out of 16 PRL-nonresponsive neurons were responsive to either or all SP. OT, or T R H (see Fig. 5d and 7a).

h@uromodulatory Actions of LHRII and SP Luteinizing hormone-releasing hormone and SP were markedly contrasted in their transmitter-like actions on PAG neurons: SP excited PAG neurons (Figs. 2a-d and 7a'), whereas L H R H virtually never acted this way (Figs. 5d and 7a). Reasoning from the behavioral effects of L H R H , it was necessary to search for modulation of responses of PAG neurons to lordosis-relevant neurotransmitters, G A B A and NE, using O V X + E tissue preparations. During experiments that involved recording from single neurons for up to 8 h, it was found that both L H R H and SP modulated PAG neuronal responses (Table 2). Luteinizing hormonereleasing hormone could either potentiate (Fig. 7b) or attenuate (Fig. 7c) inhibitory responses to T H I P (GABAA agonist) and baclofen (GABAB agonist), and tended to potentiate both excitatory and inhibitory responses to NE (not shown). These L H R H effects persisted over a long period of time (up to 50-70 min, Fig. 7b-c). Substance P at 1 riM, which excited only 10% of tested neurons in O V X + E preparations (Table 1 and Fig. 7a'), also had long-lasting neuromodulatory actions (Table 2, Fig. 7c). Substance P potentiated T H I P and baclofen effects at a dose (1 nM) rarely effective for direct excitatory actions. It also tended to potentiate excitatory actions of NE (Fig. 7d) and attenuate inhibitory actions of NE (not shown). DISCUSSION

We found that SP excites PAG neurons in a robust manner, which suggests that SP acts as a neurotransmitter in the PAG. Microinfusion of SP has been shown to facilitate lordosis in the PAG (6). Previous anatomical studies also showed that 17% or 20% of SP-containing V M N neurons project to PAG in rats (5) or guinea pigs (30), respectively. These findings led to the idea that SP originating in the V M N facilitates lordosis by acting on PAG neurons. Our present results provide electrophysiological support for this idea. Together with previous neuroanatomical

972

OGAWA,

KOW AND PFAFF

spikes/sec

=pikes/sec 1=

a'

spikes/see 1

spikes/sec I

I~Q e . u l

10

P

iated

8

Lged)

Baclofen (Prolonged)

Potentiated

J

310

3 2 ~ in

6 4 2

°,o

3OO

splkecJsec

NE 2.5pM

10

NE NOChange

RE

, 0 0

NE Potentiated

Potentiated

i

NE Recovered

I

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 20 30 40 60 70 80 gO

Potentiated

10

Pote~tia

5

130

J~ 140

• .............. 110

, 120

Recovering

N~C~

o ol I .............

100

TsT,

ale

g

v 150

160

170

180

190

200

210mi n

FIG. 7. Neuromodulatory vs, direct neurotransmitter-like actions of LHRH and SP on PAG neurons. All units were recorded from the middle third of the dorsal PAG in estrogen-treated tissue preparations. (a and a') LHRH (100 nM) and PRL (l /~M) failed to affect spontaneous activity of this neuron (a), whereas SP (10 nM) could repeatedly excite it. Note SP at 1 nM had no such excitatory effect on this unit (a'). (b) LHRH (100 riM) potentiated (prolonged) the inhibitory actions of THIP (40 #M) and baclofen (400 riM), This neuromodulatory action of LHRH lasted about 50 rain: inhibitory actions of baclofen and THIP eventually recovered to pre-LHRH levels. Note that it was estimated (19) that concentration of an applied agent in the recording chamber reached a peak transiently at 60 s, then dissipated quickly and was almost nondetectable in 5 rain. (c) In this case, LHRH ( 100 nM) attenuated inhibitory actions of THIP (4 uM) and baclofen (200 nM). After LHRH application, the inhibitory action of THIP became smaller and smaller and was then completely absent. It started to recover about 70 min after LHRH. The inhibitory action of baclofen was less affected. Application of 50/~l saline did not attenuate inhibitory actions of THIP or baclofen, indicating that modulation observed after LHRH application was not a simple effect of the injection. Substance P at 1 n M also modulated (potentiated) inhibitory actions of THIP and baclofen in the same neuron. (d) SP (1 riM) potentiated excitatory action of NE in this silent unit. Numbers marked with * indicate the numbers of spikes evoked by NE. After single injection of SP, 27% more spikes were evoked by NE, and after double injection of SP, three time more spikes were evoked by NE. Note the apparent recoveries in both cases. Note also that neurons (c) and (d) were stable for a long period of time.

