Antigen-induced synaptic plasticity in sympathetic ganglia from actively and passively sensitized guinea-pigs

Journal of the

ELSEVIER

Journal of the Autonomic Nervous System 61 (1996) 139-144

Autonomic Nervous System

Antigen-induced synaptic plasticity in sympathetic ganglia from actively and passively sensitized guinea-pigs Aline Alice Cavalcante de Albuquerque, Jose Henrique Leal-Cardoso, Daniel Weinreich * Department of Pharmacology and Experimental Therapeutics, School of Medicine, University of Maryland at Baltimore, 655 West Baltimore Street, Baltimore, MD 21201, USA Received 3 January 1996; revised 28 May 1996; accepted 5 June 1996

Abstract

Alterations in synaptic efficacy induced by antigen challenge to isolated superior cervical ganglia (SCG) were monitored by measuring the magnitude of the postganglionic compound action potential (CAP) elicited by electrical stimulation of the cervical sympathetic nerve trunk. Antigen-induced changes in the CAP were measured in SCG removed from actively and from passively sensitized guinea-pigs. Additionally, some SCG were sensitized in vitro by incubating naive ganglia 24 h in serum obtained from actively sensitized animals. Histamine released from SCG upon specific antigenic challenge was measured to assess the effectiveness of the two forms of sensitization. Challenging SCG isolated from passively or actively sensitized animals with the sensitizing antigen, ovalbumin (OVA), produced a sustained potentiation of the CAP lasting longer than 30 min (antigen-induced long-term potentiation, A-LTP) and a net increase in histamine release. Neither the magnitude nor duration of A-LTP induced by passive sensitization differed significantly (p < 0.05) from results after active sensitization. The existence of A-LTP in SCG following passive sensitization indicates that the afferent limb of the immune system is not required for the development of this phenomenon and that the immune cells and the mediators responsible for A-LTP are resident to sympathetic ganglia. Keywords: Long-term potentiation; Synaptic transmission; Autonomic ganglia; Superior cervical ganglion; Mast cell; Immediate hypersensitivity

1. Introduction

Interaction of antigen with mast c~ell-bound antibodies and the consequent release of inflammatory mediators are generally thought to be the pivotal events in an allergic response. Although our knowledge regarding the mechanism of mast cell activation is rapidly progressing, little is known about the neurophysiological effects this activation has in the various tissues in which mast cells reside. Autonomic ganglia are of particular interest in this regard because they are enriched with mast cells [10,15] and they represent a key control point for balance of autonomic tone. Furthermore, sympathetic ganglia, specifically the superior cervical ganglion (SCG), participate in late phase pulmonary inflammation subsequent to the induction of an anaphylactic reaction [8]. Despite the known presence of mast cells in autonomic ganglia and their documented role

* Corresponding author. Tel.: + 1 410 7065833; fax: + 1 410 7060032.

in anaphylaxis, their function within autonomic ganglia still remains enigmatic. To study the potential functional interaction between mast cells and neurons in autonomic ganglia, we have developed an in vitro model based upon the classical Shultz-Dale technique. Adult male guinea-pigs are actively sensitized to a foreign antigen (chick ovalbumin, OVA). Twenty one days later they are killed and their SCG are removed for study in vitro. Upon specific antigen challenge with OVA (10 /zg/ml), isolated SCG release a variety of preformed and newly synthesized inflammatory mediators including histamine, leukotrienes (LTC 4) and prostaglandins (PGE 2, PGD 2, 911/3-PGF 2, PGF2~, and TxB 2 [ 14,17]; concomittantly, the number of toluidine blue stainable mast cells in the SCG are significantly reduced [17]. Accompanying these histochemical and biochemical changes, antigen challenge produces physiological alterations in the SCG that range from a transient increase in neuronal excitability lasting many minutes [4] to a profound and long-lasting enhancement in the efficacy of ganglionic synaptic neurotransmission lasting many hours

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A.A. Cavalcante de Albuquerque et al. / Journal of the Autonomic Nervous System 61 (1996) 139-144

