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Thymocyte-neuron antigenic correlation: Anti-thymocyte membrane antibodies modify bioelectrical activity of Helix pomatia neurons
Immunology Letters, 1 (1980) 275-279 © Elsevier/North-HollandBiomedicalPress
T H Y M O C Y T E - N E U R O N ANTIGENIC CORRELATION: ANTI-THYMOCYTE MEMBRANE ANTIBODIES MODIFY BIOELECTRICAL ACTIVITY OF H E L I X POMA TIA NEURONS B. D. JANKOVI~, V. SAVI~, ~. gOLTES, J. HORVAT and Kosana MITROVI~ Immunology Research Center, VojvodeStepe 458, 11000 Belgrade, Yugoslavia (Accepted 4 March 1980)
1. Summary The snarl (Helix pomatia) suboesophageal gang[ionic complex treated in vitro with rabbit anti-rat thymocyte membrane (ATM) antibodies was used as an immunoneurological model to study the functional relationship between the thymocyte and neuron. Intracellular recordings were made from pacemaker (spontaneously active) and non-pacemaker (silent) neurons before and after application of ATM antibodies. ATM antibodies induced significant changes of the membrane potential, spike amplitude and firing patterns of both pacemaker and non-pacemaker neurons. This is the first demonstration that antithymocyte antibodies are capable of modifying the bioelectrical activity of the neuron.
2. Introduction
Numerous experiments, performed on different animal species, have established a cross-reactivity between antigens from the brain tissue and thymocyte surface membrane. The chemistry of these common antigenic determinants is still a matter of dispute [1-3]. So far, studies of the brain-thymocyte antigenic relationship were confined to the structural and chemical characterization of thymocytes and T-cells, using anti-brain sera as a substitute for anti-theta sera. Since the brain-thymocyte antigen(s) appears to be mainly associated with neuronal membranes and fibres [4], one is tempted to assume that these antigens (or closely related antigens) could mediate reac-
tions which occur on the neuronal membrane. Therefore, the present immunoneurological experiment was designed to answer a rather elementary question: whether anti-thymocyte antibodies are capable of inducing essential changes in bioelectrical activity of the neuron?
3. Materials and methods
3.1. PreparatiOn of rabbit anti-rat thymocyte membrane (A TM] serum Thymuses from Wistar rats perfused with saline were used for the preparation of thymocyte suspensions in medium 199. Anti-rat thymocyte serum [5] was produced in rabbits and absorbed with rat erythrocytes, liver cell membranes [6], foetal liver [7], purified B-lymphocytes [8] and insolubilized IgM and IgG [9]. The immunoglobulin fraction [10] isolated from this antiserum exhibited cytotoxic (80% dead cells at a 1:1024 dilution) and immunofluorescent (about 97% stained cells) activity against rat thymocytes, but was completely inactive for rat B-lymphocytes in cytotoxicity [11] and immunofluorescence [12] assays. In order to obtain anti-rat thymocyte membrane (ATM) antibodies, 200 mg of lyophilized rabbit antirat thymocyte immunoglobulin fraction [10] was dissolved in 0.1 M phosphate buffer and mixed with immunoabsorbent [9]. Cross-linked solubilized rat thymocyte membranes [ 13] and bovine serum albumin served as the immunoabsorbent. ATM antibodies were eluted [9] from immunoabsorbent, pressureconcentrated and lyophilized. 275
3.2. Electrophysiological assays The snails (Helix pomatia) were collected locally and maintained for 2 - 3 weeks at 20°C and on a standard diet until required. The suboesophageal ganglionic complex [14,15] was dissected and mounted in a small recording chamber containing Ringer solution [15]. The nerve ends were aspirated into Ag-AgC1 glass electrodes. The glass microelectrode filled with 2.5 M KC1 ( 7 - 1 8 m~2), connected with the input o f a Grass P16 amplifier, was inserted into the neuron [ 14]. The left pallial nerve was stimulated with a single pulse of 6 V and 2 msec duration or a train of stimuli (6 V, 2 msec, 4 impulses/0.5 sec) using a Grass $88 stimulator and Grass stimulus isolation unit (SIU5). The current of 1 . 5 - 2 × 10 -9 A o f 5 - 1 0 msec duration was injected through the stimulatingrecording microelectrode connected to a bridge circuit. Permanent recordings were made simultaneously using aTectronix 502A oscilloscope and Beckman RM
Dynograph pen recorder before and after application of ATM antibodies. The experiment consisted o f 3 phases: recording of bioelectrical activity in Ringer solution; application o f 750/.tg o f ATM antibody protein (or application o f 750 #g o f a control protein); and washing o f the ganglion with Ringer solution and recording: The circulation o f Ringer solution was stopped during the administration o f ATM antibodies or control protein solutions. This 3-step procedure was repeated when necessary.