NEUROPEPTIDES AND PAG NEURONAL ACTIVITY

973

TABLE 2 SUMMARY OF NEUROMODULATORY ACTIONS OF LHRH (100 nM) AND SP (1 riM) ON THIP, BACLOFEN AND NE RESPONSES IN OVX + E PREPARATIONS LHRH Transmitter Agents

THIP (GABAAagonist) Baclofen (GABABagonist) NE NE

Substance P

Response Direction

Potentiated

Attenuated

No Change

Potentiated

Attenuated

No Change

Inhibition Inhibition Excitation Inhibition

6 3 2 3

5 5 1 1

2 4 4 0

5 3 3 0

0 1 I 2

2 2 3 0

findings of SP-containing terminals (23,28) and SP receptors (25,54) in the PAG, we can hypothesize that endogenous SP, by influencing neuronal activity of PAG neurons, may play a major role for PAG control of lordosis in receptive females. Importantly, the SP antagonist DPDT-SP had only a weak antagonistic effect on SP excitation (Fig. 2c-d), consistent with its ineffectiveness for antagonizing lordosis-facilitatory effects of SP in the PAG (6). Substance P was found here to act, also, as a neuromodulator in the PAG of estrogen-primed rats. Neuromodulatory actions of SP have been reported electrophysiologically in cortical neurons (20) and spinal neurons (7,36,53). Modulations of GABAergic agonists and NE actions by SP were unimodal: SP potentiated inhibitory actions of THIP or baclofen in all or most of the modulated PAG neurons. This observation is consistent with the facts that both SP (6) and GABAA agonist (27) infused into the PAG facilitate lordosis in estrogen-primed female rats. Thus, it may be hypothesized that lordosis-facilitatory actions of SP involve potentiation of the endogenous GABAergic system by SP. A recent study has shown that SP evokes release of GABA in the spinal cord (40). It should be also noted that modulatory actions of SP were long lasting. This appears consistent with behavioral effects of SP, which appeared as early as 5 min after infusion and lasted for at least 3 h (6). Sbustance P also tended to potentiate excitatory actions of NE and attenuate inhibitory actions of NE, making net NE effects more excitatory. Since SP could exert a neuromodulatory action at a dose rarely effective for direct excitatory actions, it seems likely that SP may play a behaviorally important role as a neuromodulator in the PAG. Oxytocin also was found to act as an excitatory neurotransmitter in the PAG. Since previous binding studies reported high levels of AVP binding sites but only very few OT binding sites in the PAG (52), this finding was not anticipated. One possibility was that OT might not act on the recorded neurons in the PAG but on some neurons with high levels of OT binding sites located outside of the PAG. However, we found that the excitatory action of OT was not abolished by blockade of synaptic transmission with high Mg2+ and low Ca 2+ ACSF (Fig. 3b). This indicates that OT indeed acts on the neurons recorded within the PAG. The other possibility was OT might act through AVP receptors in PAG instead of through OT receptors. We tested this possibility by i) comparing excitatory actions of OT with those of the OT-specific agonist, TG-OT, and those of AVP (Figs. 3c and 4), and ii) challenging them with an OT-specific antagonist, ETOV, and V,- or V2-receptor antagonists (Fig. 5a-c). Since it was found that responses to OT were i) well correlated with the response to TG-OT (r = 0.976) but not with that to AVP (r = 0.064), and ii) were blocked by OT-specific antagonists, ET-OV, but not by V~- or V2-receptor antagonists, we also could eliminate the second possibility. All these data indicate that OT acts as an

