Effects of chemical stimulation in the periaqueductal gray on vocalization in the squirrel monkey

Brain Research Bulletin. Vol. 32, pp. 143-151,1993 Printed in the USA. All rights reserved.

0361-9230/93$6.00 + .oo Copyright0 1993Pergamon Press Ltd.

Effects of Chemical Stimulation in the Pe~aqueduc~l Gray on Vocalization in the Squirrel Monkey C.-L. LU* AND U. JijRGENSf

*Second Military Medical College, Shanghai, P.R. China f-German Primate Center, Kellnerweg 4, 3400 Glittingen, Germany Received 8 July 1992; Accepted 8 March 1993 LU, C.-L. AND U. JURGENS. E&m of chemical sGmdalion in the periaqueductalgrayon vocalizationin the squirrelmonkey. BRAIN RES BULL 32(2) 143-I 51, 1993.-Twenty-nine agonists and 32 antagonists of more than 10 transmitters known to be present in the ~~aqu~uctal gray (PAG) have been injected into the squirrel monkey’s PAG in order to test their effects on spontaneous vocalization at sites yielding vocalization with electrical stimulation. Vocalization could be elicited with the glutamate agonists s~ium-L-~utamate, L-aspartic acid, L-homocysteic acid, N-methyl-~aspa~ic acid, quisqualic acid, and kainic acid, the chohnergic agonists acetyicholine, carbachol, and muscarine, the monoaminergic agonist histamine, and the GABA antagonists bicuculline and picrotoxin. No vocalizations could be obtained with agonists of dopamine, noradrenaline, adrenaline, serotonin, GABA, glycine, nicotinic receptors, and endogenous opioids, as well as with antagonists of glutamate, acetylcholine, dopamine, noradrenaline, adrenaline, serotonin, histamine, glycine, GABA-B, 6- and r-receptors. Blocking of spontaneous vocalization was obtained with the nonspecific glutamate antagonist kynurenic acid and the GABA-A receptor agonist muscimol. The results indicate that the production of vocalization depends upon the activation of glutamatergic synapses in the PAG. GABAergic afferents seem to have a tonic inhibitory control on the periaqueductal vocalization mechanism, while acetylcholine and histamine seem to exert only a transient m~ulato~ control. Vocalization

THE ~~aquedu~ta1

Periaqueductal gray

gray of the midbrain relay station of the v~alization-controlling

Squirrel monkey

Glutamate

Acetylcholine

GABA

Histamine

related peptide (63), and vasoactive intestina1 poi~ptide ( 13,25), all possibly contribute to the neural activity within this structure. In order to find out which of these substances may be specifically involved in the control of vocal behavior, we have injected in the present study 6 I agonists and antagonists of more than 10 different neurotransmitterslneuromodulators into the periaqueductal gray and studied their effects on spontaneous vocalization in the squirrel monkey.

seems to be a crucial system in mammals.

Its electrical stimulation has been reported to yield vocalization in many species (5,18,40,48,50,64,68,73). Its destruction causes mutism (I, 15,2 1,4 1,64). Single-unit recording reveals numerous cells, the activity of which is vocalization correlated (44). Neuroanatomically, the periaqueductal gray receives afferents from virtuaily all other brain areas yielding vocalization when electrically stimulated (41); its e&rents project to the region of the nucl. retroambiguus where the motor coordination of laryngeal, respiratory and articulator-y movements is assumed to take place (35). ImmunohistochemicaI and autoradiographic studies have identified a number of ne~otransmitte~ and tmnsmi~er-binding sites within the ~~aqueductal gray. From these studies, it appears that excitatory amino acids (I 1,12,23,33,53,55), GABA ( IO,1 1,22), glycine (6) acetylcholine (5 1,66), dopamine ( 16,19), noradrenaline (57) adrenaline (57), serotonin (59,67), histamine (2,17,37), endogenous opioids (36,38,49,60,74), cholecystokinin (38), somatostatin (65), neurotensin (62,69), substance P (32,45,54), neuropeptide Y (14) galanin (43), calcitonin gene-

METHOD

The experimental subjects were 29 captivity-bred squirrel monkeys (Suimiri sciureus) with a body weight of 530 to 900 g. Under deep narcosis (40 mg/kg pentobarbital sodium), a midline incision was made into the scalp and a 20 X 20 mm platform with 6 to IO electrode guides was fixed in a stereotaxic operation on the skull. For this fixation, four T-shaped openings were made into the skull using a dental drill. Stainless steel screws were inserted into the openings with the heads placed between dura and skull. The screws were anchored by the help of nuts and dental cement. Six to 10 additional small holes were drilled at

r To whom requests for reprints should be addressed.