4. Results
4.1. Binding of A TM antibodies to neurons That ATM antibodies indeed attached to neurons o f the snail ganglionic complex was demonstrated by immunofluorescence (Figs. 1 - 3 ) .
Y
Fig.1. Brightly stained rat thymocytes. Fig.2. A section of rat cerebrum. Specific fluorescence of neuronal membranes, axons and nerve endings. Note unstained nerve cell bodies. Fig. 3. A section of the snail suboesophageal ganglionic complex. Fluorescence is concentrated to membranous structures. Note negative shadows of ganglion cell bodies. Figs. 1-3. Illustrate specific staining of tat thymocytes and brain, and snail ganglionic complex treated with rabbit ATM antibodies and fluorescein-conjugated sheep anti-rabbit lgG. Negative staining was obtained in controls: ATM without conjugate; conjugate alone; non-immune rabbit serum; ATM absorbed with rat thymocytes; and 'blocking' test (blocking of fluorescence by non-conjugated sheep anti-rabbit IgG). Negative staining was also obtained with sections of the rat brain and cervical ganglion, and snail ganglion after exposure to ATM reagent absorbed with rat brain, rat cervical ganglion or marl suboosophageadganglion. However, ATM reagent thus absorbed was still capable of staining specifically rat thymoeytes. This would indicate that a set of antigens specific to rat thymocytes was active in the course of production of ATM antibodies in rabbits. 276
4.2. Influence o f A TM antibodies on bioelectrical
activity o/snail neurons Intracellular recordings were made from 30 spontaneously firing (pacemaker) neurons and 4 silent (non-pacemaker) neurons. The membrane potential ( - 3 5 to - 4 5 mV), spike amplitude ( 6 0 - 8 0 mV) and firing patterns were principal parameters. As a rule, ATM antibodies changed the bioelectrical activity o f both pacemaker and non-pacemaker neurons. In the case of a silent cell, polyspike activity was evoked by ATM antibodies (Fig. 4). In the case of a neuron with spontaneous action potentials, ATM antibodies induced first an excitatory spike activity of varying duration and membrane depolarization, and then inhibition o f spontaneous spike generation (Fig. 5). The cells were depolarized by 5 mV (at time 0, i.e. immediately after the addition of ATM antibodies) to 20 mV (30 min after the addition of ATM antibodies). Action potentials disappeared promptly or became gradually smaller until they finally vanished. In the presence of ATM antibodies, electrical stimulation o f the pallial nerve and the neuron failed to evoke pace-
maker discharges and to influence antibody-induced membrane depolarization (Figs. 4 and 5). However, 1 1 - 2 4 min after washout o f ATM antibodies (removal o f ATM protein) the action potential sometimes reappeared although their amplitudes were smaller ( 4 5 - 5 5 mV) compared to those recorded before administration of ATM antibodies. After washing, the resting potential approached ( - 2 5 to - 3 5 mV) the control level slightly. The second exposure o f the ganglion to ATM antibodies was accompanied by drastic and irreparable blocking of the spontaneous spiking and by permanent membrane depolarization. In control experiments (see: legends for Figs. 4 and 5), bioelectrical patterns o f pacemaker and nonpacemaker neurons were not affected by protein solutions lacking ATM antibodies. Thus, bioelectrical
4
#st I
('st 2
('st 3
B