excitatory neurotransmitter on PAG neurons through OT receptors, the first demonstration of OT-responsive neurons in the PAG. Furthermore, we could detect possible estrogen effects on responsiveness to OT, a trend toward increases in proportion of OT-responding neurons and response size. Further analysis revealed that the trend toward more OT-responsive neurons in the estrogen-treated group was mainly due to appearance of neurons responsive only to OT (Fig. 6). Unlike the VMN, where OT facilitates lordosis in estrogen- and progesterone-treated rats (45,46), however, the behavioral functions of OT in the PAG have not been well established. Although PAG infusion of OT was found to be ineffective in facilitating lordosis in estrogentreated female rats (3,10), more studies under different hormonal and stimulus conditions are needed. Excitation of PAG neurons by TRH is consistent with the finding in a previous in vivo experiment in which 56% of the neurons were excited in ovariectomized female rats (44). Regional distribution of TRH-responsive neurons was different from that of SP- or OT-responsive neurons (Fig. 1). Thyrotropinreleasing hormone-responsive neurons were more frequent in the dorsal and caudal PAG, in contrast to OT-responsive neurons, which were distributed more in the ventral and rostral PAG. Substance P-responsive neurons, on the other hand, were distributed more or less evenly throughout the PAG. Also, TRHresponsive neurons were not always SP or OT responsive (Fig. 6). These findings may suggest that although SP, OT, and TRH all have neurotransmitter-like excitatory actions on PAG neurons, they affect a different population of PAG neurons from each other. Prolactin had very minor effects on PAG neuronal activity, consistent with a previous in vivo study (4). Luteinizing hormone-releasing hormone, also, had no direct effects on spontaneous activity of PAG neurons. This result contrasts with previous studies. Chan et al. (4) reported from an in vivo study that iontophoretically applied LHRH affected (mainly inhibited) half of the PAG neurons in estrogen + progesterone-primed ovariectomized rats but had no effect in nonprimed rats. During an in vitro experiment, it was found that LHRH at 250 nMaffected (mainly excited) half of the medial preoptic area (MPOA) neurons sampled in both estrogen-primed and nonprimed ovariectomized rat preparations (33). Since LHRH has strong behavioral effects in the PAG (37,38,43,50), the absence of a direct transmitter action was surprising. One possibility is that in the PAG, LHRH may act mainly as a neuromodulator. In fact, we found that in estrogen-treated preparations, LHRH at 100 nM, which virtually never affected baseline firing rates of PAG neurons, could modulate inhibitory actions ofGABAergic agonists and excitatory and inhibitory actions of NE. Notably, this type of action lasted for long periods of time, up to 60 min. In contrast to neuromodulatory actions of SP, modulations of GABAergic

974

OGAWA, KOW AND PFAFF

agonist by L H R H were not always in the same direction. Although m e c h a n i s m s o f this heterogeneity are u n k n o w n , it is hypothesized that strong lordosis-facilitatory effects o f L H R H in the PAG may involve modulatory actions o f L H R H on the endogenous GABAergic system, since PAG infusion o f a GABAA agonist facilitates lordosis and a GABAA antagonist inhibits lordosis in estrogen-primed female rats (27). The fact that L H R H acts on PAG neurons p r e d o m i n a n t l y in a n e u r o m o d u l a t o r y m a n n e r may be related to the finding that endogenous L H R H

released in the PAG seems to affect PAG neurons in a nonsynaptic way by diffusion through the intercellular space (2). ACKNOWLEDGEMENTS The authors are grateful to Dr. M. Harrison and Mr. B. Stromquist for their helpful suggestions in developing the data acquisition and analysis software, and Dr. S. Schwartz-Giblin for her important contribution to this project. Supported by NIH grant HD-05751.