143

144

LU AND JURGENS

I

TABLE VOCALIZATION-INDUCING

SUBSTANCES

TESTED

Substances Tested

Category

Doses (nmol)

Sites Tested

voc. sites

Mono-sodium-L-glutamate (Glu) L-Aspartic acid (Asp) L-Homocysteic acid (HCA) N-Methyl-r%aspartic acid (NMDA) Quisqualic acid (Quis) Kainic acid (Kain) Kynurenic acid DL-2-Amino-5-phosphonovaleric acid L-Glutamic acid diethyl ester o_Glutamylaminomethylsulfonic acid 6-Cyano-7-nitroquinoxaline-2,3-dione DL-2-Amino-4-phosphonobutyric acid (APB)

Transmitter Transmitter Transmitter EAA* agonist EAA* agonist EAA* agonist EAA antagonist NMDA antagonist Non-NMDA antagonist Non-NMDA antagonist Non-NMDA antagonist EAA antagonist/EAA agonist

0,07-59 I 0,09-376 0,02-50 0,0007- 150 0,0004-53 0,0002-35 3-132 0,3-63 4-83 4-66 0.4-43 27-7 I

I7 8 57 27 I7 23 57 35 I3 I3 13 9

II 7 43 24 12 16 0 0 0 0 0 5

y-Aminobutyric acid (GABA) Muscimol hydrobromide Piperidine-4-sulfonic acid Imidazole-4-acetic acid Nipecotic acid Baclofen Bicuculline methiodide (Bicuc) Picrotoxin (Picro) Phaclofen

Transmitter GABA-A agonist GABA-A agonist GABA-A agonist GABA agonist GABA-B agonist GABA-A antagonist GABA-A antagonist GABA-B antagonist

29-485 0.03-5 I 40-9 I 0.7-68 78 O,l-47 0.05-I 0,08-0.8 24-40

35 45 2 9 I2 I3 48 24 5

0 0 0 0 0 0 25 I3 0

Glycine Strychnine hydrochloride

Neurotransmitter Glycine antagonist

13-666 I-34

40 39

0 0

Acetylcholine chloride (Ach) Carbachol (Carb) Muscarine chloride (Must, M) Nicotine ditartrate (Nicot, N) Physostigmine sulfate Atropine sulfate Scopolamine hydrochloride Quinuclidinyl benzilate Benztropine mesylate Pirenzepine dihydrochloride Mecamylamine hydrochloride

Transmitter Ach agonist Ach agonist Ach agonist Ach agonist M antagonist M antagonist M antagonist M antagonist M 1 antagonist N antagonist

55-l IO 5,5-55 48 I l-22 15 7-37 3-74 6-30 25 24-30 5-123

6 57 8 I2 4 52 38 I8 4 5 35

I 32 2 0 0 0 0 0 0 0 0

Noradrenaline bitartrate Phenylephrine hydrochloride Clonidine hydrochloride Metaraminol bitartrate Isoproterenol hydrochloride Phentolamine mesylate Phenoxybenzamine hydrochloride Benextramine tetrahydrochloride Tolazoline Prazosin hydrochloride Yohimbine hydrochloride Propranolol hydrochloride

Transmitter al-Agonist N2-Agonist a-Agonist @-Agonist n-Antagonist a-Antagonist a-Antagonist
2-157 32 3-47 16-32 20-101 2-27 29 I4 5-64 2-12 3-26 17-42