Fig. 4. The snail suboesophageal ganglionic complex: a silent neuron. A: stimulation of the nerve trunk (st 1) provoked a short-lasting beating spike activity. Direct stimulations of the neuron (st 2 and st 3) elicited a single spike. B: the same neuron. Application of ATM antibodies (Ab) induced a burst of high density and subsequent membrane depolarization. C: the same neuron 27 rain after addition of antibodies. A single pulse (st 4) and a train of stimuli (st 5) through the left pMiiM nerve, and direct stimulation via microelectrode (st 6) did not elicit action potentials. Fig. 5. The snail suboesophageal ganglionic complex: a neuron with spontaneous beating spike activity. A and B: single stimulus (st 1) evoked a 5 seclong inhibition, whereas train of stimuli (st 2) elicited a 10 sec-long inhibition. C: the same neuron. Application of ATM antibodies (Ab): initial excitation of about 4 sec duration was followed by inhibition of spontaneous discharges. D: The same neuron 24 rain after antibody addition. Single stimulus (st 3) and train of stimuli (st 4) failed to generate spontaneous firing. The control solutions: ATM reagent absorbed with rat thymocytes; ATM absorbed with rat brain; ATM absorbed with snarl ganglionic mass; ATM absorbed with rat cervical ganglion; ATM reagent from which antibodies were removed b y a goat anti-rabbit IgM and IgG serum; and non-immune rabbit Ig. All these reagents were also absorbed with viable rat B4ymphocytes [8]. These control protein solutions failed to affect bioelectrieal patterns of pacemaker and non-pacemaker neurons of the snail suboesophageal ganglionic complex.
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277
records described hire leave little if any doubt as to the observation that A I M antibodies can cause profound modification in electrical activity of the snail neuron.
of the neuron and its associates. The function of Tlymphocytes in the cerebrospinal fluid of individuals with a variety of neurological disorders [27] should also be taken into consideration.
5. Discussion
Acknowledgement
In earlier studies, we demonstrated in the rat the shared antigenicity between brain cells and superior cervical ganglion [ 16], between brain subcellular fractions and thymocytes [ 17], and between superior cervical ganglion and thymocytes [18]. Recently, we described changes in the bioelectrical activity of the snail (Helix pomatia) ganglionic neuron evoked by rabbit anti-rat brain microsome antibodies [19]. Besides, preliminary experiments (immunofluorescence assays) from our laboratory showed that rabbit anti-rat cervical ganglion antibodies stained specifically the snail suboesophageal ganlionic complex. On these grounds, it was logical to assume that bioelectrical abnormalities should be expected to appear following treatment of the snarl ganglionic neuron with ATM antibodies. The manner whereby antibody molecules influence the neuronal membrane and cause a cell depolarization is not known [20]. It seems that this depolarization is sufficient to explain both the initial excitation of the neurons and the subsequent inhibition (presumably due to sodium channels inactivation). The present in vitro experiments showed that antithymocyte antibodies induced profound modification of bioeleetrical activity of the snail neuron. These results may have an important bearing upon the pathogenesis of immunoneurological [21] and immunopsychiatric [22,23] diseases in humans. It has been postulated that in schizophrenic patients there is a unique antibody ('taraxein') which interacts with brain tissue and produces behavioural aberrations [24]. Besides, a serum globulin ('antibody') cross-reacts with the human thymus [25]. In patients with multiple sclerosis, both anti-brain and lymphocytotoxie antibodies are present in sera, although the role of lymphocytotoxic antibodies in the development of disease is still unclear [26]. Nevertheless, the possibility remains that anti-lymphocyte antibodies may join anti.brain antibodies in triggering a series of events which lead to metabolic and other disturbances
This study was supported by grants from the Republic of Serbia Research Fund, Belgrade.