REFERENCES 1. Akesson, T. R.; Micevych, P. E. Estrogen concentration by substance P-immunoreactive neurons in the medial basal hypothalamus of the female rat. J. Neurosci. Res. 19:412-419; 1988. 2. Buma, P. Characterization ofluteinizing hormone-releasing hormone fibers in the mesencephalic central grey substance of the rat. Neuroendocrinology 49:623-630; 1989. 3. Caldwell, J. D.; Jirikowski, G. F.; Greet, E. R.: Pedersem C. A. Medial preoptic area oxytocin and female sexual receptivity. Behav. Neurosci. 103:655-662; 1989. 4. Chan, A.; Dudley, C. A.; Moss, R. k Hormonal modulation of the responsiveness of midbrain central gray neurons to LH-RH. Neuroendocrinology 41 : 163-168:1985. 5. Dornan, W. P.; Akesson, T. R.: Micevych, P. E. A substance P projection from the VMH to the dorsal midbrain central gray: Implication for lordosis. Brain Behav. Bull. 25:791-796; 1990. 6. Dornan, W. P.; Malsbury, C. W.; Penney, R. B. Facilitation of lordosis by injection of substance P into the midbrain central gray. Neuroendocrinology 45:498-506; 1987. 7. Dougherty, P. M.; Willis, W. D. Enhancement of spinothalamic neuron responses to chemical and mechanical stimuli following combined micro-iontophoretic application of N-methyl-D-aspartic acid and substance P. Pain 47:85-93; 1991. 8. Femano, P. A.; Schwartz-Giblin, S.; Pfaff, D. W. Brain stem reticular influences on lumbar axial muscle activity. I. Effective sites. Am J. Physiol. 246:R389-R395: 1984. 9. Femano, P. A.; Schwartz-Giblin, S.; Pfafl, D. W. Brain stem reticular influences on lumbar axial muscle activity. II. Temporal aspects. Am. J. Physiol. 246:R396-R401; 1984. 10. Harlam R. E.; Shivers, B. D.; Pfaff, D. W. Midbrain microinfusions ofprolactin increase the estrogen-dependent behavior, lordosis. Science 219:1451-1453; 1983. 11. Harlan, R. E.; Shivers, B. D.; Fox, S. R.; Kaplove, K. A.; Schachter, B. S.: Pfafl; D. W. Distribution and partial characterization of immunoreactive prolactin in the rat brain. Neuroendocrinology 49:722: 1989. 12. Hrkfelt, T.; Fuxe, K.; Johansson, O.: Jeffcoate, S.: White, N. Thyrotropin releasing hormone (TRH)-containing nerve terminals in certain brain stem nuclei and in the spinal cord. Neurosci. Lett. 1: 133-139: 1975. 13. Hsueh, A. J. W.; Schaeffer, J. M. Gonadotropin-releasing hormone as a paracrine hormone and neurotransmitter in extra-pituitary sites. J. Steroid Biochem. 23:757-764: 1985. 14. Jennes, L.; Dalati, B.; Corm, P. M. Distribution of gonadotropinreleasing hormone agonist binding sites in the rat central nervous system. Brain Res. 452:156-164; 1988. 15. Kow, L.-M.; Pfaff, D. W. Vasopressin excites ventromedial hypothalamic glucose-responsive neurons in vitro. Physiol. Behav. 37: 153-158; 1986. 16. Kow, L.-M.; Pfaff, D. W. Responses of ventromedial hypothalamic neurons in vitro to norepinephrine: Dependence on dose and receptor type. Brain Res. 413:220-228: 1987. 17. Kow, L.-M.; Pfaff, D. W. Transmitter and peptide actions on hypothalamic neurons in vitro: Implications for lordosis. Brain Res. Bull. 20:857-861; 1988. 18. Kow, L.-M.; Pfaff, D. W. Neuromodulatory actions of peptides. Annu. Rev. Pharmacol. Toxicol. 28:163-188; 1988.