33 6 6 8 I9 5 5 4 28 5 9 23

0 0 0 0 0 0 0 0 0 0 0 0

Dopamine hydrochloride (D) Apomorphine hydrochloride Haloperidol Pimozide

Transmitter D agonist D antagonist Dz antagonist

13-105 0.3-33 3-67 22

6 33 31 I

0 0 0 0

Serotonin creatinine sulfate (5-HT) Quipazine dimaleate Cyproheptadine hydrochloride Methysergide maleate

Transmitter 5-HT agonist 5-HT antagonist 5-HT antagonist

13-52 2-22 3-46 8-2 I

24 26 30 II

0 0 0 0

continued

VOCALIZATION

AND

PERIAQUEDUCTAL

GRAY

145

TABLE

1

CONTINUED Substances Tested

Category

Histamine dihydrochloride (Hist, H) Diphenhydramine hydrochloride Chlorpheniramine maleate Promethazine hydrochloride Cimetidine

Transmitter H, antagonist H1 antagonist H1 antagonist H2 antagonist

Morphine sulfate Naloxone hydrochloride

Opioid agonist Opioid antagonist

Doses (nmol)

Sites Tested

Voc. Sites

7-136 27-86 3-26 16-78 24-40

49 39 13 18 8

20 0 0 0 0

2-31 3-69

35 34

0 0

* EAA = excitatory amino acid.

sites at which cannula implantation was intended. Then the skin was closed with multiple sutures and the srews were exteriorized through small incisions made at the pressure points of the screws. The platform with the electrode guides was fixed to the screws with dental cement (47). After 1 week of recovery, electrodes were implanted into the periaqueductal gray under local anesthesia (2% xylocaine). The implantation as well as the following intracerebral injections were carried out with the animals sitting in a primate chair. The periods between the implantations and successive injection tests were spent by the animals in their home cage. The implanted electrodes consisted of a stainless steel tube of 0.47 mm outer diameter containing a teflon-coated stainless steel wire of 0.12 mm. The wire protruded from the tube for 2 mm and was uninsulated at the tip for 1 mm. The electrode was lowered in 1 mm steps through the periaqueductal gray. After each step, the elicitability of vocalization was tested using electrical stimulation with biphasic rectangular pulses of 30 Hz, 1 ms and 20-200 PA. When a site was found, the electrical stimulation of which yielded vocalization, the electrode tube was fixed with dental cement at the platform, the electrode wire was replaced by the cannula of a microsyringe, and different transmitter agonists and antagonists were injected. The intervals between successive injections varied, depending upon the substance and effect. In cases in which an injection had no effect and the following injection differed only in dose, but not type of substance, intervals were one to 2 h. Maximally, six injections were made per day. In cases of short-

TABLE PERCENT

2

OF SITES YIELDING VOCALIZATION SUBSTANCES

WITH TWO RESULTS

Vocalization-Inducing Substances

Substance Tested Voc With

GlU

GlU HCA NMDA Quis Kain Bicuc Carb Hist

69 62 73 69 83 88 80

HCA

NMDA

Quis

Kain

Bicuc

Carb

Hist

100

100 100

100 75 91

100 80 100 100

100 54 63 100 78

75 67 63 78 64 60

80 53 60 80 75 52 42

77 100 100 88 93 100

100 100 100 92 100

86 67 88 67

64 78 60

lasting effects or changes in substance, injections followed each other by at least 5 h. In cases of long-lasting effects, injection intervals were 17 to 24 h. After each injection, reproducibility of vocalization was tested by electrical stimulation of the injection site. For this purpose, the cannula was replaced by an electrode wire introduced into the chronically implanted stainless steel tube. If electrical stimulation did not produce vocalization 24 h after injection, 27 nmol homocysteic acid was injected into that site, thus, testing for chemical elicitability of vocalization. If neither electrical nor chemical stimulation yielded vocalization any more, the preceding injection test was rejected and a new PAG electrode was implanted. Up to four different vocalization-eliciting sites were tested per animal. All electrically and chemically elicited vocalizations were recorded on tape. Injection volumes were 200 nl throughout the test. The substances were dissolved in sterile water, wherever possible. Aspartic acid, quisqualic acid, kainic acid, kynurenic acid, 2-amino-Sphosphonovaleric acid, 2-amino-4-phosphonobutyric acid and baclofen were dissolved in 1 M NaOH and then neutralized with 0.1 M HCl. Haloperidol and cyproheptadine were dissolved in cont. acetic acid and neutralized with 0.1 M NaOH. 6-Cyano-7-nitroquinoxaline-2,3dione was dissolved in dimethylsulfoxide. Prazosin was dissolved in 50% methanol. Dopamine and apomorphine were dissolved in 0.1% ascorbic acid. At the end of the experiment, the animal was deeply narcotized with 40 mg/kg pentobabital sodium and perfused with physiological saline followed by 4% formaldehyde. The brains were removed, cut at 30 pm on a freezing microtome. Alternate sections were stained with Luxol fast blue/Nuclear fast red and cresyl violet. The injection positions were determined by the aid of the stereotaxic atlases of Gergen and MacLean (3 1) and Emmers and Akert (27).