278
References [1 ] Vitetta, E. S., Boyse, E. A. and Uhr, J. W. (1973) Eur. J. ImmunoL 3,446-453. [2] Morris, R. J., Letarte-Muirhead, M. and Williams, A. F. (1975) Eur. J. Immunol. 5,282-285. [3] Inokuchi, Y. and Nagal, Y. (1979) Molec. ImmunoL 16, 791-796. [4] Barclay, A. N. and Hyd~n, H. (1979) J. Neurochem. 32, 1583-1586. [5] Isakovi~, K., Mitrovi~, K., Markovi~, B. M., Raj~evi~,M. and Jankovi6, B. D. (1975) J. Immunol. Meth.7, 359-369. [6] Hudson, L. and Roitt, I. M. (1973) Eur. J. Immunol. 3, 63-67. [7] FiUppi, J. A., Rheins, M. S. and Nyergens, G. A. (1976) Transplantation 21,124-128. [8] Jankovi6, B. D., Horvat, J. and Mitrovi6, K. (1979) Experientla 35, 1393-1395. [9] Temynck, T. and Avrameas, S. (1976) in: Immuneabsorbents in Protein Purification (Ruoslahti, E. Ed.) pp. 29-35, University Park Press, Baltimore. [10] Garvoy, J. S., Crornex,N. E. and Suudorf, D. H. (1977) Methods in Immunology. pp. 218-219, W. A. Benjamin, London. [11] Konda, S., Stookert, E. and Smith, R. T. (1973) Cell. Immunol. 7,275-289. [12] Jankovi6, B. D., Horvat, J., Mitrovi6, K. and Mostarica, M. (1977) Immunoehemistry 14, 75-78. [13] Letarte-Muirhead, M., Aoton, R. T. and Williams, A. F. (1974) Biochem. J. 143, 51-61. [14] Kerkut, G. A., Ralph, K., Walker, R. J., Woodruff, G. N. and Woods, R. (1970) in: Excitatory Synapti¢ Mechanims (Andersen, P. and Jansen, J. K. S. Eds.) pp. 105-117, University Olso Press, Osio. [15] Loker, J. E., Kerkut, G. A. and Walker, R. J. (1975) Comp. Biochem. Physiol. 50A, 443-452. [16] Jankovi~, B. D., Horvat, J. and Mitrovi6, K. (1977) J. Neurochem. 29,605-609. [17] Jankovi~, B. D., Horvat, J., Mitrovi6, K. and Mostarica, M. (1977) Scan& J. Immunol. 6,843-847. [18] Jankovi~, B. D., Horvat, J., Mitrovi6, K. and Mostarica, M. (1977) Experientia 33, 1526-1527.
[19] Soltes, S., Savi6, V., Horvat, J. and Jankovi6, B. D. (1979) Period. Biol. 81,217-218. [20] Jankovi6, B. D. (1972) in: Macromolecules and Behavior (Gaito, J. Ed.) pp. 99-130, Appleton-Century-Crofts, New York. [21] Paterson, P. Y. (1979) Rev. Infect. Dis. 1,468-482. [22] Jankovi6, B. D., Jakuli6, S.and Horvat, J. (1977) Lancet 2, 219-220. [23] Jankovi6, B. D., Jakuli6, S. and Horvat, ]. (1980) 0in. Exp. Immunol. (in press).
[24] Heath, R. G. and Krupp, I. M. (1967) Arch. Gen. Psychiat. 16, 1-33. [25] Baton, M., Stern, M., Anavi, R. and Witz, I. P. (1977) Biol. Psychiat. 12, 199-219. [26] Schocket, A. L., Weiner, H. L., Walker, J., McIntosh, K. and Kohler, P. F. (1977) Clin. Immunol. Immunopath. 7, 15-23. [27] Levinson, A. I., Lisak, R. P. and Zweiman, B. (1979) Neurology 26,693-695.
