GABA and specific GABA binding sites in brain nuclei associated with vagal outflow

GABA Neurorransmission Bruin Research Bullerin,

Vol. 5, Suppl.

2, pp. 325-328.

in the

Printed

U.S.A

GABA and Specific GABA Binding Sites in Brain Nuclei Associated with Vagal Outflow’ KAREN GALE, BETTY L. HAMILTON, SANDRA C. BROWN,2 WESLEY JANETTE DIAS SOUZA AND RICHARD A. GILLIS Departments

of Pharmacology

P. NORMAN,

and Anatomy, Georgetown University, Schools of Medicine and Dentistry, Washington, D. C. 20007.

GALE, K., B. L. HAMILTON, S. C. BROWN, W. P. NORMAN, .I. DIAS SOUZA AND R. A. GILLIS. GABA and specific GABA binding sites in brain nuclei associated with vagal outjlow. BRAIN RES. BULL. 5 Suppl. 2, 325-328, 1980.GABA levels and specific (sH) GABA binding were determined in several nuclei of cat brain. Since previous pharmacological studies (DiMicco et al. [3]) suggested that nucleus ambiguus (NA) may be the site of a GABA-receptor mediated inhibition of vagal outflow to the heart, we were interested in comparing the GABA content and density of ?H) GABA binding in NA with that of other nuclei known to contain GABAergic synapses. The GABA content of NA was 21.2 ? 2.4 nmol/mg protein, similar to that found in the caudate nucleus (28.4 2 2.9 nmohmg protein) and 2.5 fold higher than the GABA content of the surrounding reticular nuclei. In frozen-thawed and Triton X-100 treated membranes prepared from NA, specific GABA binding was 98 ? 28 fmol/mg protein when measured using 30 nM (3H) GABA. This was more than 3 fold higher than binding obtained in surrounding reticular tissue and approximately half the value obtained in substantia nigra. GABA content of paired right and left NA was nearly equal; however specific GABA binding of paired right and left NA differed markedly, with the right NA usually exhibiting greater specific binding than the left NA. Retrograde degeneration of vagal fibers of NA by intracranial sectioning of the right vagal trunk decreased the asymmetry in GABA binding of paired right and left NA. Asymmetry was also noted in the percent of the reflex-induced bradycardic response mediated by each vagus nerve. These results suggest that GABAergic synapses may be present in NA, and that some of the postsynaptic receptors for GABA may be associated with vagal efferents. The bilateral asymmetry in the physiological reflex-induced response coupled with the bilateral asymmetry in GABA binding in NA suggests that the degree of vagal activity emanating from NA may be determined by the density of GABA receptors. GABA content

Specific 3H-GABA binding

Cat brain

WE have recently obtained evidence in cats that nucleus ambiguus may be the site of GABA-receptor mediated inhibition of vagal outflow to the heart [3]. This was shown by microinjecting nanogram amounts of the GABA receptor antagonist, bicuculline, into nucleus ambiguus (NA) and obtaining pronounced cardiac slowing mediated by the vagus nerves. Furthermore, microinjection of the specific GABAreceptor agonist, muscimol, into NA was found to counteract the parasympathetic stimulation elicited by bicuculline. Finally, inhibition of GABA synthesis by microinjection of isoniazid into NA also induced pronounced cardiac slowing mediated by the vagus nerves. As in the case of the bicuculline-induced response, this effect of isoniazid was counteracted by microinjecting muscimol into NA. These data indicate that GABA receptors probably are present in NA and that GABA may function as an inhibitory neurotransmitter at this site. The purpose of the present study was to obtain biochemical support for the presence of GABAergic synapses in NA by measuring GABA content and spe-

Nucleus ambiguus

Central vagal outflow

cific GABA binding sites in the nucleus. An attempt was also made to relate a physiological response elicited from NA, namely reflex-induced vagal bradycardia, to the biochemical data obtained from analyzing specific GABA binding sites of paired right and left nucleus ambiguus. METHOD

Tissues were taken for analysis of GABA content and specific GABA binding from adult cats unselected as to age and sex, anesthetized with either alpha-chloralose, 70-80 mg/kg (IV) or pentobarbital, 35 mg/kg (IP). Most of our cats had been used in other unrelated experiments which, in our judgment, would not have an effect on either the GABA content or binding levels. Animals subjected to either sectioning of the right vagal trunk or to reflex testing with phenylephrine as described below were not utilized for any other purpose. Brain tissues were rapidly removed from the anesthetized

‘This work was supported by a Grant-in-Aid from the American Heart Association with funds contributed in part by the Nation’s Capitol Affiliate, and by a grant from the U.S.P.H.S. DA 02206. *Dr. Sandra C. Brown’s address is: Bureau of Drugs, Division of Cardiorenal Drug Products, Food and Drug Administration, Rockville, MD 20852.