68 79

59

Only substances that were tested with each other at least three sites are listed. Abbreviations, see Table 1.

Table 1 lists the substances and doses tested, the number of injection sites studied with each substance, and the number of sites yielding vocalization. It can be seen that vocalization was obtained with glutamate agonists, cholinergic agonists histamine, and GABA antagonists. No vocalizations could be elicited with catecholaminergic, serotonetgic, GABAergic, glycinergic, and opioidetgic agonists, nor with antagonists of any kind except GABA antagonists. In the case of the glutamate agonists, all excitatory amino acids tested were effective. In contmst, cholinergic agonists were effective only if they activated muscarinic receptors, i.e., activation of nicotinic receptors, was ineffective. GABA antagonists yielded vocalization only ifthey blocked GABA-A receptors; GABA-B receptor-blockade was again ineffective.

146

LU AND JijRGENS

Al

A2

GGB,

/-

v

cos /

FIG. I. Distribution of test sites. Each dot represents a site of the electrical stimulation that yields vocalization. Each site was tested with at least one representative of Glu agonists, Ach agonists, GABA-A antagonists and Hi&. Dot labeling: G: vocalization with at least one Glu agonist; C: vocalization with carbachol; B: vocalization with Bicuc or Picro; H: vocalization with Hist; X: no vocalization by chemical stimulation. Abbreviations of brain structures: AR: area praetectalis; BC: brachium conjunctivum; BP: brachium pontis; CC: corpus callosum; CI: capsula interna; Cd: nut. caudatus; Cf: nucl. cuneiformis; Col: colliculus inferior; CoS: colliculus superior; CP: commissura posterior; CT: corpus trapezoideum; DC: nucl. dorsalis tegmenti (Gudden); DR: nucl. dorsalis raphae; Ep: epiphysis; FLM: fasciculus longitudinalis medialis; FRP: formatio reticularis pontis; FRPc: formatio reticularis pontis caudalis; FRPo: formatio reticularis pontis oralis; FRTM: formatio reticularis tegmenti mesencephali: CC: griseum centrale = periaqueductal gray; CL: corpus geniculatum laterale; GM: corpus geniculatum mediale; GPO: griseum pontis; H: habenula; Hip: hippocampus; IP: nucl. interpeduncularis; LC: locus coeruleus; Lim: nucl. limitans; LM: nucl. dorsalis lemnisci lateralis; LLv: nucl. ventralis lemnisci lateralis; LM: lemniscusmedialis; LP: nucl. lateralisposteriorthalami; Md: nucl. medialisdorsalisthalami; NCS: nucl.centralis superior; NCT: nucl. trapezoidalis; NR: nucl. ruber, N III: nucl. oculomotorius; N IV: nucl. trochkaris; OS: oliva superior; P: nucl. posterior thalami; Pbl: nucl. parabrachiahs lateralis; PbM: nucl. parabrachialis medialis: Pg: nucl. pambigeminalis; Pul: nucl pulvinaris inferior, PuM: nucl. pulvinaris media& Py: tractus pyramidalis; P V: nucl. principalis n. trigemini; RTP: nucl. reticularis tegmenti pontis; SN: substantia nigra: ST: stria terminalis; VR: nucl. ventralis raphae; V: n. txigeminus.