279
T H Y M O C Y T E - N E U R O N ANTIGENIC CORRELATION: ANTI-THYMOCYTE MEMBRANE ANTIBODIES MODIFY BIOELECTRICAL ACTIVITY OF H E L I X POMA TIA NEURONS B. D. JANKOVI~, V. SAVI~, ~. gOLTES, J. HORVAT and Kosana MITROVI~ Immunology Research Center, VojvodeStepe 458, 11000 Belgrade, Yugoslavia (Accepted 4 March 1980)
1. Summary The snarl (Helix pomatia) suboesophageal gang[ionic complex treated in vitro with rabbit anti-rat thymocyte membrane (ATM) antibodies was used as an immunoneurological model to study the functional relationship between the thymocyte and neuron. Intracellular recordings were made from pacemaker (spontaneously active) and non-pacemaker (silent) neurons before and after application of ATM antibodies. ATM antibodies induced significant changes of the membrane potential, spike amplitude and firing patterns of both pacemaker and non-pacemaker neurons. This is the first demonstration that antithymocyte antibodies are capable of modifying the bioelectrical activity of the neuron.
2. Introduction
Numerous experiments, performed on different animal species, have established a cross-reactivity between antigens from the brain tissue and thymocyte surface membrane. The chemistry of these common antigenic determinants is still a matter of dispute [1-3]. So far, studies of the brain-thymocyte antigenic relationship were confined to the structural and chemical characterization of thymocytes and T-cells, using anti-brain sera as a substitute for anti-theta sera. Since the brain-thymocyte antigen(s) appears to be mainly associated with neuronal membranes and fibres [4], one is tempted to assume that these antigens (or closely related antigens) could mediate reac-
tions which occur on the neuronal membrane. Therefore, the present immunoneurological experiment was designed to answer a rather elementary question: whether anti-thymocyte antibodies are capable of inducing essential changes in bioelectrical activity of the neuron?
3. Materials and methods
3.1. PreparatiOn of rabbit anti-rat thymocyte membrane (A TM] serum Thymuses from Wistar rats perfused with saline were used for the preparation of thymocyte suspensions in medium 199. Anti-rat thymocyte serum [5] was produced in rabbits and absorbed with rat erythrocytes, liver cell membranes [6], foetal liver [7], purified B-lymphocytes [8] and insolubilized IgM and IgG [9]. The immunoglobulin fraction [10] isolated from this antiserum exhibited cytotoxic (80% dead cells at a 1:1024 dilution) and immunofluorescent (about 97% stained cells) activity against rat thymocytes, but was completely inactive for rat B-lymphocytes in cytotoxicity [11] and immunofluorescence [12] assays. In order to obtain anti-rat thymocyte membrane (ATM) antibodies, 200 mg of lyophilized rabbit antirat thymocyte immunoglobulin fraction [10] was dissolved in 0.1 M phosphate buffer and mixed with immunoabsorbent [9]. Cross-linked solubilized rat thymocyte membranes [ 13] and bovine serum albumin served as the immunoabsorbent. ATM antibodies were eluted [9] from immunoabsorbent, pressureconcentrated and lyophilized. 275
3.2. Electrophysiological assays The snails (Helix pomatia) were collected locally and maintained for 2 - 3 weeks at 20°C and on a standard diet until required. The suboesophageal ganglionic complex [14,15] was dissected and mounted in a small recording chamber containing Ringer solution [15]. The nerve ends were aspirated into Ag-AgC1 glass electrodes. The glass microelectrode filled with 2.5 M KC1 ( 7 - 1 8 m~2), connected with the input o f a Grass P16 amplifier, was inserted into the neuron [ 14]. The left pallial nerve was stimulated with a single pulse of 6 V and 2 msec duration or a train of stimuli (6 V, 2 msec, 4 impulses/0.5 sec) using a Grass $88 stimulator and Grass stimulus isolation unit (SIU5). The current of 1 . 5 - 2 × 10 -9 A o f 5 - 1 0 msec duration was injected through the stimulatingrecording microelectrode connected to a bridge circuit. Permanent recordings were made simultaneously using aTectronix 502A oscilloscope and Beckman RM
Dynograph pen recorder before and after application of ATM antibodies. The experiment consisted o f 3 phases: recording of bioelectrical activity in Ringer solution; application o f 750/.tg o f ATM antibody protein (or application o f 750 #g o f a control protein); and washing o f the ganglion with Ringer solution and recording: The circulation o f Ringer solution was stopped during the administration o f ATM antibodies or control protein solutions. This 3-step procedure was repeated when necessary.