Copyright

0 1980 ANKHO

International

Inc.-0361-9230/80/080325-04$00.90/O

TABLE

1

GABA LEVELS AND SPECIFIC GABA BINDlNG REGIONS OF THE CAT BRAIN

Brain region examined

GABA content (nmolesimg protein)

1. Substantia nigra

63.8 -c 10.6 (4)

2. Dorsal motor nucleus of the vagus plus nucleus tractus solitarius

35.1 +

3. 4. 5. 6.

28.4 24.8 21.1 17.0

Caudate nucleus Cerebral cortex Nucleus amibiguus Cerebellar cortex

OH-GABA-binding (fmoles/mg protein) 45

(8)

2.4 (4)

85 IT 19

(4)

~tr. 2.9 (3) ” 3.0 (4) i 2.4(4) It- 1.8 (4)

955 rt 235 98 t 28 1211 ;t 154

(4) (9) (6)

7. External cuneate nucleus

9.7 ?I 2.5 (4)

8. Lateral reticular nucleus

8.6 r

9. Area around nucleus ambiguus

IN SEVERAL

2.0 (4)

-

199 +

71 27 ”

4.5(4) 13

(6)

Values represent mean 2 SEM. Numbers in parentheses indicate number of experiments.

animal and the brainstem was immediately frozen using dry ice. Small blocks of the cerebral and cerebellar cortices and the head of the caudate were dissected out and frozen. Tissue samples were stored at -80°C and assayed within one to two weeks for either GABA content or binding. For the measurement of GABA content, the brainstem was sectioned at 200 I_Lusing a cryostat. The following tissue samples were selectively punched from these frozen sections using a 13 ga needle with a blunt tip: substantia nigra, NA, dorsal motor nucleus of the vagus and nucleus tractus solitarious area (DMV-NTS area), lateral reticular nucleus, and the external cuneate nucleus. GABA was extracted in 10-20 volmnes of 0.4N HClO, and measured by the enzymaticfluorometric method of Okada and colleagues 181except that the 60” heating step was omitted. Tissue blanks (without GABase) and reagent blanks (without tissue) were run with each assay and found to be equivalent. Each brain sample was assayed in duplicate along with GABA standards. For the binding assays the tissue samples were obtained using a more rapid technique, since ahowing the tissue to transiently thaw did not affect binding values. The frozen brainstem was sectioned at 1 mm using a freezing microtome and the various nuclei were punched from these very thick sections, again using a 13 pa needle. This method greatly reduces the number of sections and the thicker punches are much easier to handle. Crude membrane fractions from different brain areas were prepared according to the method of Enna and Snyder [4] as modified by Toffano and coworkers [ 111. This method, which involves sequential freeze ing and thawing, followed by Triton X-100 treatment of the membranes (0.01% at 37” for 1 hr), and extensive washing, rest&s in the removal of a memb~-aid protein ~bitor of the high affinity binding for GABA 1111. Binding was

measured in the presence of 30 nM (3H) GABA, with and without IO-” M cold GABA. Proteins were measured by the method of Lowry and colleagues 171. To determine the ~ont~bution of the right and left vagal nerves in reflex-induced vagal bradycardia, cats were anesthetized with alpha-chloralose, and a femoral artery and vein c~nulated for measurement of arterial pressure and systemic administration of drugs, respectively. The trachea was also cannulated and animals were artificially respired with room air. Rectal tem~~ture was monitored and maintained between 37” and 38°C by an infrared lamp. Blood pressure and lead II of the ECG were monitored continuously on a Grass polygraph. Reflex-induced vagal bardycardia was produced by administering a rapid IV injection of pressor doses of phenylephrine (6 to 75 pLg/kg). Once a stable reflex bradycardia was obtained to a given dose of phenyleph~e administered at 5 min intervals, either the right cervical vagus (5 animals) or the left cervical vagus (4 animals) nerve trunks was sectioned and phenylep~e-induced reflex responses repeated. Again, once a stable (although usually attenuated) reflex bradycardia was obtained the remaining vagus nerve trunk was sectioned and pheny~p~e readministered. Data obtained from these experiments enabled us to determine the percent of phe~yleph~ne-induced reflex bradycardia that was mediated by right and left vagus nerves. Six animals were subjected to denervation of the right Xth cranial nerve for the purpose of producing retrograde degeneration of cell bodies in the right nucleus ambiguus. Denervation of the Xth cranial nerve was performed asepticx&y under ketamine anesthesia (30 mg&g, II+@. In some anids anesthesia was supplemented with pentobarbital, 5 m&g,