D@rential I@ctiveness Table 2 shows that the most effective substance in vocalization production was NMDA (for abbreviations see Table 1). All sites yielding vocalization with Glu, HCA, Quis, Kain, Bicuc, Hist, and 92% of those producing vocalization with Carb yielded also vocalization with NMDA. But only 62% of the NMDA-vocalization sites produced vocalization with Glu; only 63% of the NMDA-positive sites produced vocalization with Bicuc and

Carb; only 77% of the NMDA-vocalization sites were positive with HCA. The least effective substance was histamine, with only 60% of the NMDA-positive sites producing vocalization with histamine. Within the group of excitatory amino acids the relative effectiveness raised from Glu via Quis, Kain, HCA to NMDA (Asp was not tested at a great enough number of sites). The excitatory amino acids as a group were more effective than Bicuc, Carb, and Hist. Within the latter, Bicuc was more effective than Carb, and Carb was more effective than Hist.

VOCALIZATION

AND PERIAQUEDUCTAL

147

GRAY

substance classes from the dorsal as well as ventral, medial as well as lateral, and rostra1 as well as caudal PAG. The combination of substances being effective at a specific injection site often changed within small distances. It should be kept in mind, however, that closely spaced injection sites in Fig. 1 always stem from different animals; in one and the same animal the minimal distance between injection sites was 2 mm. No vocalizations could be obtained from the most rostral and most caudal part of the PAG. If vocalization was obtained from the PAG by electrical stimulation, vocalization always could be obtained with chemical stimulation as well. In other words, all sites being responsive to electrical stimulation but remaining mute with chemical stimulation were located outside of the PAG. While Table 2 characterizes the effectiveness of the different substances by mutually comparing their ability to induce vocalization, Table 3 presents data on three additional criteria of effectiveness, namely, latency of vocalization induction, duration of vocalization, and threshold dosis. It can be seen that the shortest latencies were obtained with excitatory amino acids which produced vocalization within less than half a second at some sites. The time resolution of our method (0.2 s) does not allow us to further differentiate the latencies within the group of excitatory amino acids. While two of the carbachol vocalization latencies came close to the excitatory amino acid ones, there was a clear increase in minimal latency in GABA-A antagonists and histamine. Table 3 also reveals an enormous range of minimal latencies for one and the same substance, depending upon the injection site. Maximal vocalization duration again was very much dependent upon the injection site, apart from substance and dose. Duration was relatively short with the endogenous transmitters, Glu, Asp, HCA, and Ach. It reached 1 h and more with the artificial Glu agonists Kain and Quis, and the GABA antagonists Bicuc and Picro. The doses necessary for the elicitation of vocalization are lowest for the synthetic Glu agonists Quis, NMDA, and Kain, followed by the endogenous Glu agonists HCA, Glu, and Asp. The range of effective doses of GABA antagonists overlaps with that of Glu and Asp. Very high doses were necessary, if vocalization was to be induced by Ach, Carb, Must, Hist, and APB.

TABLE 3 DATA ON ADDITIONALCRITERIA Vocalization Latency (s)

Duration (min)

Threshold (nmol)

Asp HCA NMDA Quis Kain APB Bicuc Picro

0.3-72 0.3-12 0.3-240 0.3-360 0.3-900 0.3-600 l-330 2-660 6-900

0.03-3 0.08-4 0.08-14 0.1-25 0.03-124 0.2-60 2-4 6-194 0.08-60

0.1-29 0.2-38 0.04-4 1 0.007-17 0.003-0.03 0.01-0.2 27-55 0.1-I 0.2-0.8

Ach Carb Must Hist

2 0.5-1380 2-230 5-840

2 0.1-47 1-12 0.5-49

SubstancesTested GIU

110 5-55 48 7-109

Latency: minimal time between injection and vocalization at a specific site, irrespective of the dose used. Duration: maximal time of vocalization per injection at a specific site, using different doses. Threshold: minimal dose of a substance producing vocalization at a specific site.