4. Results
4.1. Binding of A TM antibodies to neurons That ATM antibodies indeed attached to neurons o f the snail ganglionic complex was demonstrated by immunofluorescence (Figs. 1 - 3 ) .
Y
Fig.1. Brightly stained rat thymocytes. Fig.2. A section of rat cerebrum. Specific fluorescence of neuronal membranes, axons and nerve endings. Note unstained nerve cell bodies. Fig. 3. A section of the snail suboesophageal ganglionic complex. Fluorescence is concentrated to membranous structures. Note negative shadows of ganglion cell bodies. Figs. 1-3. Illustrate specific staining of tat thymocytes and brain, and snail ganglionic complex treated with rabbit ATM antibodies and fluorescein-conjugated sheep anti-rabbit lgG. Negative staining was obtained in controls: ATM without conjugate; conjugate alone; non-immune rabbit serum; ATM absorbed with rat thymocytes; and 'blocking' test (blocking of fluorescence by non-conjugated sheep anti-rabbit IgG). Negative staining was also obtained with sections of the rat brain and cervical ganglion, and snail ganglion after exposure to ATM reagent absorbed with rat brain, rat cervical ganglion or marl suboosophageadganglion. However, ATM reagent thus absorbed was still capable of staining specifically rat thymoeytes. This would indicate that a set of antigens specific to rat thymocytes was active in the course of production of ATM antibodies in rabbits. 276
4.2. Influence o f A TM antibodies on bioelectrical
activity o/snail neurons Intracellular recordings were made from 30 spontaneously firing (pacemaker) neurons and 4 silent (non-pacemaker) neurons. The membrane potential ( - 3 5 to - 4 5 mV), spike amplitude ( 6 0 - 8 0 mV) and firing patterns were principal parameters. As a rule, ATM antibodies changed the bioelectrical activity o f both pacemaker and non-pacemaker neurons. In the case of a silent cell, polyspike activity was evoked by ATM antibodies (Fig. 4). In the case of a neuron with spontaneous action potentials, ATM antibodies induced first an excitatory spike activity of varying duration and membrane depolarization, and then inhibition o f spontaneous spike generation (Fig. 5). The cells were depolarized by 5 mV (at time 0, i.e. immediately after the addition of ATM antibodies) to 20 mV (30 min after the addition of ATM antibodies). Action potentials disappeared promptly or became gradually smaller until they finally vanished. In the presence of ATM antibodies, electrical stimulation o f the pallial nerve and the neuron failed to evoke pace-
maker discharges and to influence antibody-induced membrane depolarization (Figs. 4 and 5). However, 1 1 - 2 4 min after washout o f ATM antibodies (removal o f ATM protein) the action potential sometimes reappeared although their amplitudes were smaller ( 4 5 - 5 5 mV) compared to those recorded before administration of ATM antibodies. After washing, the resting potential approached ( - 2 5 to - 3 5 mV) the control level slightly. The second exposure o f the ganglion to ATM antibodies was accompanied by drastic and irreparable blocking of the spontaneous spiking and by permanent membrane depolarization. In control experiments (see: legends for Figs. 4 and 5), bioelectrical patterns o f pacemaker and nonpacemaker neurons were not affected by protein solutions lacking ATM antibodies. Thus, bioelectrical
4
#st I
('st 2
('st 3
B