[4,18]. This latter phenomenon is designated antigen-induced long-term potentiation (A-LTP) because it resembles neurogenic LTP in this ganglia [2,3,18]. Neither treatment of ganglia from sensitized animals with non-sensitizing antigens nor application of OVA to SCG removed from control animal, produces observable release of mediators, changes in mast cell numbers or measurable electrophysiological changes [16]. Thus, the observation that specific antigen challenge releases known mast cell mediators and reduces the number of stainable ganglionic mast cells suggests that immunological activation of ganglionic mast cells is an integral step for the antigen-induced physiological changes. It is still unclear, however, whether the antigen-induced neurophysiological changes observed are indeed secondary to homocytotropic antibody generated during active sensitization or whether the changes observed are attributable to cellular immune responses associated with active sensitization. Active sensitization requires the coordinated actions of numerous cells (including lymphocytes) that utilize a variety of cytokines and their receptors. It is now recognized that many of these immune cytokines and their receptors are also expressed in nervous tissue where they can serve as neurotrophic factors regulating neural growth, survival and gene expression [7,11 ]. Thus the electrophysiolgical changes we observe following antigen challenge to SCG removed from actively sensitized animals could reflect alterations in neural function wrought by the process of active sensitization. To bypass the afferent limb of the immune system and thereby test whether some aspect of the cellular immune response might contribute to the observed antigen-induced electrophysiological effects, we treat non-sensitized SCG with antigen-specific antibodies. This process (passive sensitization) can be achieved through two manipulations. Serum obtained from actively sensitized guinea-pigs can be intraperitoneally injected into naive guinea-pigs and SCG from these animals can be removed 1 to 4 days later, or alternatively, ganglia from naive animals can be isolated and then incubated directly with serum obtained from actively sensitized guinea-pigs. In the current work we use both methods of passive sensitization and tested for the presence of A-LTP and histamine release following specific antigen challenge. Our results show that passive or active sensitization produces similar physiological changes in ganglionic synaptic transmission.

2. Materials and methods 2.1. Preparation o f tissue

Male guinea-pigs (350 to 800 g) were killed by administration of CO 2. Preparation of the SCG and the composition of the Locke solution were identical to those described in [18]. Ganglia were super/used at 3 - 4 ml/min with

oxygenated Locke solution that was maintained at 3637°C. 2.2. Active and passive sensitization o f animals and ganglia 2.2.1. Active sensitization

Ovalbumin (chicken egg albumin, ovalbumin grade V; Sigma Chemical Co., St. Louis, MO, USA) was intraperitoneally injected into guinea-pigs as described previously [17]. Animals were injected with doses of ovalbumin (OVA; 10 m g / k g body weight) every other day for three doses. Ganglia were studied 21 to 50 days after the last injection. 2.2.2. Passive sensitization

Guinea-pigs were injected intraperitoneally on two consecutive days with serum (0.5 ml) obtained from actively sensitized animals. SCG were removed from these animals 1-5 days after the last injection. There was no significant difference in results obtained at 1 or 5 days. Passive sensitization in vitro was accomplished by incubating SCG from naive animals in serum (0.5 ml) obtained from actively sensitized animals for 15-24 h at (5°C). Serum from actively sensitized and from control animals was stored in aliquots at -80°C. 2.3. Histamine release studies

Histamine released from isolated SCG was quantified by automated fluorometry [12] as described by [14]. This procedure has a limit of sensitivity (two times the blank) of 10 pmol/ml. 2.4. Electrophysiological studies

Extracellular recordings of the postganglionic compound action potential (CAP) were measured with bipolar suction electrodes connected to the input stage of an AC-coupled differential preamplifier (0.1-1.0 kHz; WPI Corp.; model DAM-5A). Data was filtered at 1 kHz and when necessary sampled at 10 kHz (see [18] for details). The effect of antigen and drugs on ganglionic synaptic transmission was assessed by measuring changes in the integral of the evoked CAP. Alterations in the area of the CAP were taken as an index of the number of ganglion cells synaptically excited to spike threshold. CAPs were evoked at 0.2 Hz and 12 CAPs were collected and averaged via pClamp software (Axon Instruments Inc., Foster City, CA, USA) running on an IBM PC with a TL1 interface. Data files were analyzed off-line as described [18]. The 0.2 Hz presynaptic rate of stimulation was chosen to minimize synaptic depression [9] and activity-dependent synaptic potentiation [1-3,6,19]. The postganglionic response was made approximately one half the maximal

A.A. Cavalcante de Albuquerque et al. / Journal of the Autonomic Nervous @stem 61 (1996) 139-144

amplitude by the addition of the nicotinic antagonist hexamethonium (100-300/xM). After the CAP amplitude had declined to a steady-state baseline in the presence of hexamethonium, CAPs were monitored for 30 rain before antigen challenge.