Figure 1 shows the localization of 33 injection sites, all of which were tested with representatives of all four vocalizationeffective substance classes, that is, excitatory amino acids, GABAA antagonists, cholinergic agonists, and histamine. It can be seen that most injection sites yielded vocalization with more that one substance class. There are a few sites, however, producing vocalization only with an excitatory amino acid or a GABA-A antagonist. None of these 33 sites was effective with choline@ agents or histamine alone. Vocalization could be elicited from the PAG itself as well as from the laterally bordering tegmentum. No specific relationship between the localization of the injection site and the effectiveness of a specific substance class could be detected. That is, vocalization could be obtained with representatives of all four effective

Elicited Call Types

Most vocalization-effective injection sites yielded vocal sequences consisting of more than one call type. The sequences

TABLE 4 RELATIVE FREQUENCIESOF ELICITED CALL TYPES Glu

Asp

.

0

Purr

Trill Chuck Kecker Yap Peep Squeal Shriek Cackle Groan

HCA

NMDA

Quis

. .

0

.

. 0

.

0

0 0

. 0

;

Kain

Bicuc

Picro

Carb

Hist

. 0 .

.

0

0

0

;

0 .

0 0

.

0 . 0.

0 . . 0.

0

z

0

.

0

0 0

0

; .

;

;

;

0

;

;

The circles indicate the relative frequencies of the 10 most frequently elicited call types. Only substances with more than five vocalization-effective injection sites are listed. Large circles: call type can be obtained from at least 50% of the tested vocalization-eliciting sites. Medium circles: call type occurs at 25-49s of sites. Small circles: call type occurs at 12.5-24s of sites.

148

obtained with one and the same substance differed, depending upon injection dose and site. Higher doses usually produced more call types than lower doses. No systematic relationship could be detected between injected subarea of PAG and elicited call types. There was a relationship, however, between call type and injected substance. Table 4 shows that peeping, squealing, and shrieking, expressing alert, frustration, and defensive threat, respectively, occurred with a high probability after GABA antagonist injections [for a more detailed description of the functional significance of the different call types, see (39) and (7 I)]. Peeping, squealing, and shrieking almost never occurred after histamine injections. Histamine, on the other hand, produced trilling at every third vocalization-positive injection site, whereas this call type was never heard after picrotoxin injections and occurred at only two of 25 bicuculhne injection sites. Trilling, under normal conditions, is used to announce pleasurable events in the squirrel monkey. The most frequent call type obtained with histamine was groaning, a call expressing slight uneasiness. Groaning also was the most frequently elicited call after carbachol and excitatory amino acid injections. Carbachol effects, however, differed from histamine effects by a relatively high proportion of peep and squeal calls and a low proportion of trill and kecker calls. Kecker calls, which are normally used to recruit fellow combatants in mobbing actions against one or several outsiders, on the other hand, were quite common after excitatory amino acid injections. It can be seen from Table 4, however, that the effects of the different excitatory amino acids were not uniform. While Glu, Asp, HCA, NMDA, and Kain produced keckering and peeping quite often, these calls were only rarely heard after Quis injections. Quis injections, in contrast, yielded chuck calls at 25% of vocalization-positive sites. Chuck was never heard after Glu and Asp injections and occurred at only one of 24 NMDA injection sites. Chuck represents a low distance-contact call, normally uttered between animals with friendly relationships. Purring was another call type with an uneven distribution among excitatory amino acid reactions. It was quite common after Asp and Quis injections, but never occurred after Kain injections. Purring is considered an expression of well being. In contrast to keckering, peeping, chuck, and purring, cackling, a call expressing mild threat, showed a very uniform distribution among excitatory amino acid reactions. Altogether, Asp, HCA, and NMDA effects were more similar to each other than any ot them to Quis and Kain. The latter two, in addition, showed clearly different reaction profiles. Glutamate combined some of the effects of each of the three groups.