19. Kow, L.-M.; Johnson, A. E.; Ogawa, S.; Pfafl; D. W. Electrophysiological actions of oxytocin on hypothalamic neurons in vitro Neuropharmacological characterization and effects of ovarian steroids. Neuroendocrinology 54:526-535; 199 I. 20. Lamour, Y.; Dutar, P.: Jobert, A. Effects of neuropeptides on rat cortical neurons: Laminar distribution and interaction with the effect of acetylcholine. Neuroscience 10:107-117; 1983. 21. Lechan, R. M.; Molitch, M. E.; Jackson, I. M. D. Distribution of immunoreactive human growth hormone-like material and thyrotropin-releasing hormone in the rat central nervous system: Evidence for their coexistence in the same neurons. Endocrinology 112:877884; 1983. 22. Liposits, Z.: Setalo, G. Descending luteinizing hormone-releasing hormone(LH-RH) nerve fibers to the midbrain of the rat. Neurosci. Lett. 20:1-4:1980. 23. Ljungdahl, A.; H6kfelt, T.; Nilsson, G. Distribution of substance Plike immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals. Neuroscience 3:861-943; 1978. 24. Manaker, S.: Winokur, A.: Rostene, W. H.: Rainbow, T. C. Autoradiographic localization of thyrotropin-releasing hormone receptors in the rat central nervous system. J. Neurosci. 5:167-174: 1985. 25. Mantyh, P. W.; Hunt, S. P . Maggio, J. E. Substance P receptors: Localization by light microscopic autoradiography in rat brain using [3H]SP as the radioligand. Brain Res. 307:147-165; 1984. 26. Mantyh, P. W.: Hunt, S. P. Thyrotropin-releasing hormone (TRH) receptors: Localization by light microscopic autoradiography in rat brain using [3H][3-Me-His2]TRH as the radioligand. J. Neurosci. 5: 551-561: 1985. 27. McCarthy, M. M.: Pfafl: D. W.; Schwartz-Giblin, S. Midbrain central gray GABAA receptor activation enhances, and blockade reduces, sexual behavior in the female rat. Exp. Brain Res. 86:108-116; 1991. 28. Moss, M. S.: Basbaum, A. 1. The peptidergic organization of the cat periaqueductal gray. I1. The distribution of immunoreactive substance P and vasoactive intestinal polypeptide. J. Neurosci. 7:14371449: 1983. 29. Myers, R. D.: Metcalf, G.; Rice, J. C. Identification by microinjection of TRH-sensitive sites in the cat's brainstem that mediate respiratory, temperature and other autonomic changes. Brain Res. 126:105-115; 1977. 30. Nielson, K. H.; Blaustein, J. D. Some of the ventrolateral hypothalamic neurons containing substance P and progestin receptors project to the midbrain central gray in guinea pigs. Soc. Neurosci. Abstr. 17:434 (# 177.9): 1991. 31. Ogawa, S.; Kow, L.-M.: Pfafl; D. W. Electrophysiological responses of periaqueductal gray neurons of female rats to LHRH, substance P, oxytocin, and TRH in vitro. Soc. Neurosci. Abstr. 16:266 (#114.7): 1990. 32. Ogawa, S.; Kow, L.-M.; Pfafl~ D. W. LHRH and substance P modulate in vitro electrophysiological responses of periaqueductal gray neurons to GABA agonists and norepinephrine in estrogen-treated female rats. Soc. Neurosci. Abstr. 17:1061 (#420.2); 1991. 33. Pan, J.-T.; Kow, L.-M.; Pfaff, D. W. Neuromodulatory actions of luteinizing hormone-releasing hormone on electrical activity of preoptic neurons in brain slices. Neuroscience 27:623-628: 1988. 34. Pfaff, D. W. Estrogens and brain function: Neural analysis of a hormone-controlled mammalian reproductive behavior. New York: Springer-Verlag; 1980.