Fig. 4. The snail suboesophageal ganglionic complex: a silent neuron. A: stimulation of the nerve trunk (st 1) provoked a short-lasting beating spike activity. Direct stimulations of the neuron (st 2 and st 3) elicited a single spike. B: the same neuron. Application of ATM antibodies (Ab) induced a burst of high density and subsequent membrane depolarization. C: the same neuron 27 rain after addition of antibodies. A single pulse (st 4) and a train of stimuli (st 5) through the left pMiiM nerve, and direct stimulation via microelectrode (st 6) did not elicit action potentials. Fig. 5. The snail suboesophageal ganglionic complex: a neuron with spontaneous beating spike activity. A and B: single stimulus (st 1) evoked a 5 seclong inhibition, whereas train of stimuli (st 2) elicited a 10 sec-long inhibition. C: the same neuron. Application of ATM antibodies (Ab): initial excitation of about 4 sec duration was followed by inhibition of spontaneous discharges. D: The same neuron 24 rain after antibody addition. Single stimulus (st 3) and train of stimuli (st 4) failed to generate spontaneous firing. The control solutions: ATM reagent absorbed with rat thymocytes; ATM absorbed with rat brain; ATM absorbed with snarl ganglionic mass; ATM absorbed with rat cervical ganglion; ATM reagent from which antibodies were removed b y a goat anti-rabbit IgM and IgG serum; and non-immune rabbit Ig. All these reagents were also absorbed with viable rat B4ymphocytes [8]. These control protein solutions failed to affect bioelectrieal patterns of pacemaker and non-pacemaker neurons of the snail suboesophageal ganglionic complex.
~Ala
c
!
i
,,... Sst4
,t,st 5
Sst6
Sstl
!
I
!I!t!1!!!!!lptr!!!H!!rlrl!!'rr,rp !i I!,
~st2
II I I II I !!tr ~Ab
4O
~st 3
~st4,
i v ~0 sec
277
records described hire leave little if any doubt as to the observation that A I M antibodies can cause profound modification in electrical activity of the snail neuron.
of the neuron and its associates. The function of Tlymphocytes in the cerebrospinal fluid of individuals with a variety of neurological disorders [27] should also be taken into consideration.
5. Discussion
Acknowledgement
In earlier studies, we demonstrated in the rat the shared antigenicity between brain cells and superior cervical ganglion [ 16], between brain subcellular fractions and thymocytes [ 17], and between superior cervical ganglion and thymocytes [18]. Recently, we described changes in the bioelectrical activity of the snail (Helix pomatia) ganglionic neuron evoked by rabbit anti-rat brain microsome antibodies [19]. Besides, preliminary experiments (immunofluorescence assays) from our laboratory showed that rabbit anti-rat cervical ganglion antibodies stained specifically the snail suboesophageal ganlionic complex. On these grounds, it was logical to assume that bioelectrical abnormalities should be expected to appear following treatment of the snarl ganglionic neuron with ATM antibodies. The manner whereby antibody molecules influence the neuronal membrane and cause a cell depolarization is not known [20]. It seems that this depolarization is sufficient to explain both the initial excitation of the neurons and the subsequent inhibition (presumably due to sodium channels inactivation). The present in vitro experiments showed that antithymocyte antibodies induced profound modification of bioeleetrical activity of the snail neuron. These results may have an important bearing upon the pathogenesis of immunoneurological [21] and immunopsychiatric [22,23] diseases in humans. It has been postulated that in schizophrenic patients there is a unique antibody ('taraxein') which interacts with brain tissue and produces behavioural aberrations [24]. Besides, a serum globulin ('antibody') cross-reacts with the human thymus [25]. In patients with multiple sclerosis, both anti-brain and lymphocytotoxie antibodies are present in sera, although the role of lymphocytotoxic antibodies in the development of disease is still unclear [26]. Nevertheless, the possibility remains that anti-lymphocyte antibodies may join anti.brain antibodies in triggering a series of events which lead to metabolic and other disturbances
This study was supported by grants from the Republic of Serbia Research Fund, Belgrade.