GABA AND GABA BINDING

SITES IN BRAINSTEM

327

NUCLEI TABLE 2

COMPARISON

OF GABA LEVELS AND SPECIFIC GABA BINDING IN THE RIGHT AND LEFI’ NUCLEUS

Expt No 1 R L GABA

content ~nmoIes/mg protein

“H-GABAbinding (fmolesimg

14.4

Expt No 2 L R 18.3

12.3 14.5

19.4 21.0

12a* 16

Expt No 3 R L

Expt No 4 R L

Expt No 5 L R

Mean ?Z SEM R L

16.6

16.6 14.3

17.12 0.8

53

1302 30

14.9

17.4 49

40*

AMBIGUUS

229

100 0

102

l&Ok 1.5 42t 18

protein) *Tissue from 3 animals pooled.

IV. Each animal was placed in a stereotaxic frame and unilateral intracranial sectioning of the nerve rootlet on the right side was carried out using a lateral approach and visualized using a dissecting microscope. Prior to surgery, each animal was given atropine (0.4 mg IM to prevent excessive bronchial secretions) and dexamethasone (4 mg IM to prevent brain tissue edema). Ampicillin (TM) and dextran (IP) were given for two to three days following surgery. Tissue was taken from these animals 8 days after the denervation procedure.

TABLE 3 EFFECT OF RETROGRADE DEGENE~~ON OF EFFERENT VAGAL FIBERS ON SPECIFIC GABA BINDING IN NUCLEUS AMBIGUUS

3H-GABA-binding (fmols/mg protein) Intact side Denervated

107 5 17 82 k 27

side

Values represent means t SEM of 6 experiments. RESULTS

Table 1 lists the GABA content and values for specific f3H) GABA-binding for several regions of the cat brain including nuclei that are considered to comprise part of the central pathway controlling vagal outflow to the heart. As can be noted, NA as well as the DMV-NTS area contain a significant number of specific GABA binding sites. The GABA content of DMV plus NTS was slightly higher than the content of NA, while specific binding of each region was nearly equivalent. Relative to an area of the CNS that is rich in GABAergic synapses, namely the substantia nigra 151, GABA content of DMV-NTS area and NA was approximately 55% and 33% of the value for substantia nigra, respectively. The specific binding of GABA in these areas relative to the value for the substantia nigra was 43% and 50%, respectively. Most importantly, the specific GABA binding of the NA was more than 3-fold higher than that obtained in tissue from the reticular area surrounding NA. GABA content and specific GABA binding of cerebral and cerebellar cortex are similar to the values obtained by other investigators who have previously examined these structures [6,121. The NTS-DMV area as well as NA have been established as comprising part of the central neuronal network controlling vagal outflow to the heart. In addition, there are data which suggest that both the external euneate and the lateral reticular nucleus have an important influence on central vagal outflow 12,101. In view of this, we also measured the GABA content of these areas as well as the specific GABA binding of the lateral reticular nucleus. GABA content of both of these nuclei was less than one-half of the values obtained in NTS-DMV and NA. Specific GABA binding in the lateral reticular nucleus was less than 10% of that obtained in NA.

GABA content and specific GABA binding was also measured in the right and the left NA of five animals and the data are presented in Table 2. The right and the left NA of each animal appeared to contain equivalent amounts of GABA. However, for most animals, there was a marked difference between the right and left NA with respect to the density of specific binding sites for GABA. In 4 of the 5 animals, GABA binding was more than 3-fold higher in the right NA as compared to the left NA. In the remaining animal, the binding pattern was reversed with the left NA exhibiting the greater density of specific GABA binding. Sectioning the right vagus nerve trunk and allowing 8 days for retrograde degeneration of cell bodies in the right NA resulted in a more balanced density of specific GABA binding sites between the right and the left NA (see Table 3) as compared to the imbalance observed in animals with the right vagus trunk intact. Based on the finding of an asymmetry between specific GABA receptor binding in the right and left NA, additional experiments were performed to determine whether asymmetry also exists in the percent of phenylephrine-induced reflex vagal bradycardia mediated by right and left vagus nerves. These data are tabulated in Table 4 and indicate that in the majority of cases studied, the left vagus nerve was responsible for mediating the greatest proportion of reflex vagal bradycardia. In two cases the right vagus appeared to mediate the greatest percentage of the response. In the remaining animal, each vagus nerve was responsible for about one-half of the response. DISCUSSION