Inhibition of Voculization In one animal, it was possible to demonstrate an inibitory effect on vocalization with the unspecific glutamate antagonist kynurenic acid and the GABA-A agonist muscimol. This animal had a bilateral symmetrical placement of injection cannulae in the ventrolateral PAG at the level of the superior colliculus. Bilateral injection of 26 nmol kynurenic acid as well as of 0.5 nmol muscimol blocked spontaneous vocalization as well as vocalization induced by a visual stimulus (dummy leopard) and vocalization induced tactually (touching the flanks of the animals). Thirty-five additional agonists and antagonists tested at these sites did not block vocalization. More specifically, no inhibitory effect was observed with the cholinergic antagonists atropine, scopolamine, quinuclidinyl benzilate, benztropine, and mecamylamine, with glycine and strychnine, with all (Y- and /3-adrenergic agonists and antagonists tested, with dopamine agonists and antagonist, serotonin agonists and antagonists, histamine antagonists (histamine itself yielded vocalization), mor-

LU AND JURGENS

phine, naloxone, and glutamic acid diethyl ester. The same sites produced vocalization when injected with homocysteic acid, carbachol, bicuculline. and histamine. DISCUSSION

In the present study, four classes of substances have been found to produce vocalization when injected into the PAG. These are: glutamate agonists, GABA antagonists, cholinergic agonists, and histamine. Among these, glutamate agonists take an outstanding position. All sites within the PAG yielding vocalization with electrical stimulation also yielded vocalization with NMDA, the most effective glutamate agonist. In contrast, less than 60% of the electrical vocalization sites were effective with cholinergic agonists, histamine, and GABA antagonists. The lowest doses for the elicitation of vocalization were found with glutamate agonists. Glutamate agonists also showed the shortest latencies in vocalization production. Finally, bilateral injection of the glutamate antagonist kynurenic acid caused a reversible mutism. These observations suggest that the production of vocalization depends upon the activation of glutamate receptors in the PAG. Binding studies revealed a relatively high density of glutamate binding sites in the PAG (3,33,55,75). NMDA as well as nonNMDA binding sites were found, with the first dominating the latter by about 50% (75). Retrograde tracing studies with tritiated aspartate and wheat germ agglutinine-horseradish peroxidase injections into the PAG combined with glutamate and aspartate immunohistochemistry point to numerous glutamatergic and aspartergic projections to the PAG (4,l I, 12). Part of these projections comes from structures known to produce vocalization when electrically stimulated, such as the anterior cingulate cortex. medial amygdala, medial septum, dorsomedial hypothalamus, cuneiform nucleus, and parabrachial nuclei (40). As destruction of the PAG abolishes electrically elicitable vocalizations from all these structures, it may be concluded that the PAG represents a crucial relay station through which other structures exert their vocal control (41). The present study suggests that this control is glutamatergic and aspartergic. Excitatory amino acid-induced vocalization has been reported also for the cat PAG. According to Bandler and co-workers (7,8,9) growling and spitting can be obtained by Glu, Asp, HCA, and Kain injections into the PAG. In the cat, like in the monkey, effective sites were located throughout the PAG, that is, in ventral and dorsal, lateral and medial, rostra1 and caudal parts. Similar to the present study, all glutamate-effective sites produced vocalization with HCA but not all HCA-effective sites produced vocalization with glutamate. This finding is somewhat surprising, as HCA is assumed to exert its excitatory effect via NMDA receptors (42) while Glu acts at NMDA as well as non-NMDA receptors (52,56,58)-and. thus, should have a higher probability of being effective. A similar relationship has been found between Kain and Glu in the present study. Again, Kain is assumed to activate essentially one subtype of glutamate receptor, while Glu activates several (29.53,70). Nevertheless, Kain produces vocalization at more sites than Glu. We think the reason for this discrepancy is the different inactivation time of these substances. Glu, when injected into the brain, is quickly removed from synaptic regions by high-affinity reuptake into nerve terminals and glia (28). In contrast, the nonendogenous glutamate& agonist Kain does not have a specific uptake mechanism. It, therefore, may reach further remote structures than Glu by diffusion. In other words, we assume that there is a steeper concentration gradient for Glu than Kain in the surroundings of the injection site, thus limiting the excitatory effect of Glu to a smaller region than that of Kain. In accordance with the slower degradation