278
References [1 ] Vitetta, E. S., Boyse, E. A. and Uhr, J. W. (1973) Eur. J. ImmunoL 3,446-453. [2] Morris, R. J., Letarte-Muirhead, M. and Williams, A. F. (1975) Eur. J. Immunol. 5,282-285. [3] Inokuchi, Y. and Nagal, Y. (1979) Molec. ImmunoL 16, 791-796. [4] Barclay, A. N. and Hyd~n, H. (1979) J. Neurochem. 32, 1583-1586. [5] Isakovi~, K., Mitrovi~, K., Markovi~, B. M., Raj~evi~,M. and Jankovi6, B. D. (1975) J. Immunol. Meth.7, 359-369. [6] Hudson, L. and Roitt, I. M. (1973) Eur. J. Immunol. 3, 63-67. [7] FiUppi, J. A., Rheins, M. S. and Nyergens, G. A. (1976) Transplantation 21,124-128. [8] Jankovi6, B. D., Horvat, J. and Mitrovi6, K. (1979) Experientla 35, 1393-1395. [9] Temynck, T. and Avrameas, S. (1976) in: Immuneabsorbents in Protein Purification (Ruoslahti, E. Ed.) pp. 29-35, University Park Press, Baltimore. [10] Garvoy, J. S., Crornex,N. E. and Suudorf, D. H. (1977) Methods in Immunology. pp. 218-219, W. A. Benjamin, London. [11] Konda, S., Stookert, E. and Smith, R. T. (1973) Cell. Immunol. 7,275-289. [12] Jankovi6, B. D., Horvat, J., Mitrovi6, K. and Mostarica, M. (1977) Immunoehemistry 14, 75-78. [13] Letarte-Muirhead, M., Aoton, R. T. and Williams, A. F. (1974) Biochem. J. 143, 51-61. [14] Kerkut, G. A., Ralph, K., Walker, R. J., Woodruff, G. N. and Woods, R. (1970) in: Excitatory Synapti¢ Mechanims (Andersen, P. and Jansen, J. K. S. Eds.) pp. 105-117, University Olso Press, Osio. [15] Loker, J. E., Kerkut, G. A. and Walker, R. J. (1975) Comp. Biochem. Physiol. 50A, 443-452. [16] Jankovi~, B. D., Horvat, J. and Mitrovi6, K. (1977) J. Neurochem. 29,605-609. [17] Jankovi~, B. D., Horvat, J., Mitrovi6, K. and Mostarica, M. (1977) Scan& J. Immunol. 6,843-847. [18] Jankovi~, B. D., Horvat, J., Mitrovi6, K. and Mostarica, M. (1977) Experientia 33, 1526-1527.
[19] Soltes, S., Savi6, V., Horvat, J. and Jankovi6, B. D. (1979) Period. Biol. 81,217-218. [20] Jankovi6, B. D. (1972) in: Macromolecules and Behavior (Gaito, J. Ed.) pp. 99-130, Appleton-Century-Crofts, New York. [21] Paterson, P. Y. (1979) Rev. Infect. Dis. 1,468-482. [22] Jankovi6, B. D., Jakuli6, S.and Horvat, J. (1977) Lancet 2, 219-220. [23] Jankovi6, B. D., Jakuli6, S. and Horvat, ]. (1980) 0in. Exp. Immunol. (in press).
[24] Heath, R. G. and Krupp, I. M. (1967) Arch. Gen. Psychiat. 16, 1-33. [25] Baton, M., Stern, M., Anavi, R. and Witz, I. P. (1977) Biol. Psychiat. 12, 199-219. [26] Schocket, A. L., Weiner, H. L., Walker, J., McIntosh, K. and Kohler, P. F. (1977) Clin. Immunol. Immunopath. 7, 15-23. [27] Levinson, A. I., Lisak, R. P. and Zweiman, B. (1979) Neurology 26,693-695.
279