Data from our previous

study using mi~roinje~tion

of

328

GALE E7- AL. TABLE 4

PERCENT OF PHENY~~FHRINE-INDUCED REFLEX BRADYCARDIA MEDIATED BY RIGHT AND LEFT VAGUS NERVES

Exp. no. 1

2 3 4 5 6 7 8 9 Mean k SEM

‘5% of bradycardic response mediated by right vagus

% of bradycardic response mediated by left vagus

38 31 32 0 0 58 74 30 100 40% 11

62 69 68 100 100 42 26 70 0 602 11

bicuculline and isoniazid into NA of the cat suggested that this nucleus may be the site of a GABA receptor-mediated inhibition of vagal outflow [3]. Data from the present study confirm and extend these earlier fmdings. Examination of NA for specific GABA binding sites revealed a moderate density of these sites. In addition, measurement of the GABA content of NA indicated that this nucleus contains a significant amount of this amino acid. These results are consistent with the concept that the activity of NA cells, which project parasympathetic fibers to the heart, are normally

1. Barman, S. M. and G. L. Gebber. Picrotoxin

2. 3.

4.

5. 6.

and bicucullinesensitive inhibition of cardiac vagal reflexes. J. Pharmac. exp. Ther. 209: 67-72, 1979. Ciriello, J. and F. R. Calaresu. Vagal bradycardia elicited by stimulation of the external cuneate nucleus in the cat. Am. J. Physiol. 23% R286R293, 1978. DiMicco, J. A., K. Gale, B. L. Hamilton and R. A. Gillis. GABA receptor control of parasympathetic outflow to the heart: characterization and brain stem localization. Science 264: 1106-l 109, 1979. Enna, S. J. and S. H, Snyder. Influences of ions, enzymes, and detergent on gamma-aminobutyric acid receptor binding in synaptic membranes of rat brain. Molec. Pharmac. 13: 442-453, 1977. Fonnum, F., I. Grofova, E. Rinvik, J. Storm-Mathison and F. Waldberg. Origin and distribution of dutamate decarboxylase in the substantia nigra of the cat. Brain Res. 71:77-92,1974. Homg, J. S. and D. T. Wang. Gamma-aminobutyric acid recep tors in cerebellar membranes of rat brain after a treatment with t&on x-100. J. Neurochem. 32: 1379-1386, 1979.

under a tonic GABA input. This input may be derived from GABAergic neurons intrinsic to the NA, since the GABA content which we have measured in NA is similar to that obtained in regions where GABA is associated primarily with interneurons. Alternatively, it is possible that the NA receives an tierent GABAergic projection which may terminate in a circumscribed region within the NA. Studies are presently underway to determine whether GABA may be differentially concentrated within discrete areas of the NA. The area of NTS-DMV also exhibited a moderate amount of GABA and specific GABA binding. The NTS has been suggested to be an important site where GABA may be a neurotransmitter responsible for inhibition of reflex vagal bradycardia [I]. The DMV has been demonstrated by Satomi and colleagues [9] to contain parasympathetic neurons that innervate the intestine in the cat. Additional studies are needed to determine whether GABA may participate at this site in regulating neuronal tion. The bilateral asymmetry

control

of gastrointestinal

func-

in GABA binding of NA may be associated with the bilateraI asymmet~ seen in vagalmediation of reflex-induced bradycardia. The density of GABA binding was generally higher in the right NA, while the left NA appears most often responsible for the mediation of the largest portion of reflex-induced cardiac slowing. It is therefore tempting to speculate that the greater degree of GABAergic inhibitory tone operating in the right NA, fimctions to restrain the activity of the parasympathetic outflow on this side. In response to reflex activation, the synaptic network in the left NA may therefore serve as the path of least resistance for the mediation of vagal outflow to the heart.

7. Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. 3. Randall. Protein measurement with the Folin Phenol reagent. J. Biol. Chem. 193: 265-275, 1951. 8. Okada, Y., C. Nitsch-Hassler, J. S. Kim, I. J. Bak and R. Hassler. Role of gamma-aminobutyric acid (GABA) in the extrapyramidal motor system. ExplBrain Res. 13:514-518, 1971. 9. Satomi, H,, T. Yamamoto, H. Ise and H. Takatoma. Origins of the parasympathetic pregan@ionic fibers to the cat intestine as demonstrated by the horseradish peroxidase method. Brain Res. 151:571-578,1978. 10. Thomas, M. R. and F. R, Calaresu. Localization and function of medulkuy sites mediating vagal bradycardia in the cat. Am. J. Physiol. 226: 1344-1349, 1974. 11. Toffano, G., A. Guidotti and E. Costa. Purification of an endogenous protein inhibitor for the high affiity binding of g~ma-~nobuty~c acid to synaptic membranes of rat brain. Proc. natn. Acad. Sci. 75: 40244028, 1978. 12. Van der Heyden, J. A. M., E. R. De Kloet, J. Korf and D. H. G. Versteeg. GABA content of discrete brain nuclei and spinal cord of the rat. J, ~eur~chem. 33: W-861, 1979.