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AND PAG NEURONAL

ACTIVITY

35. Pfafl; D. W.; Schwartz-Giblin, S. Cellular mechanisms of female reproductive behaviors. In: Knobil, E.; Neill, J., et al., eds. The physiology of reproduction. New York: Raven Press; 1988:1487-1568. 36. Randic, M.; Hecimovic, H.; Ryu, P. D. Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurons. Neurosci. Lett. 117:74-80; 1990. 37. Riskind, P.; Moss, R. L. Midbrain central gray: LHRH infusion enhances lordotic behavior in estrogen-primed ovariectomized rats. Brain Res. Bull. 4:203-205: 1979. 38. Riskind, P.; Moss, R. L. Midbrain LHRH infusions enhance lordotic behavior in ovariectomized estrogen-primed rats independently of a hypothalamic responsiveness to LHRH. Brain Res. Bull. 11:481485; 1983. 39. Robbins, A.; Schwartz-Giblin, S.; Pfaff, D. W. Ascending and descending projections to medullary reticular formation sites which activate deep lumbar muscles in the rat. Exp. Brain Res. 80:463474; 1990. 40. Sakuma, M.; Yoshioka, K.; Suzuki, H.; Yanagisawa, M.; Onishi, Y.: Kobayashi, N.; Otsuka, M. Substance P-evoked release of GABA from isolated spinal cord of the newborn rat. Neuroscience 45:323330: 1991. 41. Sakuma, Y.; Pfaff, D. W. Facilitation of female reproductive behavior from mesencephalic central gray in the rat. Am. J. Physiol. 237: R278-284; 1979. 42. Sakuma, Y.; Pfaff, D. W. Mesencephalic mechanisms for integration of female reproductive behavior in the rat. Am. J. Physiol. 237: R285-290: 1979. 43. Sakuma, Y.: Pfaff, D. W. Modulation of the lordosis reflex of female rats by LHRH, its antiserum and analogs in the mesencephalic central gray. Neuroendocrinology 36:218-224; 1983. 44. Samson, W. K.; McCann, S. M.; Chud, L.; Dudley, C. A.; Moss, R. L. lntra- and extrahypothalamic luteinizing hormone-releasing hormone (LHRH) distribution in the rat with special reference to mesencephalic sites which contain both LHRH and single neurons responsive to LHRH. Neuroendocrinology 31:66-72; 1980. 45. Schumacher, M.; Coirini, H.; Frankfurt, M.; McEwen, B. S. Localized actions of progesterone in hypothalamus involve oxytocin. Proc. Natl. Acad. Sci. USA 86:6798-6801; 1989.

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46. Schumacher, M.; Coirini, H.; Pfaff, D. W.; McEwen, B. S. Behavioral effects of progesterone associated with rapid modulation of oxytocin receptors. Science 150:691-694; 1990. 47. Schwartz-Giblin, S.: Femano, P. A.; Pfaff, D. W. Axial electromyogram and intervertebral length gauge responses during lordosis behavior in rats. Exp. Neurol. 85:297-315; 1984. 48. Shivers, B. D.; Harlan, R. E.: Morrell, J. I.: Pfaff, D. W. Immunocytochemical localization of luteinizing hormone-releasing hormone in male and female rat brains. Neuroendocrinology 36:1-12; 1983. 49. Shivers, B. D.; Harlan, R. E.; Pfaff, D. W. A subset of neurons containing immunoreactive prolactin is a target for estrogen regulation of gene expression in rat hypothalamus. Neuroendocrinology 49: 23-27; 1989. 50. Sirinathsinghji, D. J. S. Modulation of lordosis behavior of female rats by corticotropin releasing factor, 3-endorphin and gonadotropin releasing hormone in the mesencephalic central gray. Brain Res. 336:45-55; 1985. 51. Sofroniew, M. V. Vasopressin, oxytocin and their related neurophysins. In: Bj6rklind, A.; H6kfelt, T., eds. Handbook of chemical neuroanatomy, vol. 4. GABA and neuropeptides in the CNS. Amsterdam: Elsevier; 1984:93-165. 52. Tribollet, E.: Barberis, C.; Jard, S.; Dubois-Dauphin, M.; Dreifuss, J. J. Localization and pharmacological characterization of high affinity binding sites for vasopressin and oxytocin in the rat brain by light microscopic autoradiography. Brain Res. 442:105-118; 1988. 53. Vincent, J.-D.; Barker, J. L. Substance P: Evidence for diverse roles in neuronal function from cultured mouse spinal neurons. Science 205:1409-1412: 1979. 54. Wolf, S. S.; Moody, Y. W.: Quirion, R.: O'Donohue, T. L. Biochemical characterization and autoradiographic localization of central substance P receptors using [*2Sl]physalaemin. Brain Res. 332: 299-307: 1985. 55. Wong, M.; Eaton, M. J.; Moss, R. L. Electrophysiological actions of luteinizing hormone-releasing hormone: lntracellular studies in the rat hippocampal slice preparation. Synapse 5:6570; 1990.