VOCALIZATION

AND PERIAQUEDUCTAL

GRAY

149

of Kain, the average vocalization time after Kain injections was 24 times longer than that after Glu injection. HCA, in contrast to Kain, is an endogenous Glu agonist (26). Nevertheless, the fact that the average vocalization time after HCA injections was three times longer than that of Glu indicates again a slower degradation of HCA in relation to Glu-and, thus, could also explain the greater number of HCA-positive vocalization sites in comparison to Glu. Vocalization has also been obtained with one Glu antagonist, namely APB. APB is assumed to inhibit glutamate release by acting at presynaptic autoreceptors (24,30). The facts that high doses of APB can depolarize neurons (24) and high doses were necessary to produce vocalization (27 nmol) suggest that the production of vocalization in the present study was due to direct excitatory effects of APB rather than to inhibition of glutamate release. Another antagonist capable of producing vocalization when injected into the PAG, is the GABA-A receptor antagonist Bicuc. As the effective dose in this case was low (0.1 nmol) and injection of the corresponding agonist muscimol had the opposite effect, causing mutism, we conclude that Bicuc vocalizations represent disinhibition responses, that is, responses due to blocking of GABAergic inhibitory synapses. In other words, vocalization can be elicited not only by direct activation of PAG neurons but also by removing a tonic inhibitory input to such neurons. Whether the disinhibition-induced vocalizations are due to a high spontaneous firing rate of specific vocalization-controlling neurons or due to a tonic excitatory input from other brain areas remains to be determined. The PAG has been shown to contain GABA-A as well as GABA-B receptors (22). However, neither the GABA-B receptor agonist baclofen nor the corresponding antagonist phaclofen had an effect on spontaneous vocalization rate. The GABAergic vocalization control, thus, seems to be exerted mainly via GABAA receptors. According to Beart et al. (1 l), GABAergic afferents to the PAG stem partly from GABA neurons within the PAG itself, partly from the bordering reticular formation, parabrachial nuclei, dorsal raphe, and substantia nigra. It is still unclear which of these inputs is/are involved in the inhibitory control of periaqueductal vocalization-inducing neurons. Cholinergic agonists form a third group of vocalization-inducing substances. In contrast to the excitatory amino acids,

high doses were necessary to produce vocalization, the reproducibility of vocalization with repetitive injections was low, and the latencies were clearly above those of glutamate agonists. Injection of choline@ antagonists did not block spontaneous vocalization. These findings suggest that Ach is not the primary excitatory transmitter of the periaqueductal vocalization mechanism but rather serves a modulatory role. This interpretation is supported by the observation that vocalization can be obtained with Must but not Nicot. The activation of muscarinic receptors, in contrast to that of nicotinic ones, leads to slowly developing depolarizations combined with a lowered conductance of voltagedependent K+ channels (58). As a result, Must does not change the spontaneous spike activity markedly, but causes a clear facilitation of excitatory inputs. In agreement with the lack of Nicot-induced vocalizations, autoradiographic binding studies reveal a lack of nicotinic, in contrast to muscarinic, binding sites in the PAG (46). The PAG receives its cholinergic input mainly from the pedunculopontine and laterodorsal tegmental nucleus (72). None of these nuclei yields vocalization when electrically stimulated in the squirrel monkey (40). The PAG itselfproduces Carb-induced vocalization not only in the squirrel monkey but also in the cat (20,34). Histamine has not yet been reported as a vocalization-inducing substance. In the present study, Hist-induced vocalizations had in common with cholinergically induced vocalization that a) the minimal latency was relatively long ( 17 times that of Glu); b) the threshold was high (about 100 times that of Glu); c) antagonists were not able to block spontaneous vocalization. These findings, together with the fact that the PAG receives its total histaminergic input from a small group of neurons in the tubero-mamillary region of the hypothalamus (6 I), a region not capable of producing vocalization when electrically stimulated, suggests that Hist, like Ach, plays only a modulatory role. In other words, histamine@ neurons feed into the vocalization system but are not part of the main line of this system. Despite the above-mentioned common characteristics of Hist- and Carbinduced vocalizations, both seem to be independent. Many Histeffective sites were ineffective with Carb, and many Car&effective sites did not yield vocalization with Hist. At sites yielding vocalization with Hist as well as Carb, vocalizations often differed.

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