2.5. Preparation and deliveo, of drug solutions Ovalbumin, histamine dihydrochloride, and hexamethonium were obtained from Sigma, St. Louis, MO. SCG were challenged with ovalbumin with the same lot number used to sensitize the animal. Drug solutions were prepared daily for experiments from concentrated ( > 10 mM) stock aliquots which were stored frozen (-20°C). Reservoirs containing oxygenated solutions of superfusate with various drugs were connected to the inflow line to the recording chamber by three-way valves which could rapidly divert flow from the main reservoir to these solutions.

141

guinea-pigs. The experiment shown in Fig. 1B illustrates a typical antigen-induced response in a SCG from a passively sensitized guinea-pig. The onset of the potentiation, the magnitude and the duration of potentiation following antigen challenge were comparable to responses observed in ganglia from actively sensitized animals. /max and 130 were 50 _+ 7.2% and 49 _ 4.4%, respectively (n = 6, Table 1). In a separate series of experiments seven SCG were passively sensitized by incubating normal ganglia for 1524 h in serum obtained from actively sensitized animals. Upon antigen challenge these ganglia showed synaptic potentiation with a time course similar to that observed with active sensitization or passive sensitization (in vivo); see Fig. 1C and Table 1. There were no significant differences ( p < 0.01) in the magnitude or duration of antigeninduced potentiation between the three groups of treatments - active sensitization, passive sensitization (in vivo) or passive sensitization (in vitro).

2.6. Statistical analysis 3.3. Control experiments' (in uiL,o and in L,itro) Data were expressed as mean _+ SE. Two samples were compared using Student' s t-test; multiple comparisons were done with parametric ANOVA, or Kruskal-Wallis ANOVA in cases where the distribution is unknown. Means were considered to differ significantly if p values for null hypothesis occurrence were < 0.05.

Three SCG were passively sensitized with OVA in vivo and three ganglia were passively sensitized with OVA in vitro. These ganglia were then challenged with bovine serum albumin (BSA, 10 /xg/ml) for 5 rain. BSA challenge did not significantly ( p < 0.001) potentiate synaptic transmission in either group (Fig. ID and Table 1).

3. Results

3.1. Characteristics of ganglionic potentiation following active sensitization

Table 1 Effect of antigen challenge on synaptic transmission in superior cervical ganglia removed from actively or passively sensitized guinea-pigs Conditions

A brief (5 min) exposure to antigen (ovalbumin, OVA; 10 /xg/ml) produced a vigorous increase in the magnitude of the postganglionic compound action potential (CAP) which reach a maximum within 5 min (Fig. 1A). At this concentration OVA produces a maximum potentiation [ 18]. The magnitude of the potentiation shown in Fig. 1A was an unusually robust response; the integral of the CAP increased about 4-fold. On the average, maximum antigen-induced potentiation of the CAP (Im~×) was 61 _+ 2.1% (n = 60, Table 1). Potentiation was also long-lasting. The fractional potentiated response 30 min after OVA (130) in the ganglion shown in Fig. 1A about 50%; for 60 ganglia the average 130 value was 56 _+ 1.8%. These values for antigen-induced potentiation are in the same range as those previously reported by our laboratory [18].

3.2. Characteristics of ganglionic potentiation .following passive sensitization (in vivo and in vitro) Antigen-induced ganglionic potentiation was also observed in ganglia from normal animals that had been injected with serum harvested from actively sensitized

Antigen

N

Potentiation

Duration (130)

(/m~) Active sensitization Passive sensitization (in vivo) Passive sensitization (in vitro) Control

OVA OVA

60 6

61 +2.1 " 50_+7.2 ~

56 _+ 1.8 ~' 49 _+4.4 "

OVA

7

55 _+7.0 ~'

47 _+5.4 ~'

BSA b OVA c

6 6

3_+ 1.7 5_+2.6

0.0_+ 0.0 0.0_+ 0.0

Values are m e a n s _ S E . Im~~ is the magnitude of potentiation of the postganglionic compound action potential (CAP) measured as the integral of the CAP. 130 is the fractional potentiated CAP amplitude 30 rain after a 5 rain application of antigen (ovalbumin, OVA; 10 txg/ml). Procedures for active, passive (in vivo) and passive (in vitro) sensitization are described in the Section 2. a Using Kruskal-Wallis ANOVA, there were no significant differences ( p < 0.01) between the means of the three groups of sensitization (active and passive in vivo and in vitro) following antigen challenge. b Control OVA experiments consisted of three animals injected with serum from naive animals (control serum) and three SCG from naive animals incubated in control serum. All six ganglia were individually challenged with 10 / z g / m l OVA for five minutes. " Control experiments consisted of three animals injected with serum from actively sensitized animals and three SCG from naive animals incubated in serum from actively sensitized animals. All six SCG were individually challenged with 10 / x g / m l bovine serum albumin (BSA).

A.A. Cavalcante de Albuquerque et al. / Journal of the Autonomic Nervous System 61 (1996) 139-144

142

PASSIVE

(in v/vo)

ACTIVE (a) CONTROL

(b) OVA

(a) CONTROL

(b) OVA

J 4o A

(b)

30

B

35

(b)

"G ~3 U)

30

g

25

~"

E

m p. O Q. m n,

25

E E

t5

v u) tO Q. v) n,

(a)

20'

q

10 -

OVA

OVA 5 0

10

I

I

I

20

30

40

10

I

I

I

I

10

20

30

40

Time (min)

PASSIVE

Time (min)

(in v/tro)

(a) CONTROL

(b) OVA

CONTROL

15 20 ]

C

~

15-

E

n,

10

OVA co D.

c o

o.

D

(b)

lO

(a) BSA

OVA I

I

I

I

I

I

I

I

I0

20

30

40

I0

20

30

40

Time (min)

Time (min)

A.A. Cavalcante de Albuquerque et al. / Journal of the Autonomic Nervous System 61 (1996) 139-144 Table 2 Effect of antigen challenge on histamine release from superior cervical ganglia removed from actively or passively sensitized guinea-pigs Condition

N

Spontaneous release (%)

Net antigen induced release ( % ) a

Active sensitization b Passive sensitization (in vivo) Passive sensitization (in vitro) Control d

61 6

< 1.0 1.7_+0.39

33 +-2.0 9 +_3.1 c

7

1.5+_0.53

l0 _+3.9 c

6

0.8+-0.11

0.5_+0.2

Values shown are means +-SE. a Released histamine is expressed as a percentage of total histamine content (released plus residual ganglion content). Ganglia were challenged with 10 /zg/ml chick ovalbumin (OVA). b Data for active sensitization taken from [14]. Characteristics of passive sensitization in vivo and in vitro delineated in Table 1 legend and in Section 2. Values significantly different (p < 0.001 ) from values for active sensitization or from control values. d Control experiments consist of three SCG removed from animals intraperitoneally injected with control serum and three SCG incubated with control serum in vitro. All six ganglia were individually challenged with OVA. OVA treatment did not significantly increase release above spontaneous release (p < 0.001 ).

In another series of experiments, three ganglia from naive animals were incubated in vitro in control serum for 24 h and then challenged with O V A (10 / ~ g / m l for 5 min). Three guinea-pigs were also injected with control serum following the same protocol used for serum obtained from actively sensitized animals (see Section 2). Two to four days later ganglia from these animals were challenged in vitro with OVA. The magnitude of the CAP did not change significantly ( p < 0.001) (Table 1). Six control ganglia (three from the O V A group and three from the B S A group described above) were tested for their ability to reveal synaptic potentiation. Superfusing these ganglia for 5 min with 10 /aM histamine produced a robust, albeit transient, potentiation of synaptic transmission (95 ± 27% increase in the C A P amplitude over control values) indicating that synaptic transmission in these ganglia was capable of plasticity [5]. 3.4. Antigen-induced histamine release f r o m actively and passively sensitized SCG W e have previously shown that exposing SCG isolated from actively sensitized guinea-pigs to the sensitizing antigen results in mast cell degranulation and the release of

143

inflammatory mediators including histamine and a variety of eicosanoids [14,17]. In the present work we determined whether histamine was released from two types of passively sensitized SCG following antigen challenge. The amount of histamine released from passively sensitized ganglia upon antigenic challenge was significantly greater ( p < 0.001) than spontaneous histamine release, Table 2. In these 13 passively sensitized ganglia (six in vivo and seven in vitro), antigenic challenge released about 10% of the total ganglionic histamine pool ( ~ 200 pmol). By contrast, O V A application to control ganglia produced no significant ( p < 0.001) elevation in histamine release above spontaneous release. For comparative purposes, Table 2 also shows the data from our previous work measuring antigen-induced histamine release from SCG removed from actively sensitized guinea-pigs [14]. Although the amounts of histamine spontaneously released from all groups of ganglia were not significantly different ( p < 0.05), the amount of histamine released from SCG following antigen challenge was significantly larger (about 3-fold; p < 0.001) in ganglia removed from the actively sensitized animals (33 versus 10%).

4. Discussion Our principle finding is that specific antigen challenge of sympathetic ganglia isolated from passively sensitized guinea-pigs produces a sustained increase in the efficacy of synaptic transmission. W e had shown previously that specific antigen challenge of sympathetic ganglia removed from actively sensitized guinea-pigs produced a sustained enhancement of synaptic transmission that we designated antigen-induced long-term potentiation (A-LTP; [18]). The data from the current work reveals that there is no significant difference in the magnitude or duration in antigen-induced ganglionic synaptic potentiation evoked by either passive or active sensitization. Thus, the existence of A - L T P in ganglia following passive sensitization indicates that the afferent limb of the immune system is not required for the development of this phenomenon and that the immune cells and the mediators responsible for A - L T P are resident to sympathetic ganglia. This result fits well with our working hypothesis [18] that ganglionic mast cell activation is required for A-LTP. The amount of histamine released from sympathetic ganglia of actively sensitized guinea-pigs by antigen chal-

Fig. 1. Time course and magnitude of antigen-induced potentiation of synaptic transmission in the superior cervical ganglion (SCG) in vitro. At the time indicated by the triangle the sensitizing antigen, chick ovalbumin (OVA, 10 /zg/ml, or control antigen), was superfused over the SCG for five minutes. Each data point represents the average magnitude (integral) of 12 evoked postganglionic compound action potentials (CAPs) evoked at 0.2 Hz. (A) Effect of antigen challenge recorded from an SCG removed from an actively sensitized animal. (B) Antigen challenge to SCG removed from passively sensitized animal (in vivo). (C) Antigen challenge to a normal SCG incubated 18 h in serum from actively sensitized guinea-pigs. (D) Two control experiments. OVA challenge (10 /zg/ml) of a normal SCG incubated in control serum for 24 h. Another SCG was removed from a guinea-pig passively sensitized to OVA (in vivo) and challenged with 10 /zg/ml bovine serum albumin (BSA). Calibration: (A) 0.5 mV, t0 ms; (B, C) 1 mV, 10 ms.

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lenge was three-fold higher than from ganglia taken from passively sensitized animals ( p < 0.001). Despite this, the magnitude and duration of A-LTP were similar in both groups. Analogous results were reported for OVA-induced contraction of tracheal rings taken from guinea-pigs actively or passively sensitized with IgG 1 antibody [13]. This result in the SCG is not surprising because we have previously observed that low concentrations of antigen (0.01 /zg/ml OVA) released only ~ 6% of the ganglionic histamine pool yet produced nondecremental A-LTP [18]. Though the duration of A-LTP elicited by this low concentration of antigen was comparable to that observed with maximal antigen challenge (10 /xg/ml), the magnitude of A-LTP was only 75% of maximum. Thus it is difficult to conclude that a smaller amount of mediator released in passive sensitization is responsible for both duration and magnitude of A-LTP. One explanation for the similarity of the magnitude of A-LTP in passively sensitized ganglia to that in actively sensitized tissue may be that the mediator(s) responsible for A-LTP is released in similar amounts in both types of sensitization. In this regard it is instructive to note that although histamine is released upon antigen challenge it is clearly not responsible for A-LTP [18] and therefore the amount of histamine released may not parallel the release of the critical mediator(s) underlying A-LTP. Finally, the ability to evoke A-LTP with passive sensitization provides considerable advantage over active sensitization for future studies of the underlying mechanisms, as animals need to be held for only a few days (or not at all) prior to the experiment.

Acknowledgements We wish to thank Ms. Jennifier O'Brien for participating in some of the experiments and Dr. Brad Undem for measurements of histamine. The authors thank Ms. Kimberly Moore for constructive suggestions on an earlier draft of this manuscript. This work was supported by NINDS grants NS22069 and NS25598 to D.W.

[3]

[4]

[5]

[6]

[7] [8]

[9]

[10] [11]

[12]

[13]

[14]

[15] [16]

[17]

[18]

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