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Antidepressant drugs potentiate the α1-adrenoceptor effect in hippocampal slices
European Journal of Pharmacology, 166 (1989) 183-191
183
Elsevier RIP 50872
Antidepressant drugs potentiate the al-adrenoceptor effect in hippoemnp~ slices M a r i a Bijak * Polish Academy of Sciences, Institute of Pharmacology, 12 Smetna, 31-343 Krakow, Poland
Received 25 August 1988, revised MS received 28 March 1989, accepted 25 April 1989
The effect of prolonged treatment with antidepressant drugs on the phenylephrine- and norepinephrine (NE)-evoked reaction in hippocampal slices was examined by extracellular recording of the spontaneous activity of CA1 layer neurons. The eq-adrenoceptor agonists, phenylephrine and methoxarnine, depressed the neuronal discharges of most of the units tested, while NE evoked both excitatory and inhibitory effects which were blocked by propranolol and phentolamine or prazosin, respectively. Imipramine, mianserin, (+)- and (-)-oxaprotiline administered subchronically (10 mg/kg p.o., twice daily for 14 days, withdrawal 48 h), potentiated the inhibitory reaction to phenylephrine. Mianserin was the only drug tested in the acute dose to effectively augment the reaction to al-adrenoceptor stimulation. Prolonged administration of mianserin and imipramine attenuated the excitatory effect to NE, which probably reflects r-receptor down-regulation; however, only mianserin, but not imipramine, enhanced the NE-induced inhibition. The observed potentiation of the al-adrenoceptor-related inhibitory reaction to phenylephrine produced by antidepressant drugs may reflect the development of the al-adrenergic system supersensitivity in the hippocampus. Antidepressants; al-Adrenoceptors; Phenylephrine; Hippocampal slice
1. Introduction
Experimental evidence suggests that alterations in the central noradrenergic system is one of the most consistent effects of treatment with antidepressant drugs. While a decrease in both the function and number of a 2- and fl-adrenoceptors after repeated administration of antidepressant drugs and electroconvulsive shock has been reported by several laboratories, it is still a matter of controversy as to whether there are changes in central %-adrenoceptors after these treatments (see Sugrue, 1983; McNeal and Cimbolic, 1986; Lipin-
* To whom all correspondence should be addressed at present address (till 31 January 1990): Max-Planck-Inst. f'firPsychiatrie, Department of Neurophysiology, Am Klopferspitz 18A, 8033 Planegg-Martinsried, F.R.G.
ski et al., 1987). Some biochemical, electrophysiological and behavioral studies have demonstrated an increase in the number and function of a 1adrenoceptors after antidepressant therapy (Rehavi et al., 1980; Menkes and Aghajanian, 1981; Menkes et al., 1983; Vetulani et al., 1984; Maj, 1984; Hong et al., 1986; Mogilnicka et al., 1987; Stockmeier et al., 1987), but there is also considerable experimental evidence that does not confirm the antidepressant-evoked alterations in the a 1adrenoceptor system (Heal 1984; Stockmeier et al., 1987). It has been suggested that the hippocampal formation, being a part of the limbic brain, is involved in the regulation of emotional and motivational functions, processes that are affected in depression (Angst and Dobler-Mikola, 1984; Post, 1986). Therefore the hippocampus m a y be an important anatomical substratum for the action of
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
184
antidepressant drugs. This suggestion is supported by reports concerning alterations in the hippocampal fl-adrenergic, dopaminergic, serotonergic, cholinergic and GABA-ergic systems after antidepressant treatment (Lerer et al., 1983; Anwyl and Rowan, 1984; Lloyd et al., 1986; Newman et al., 1987; Smia/owski and Bijak, 1987; Bijak and Smia/owski, 1988). Moreover, there is direct behavioral evidence for al-adrenoceptor supersensitivity in the hippocampus of rats treated with antidepressant drugs (Plaznik et al., 1984; Plaznik and Kostowski, 1985). It is expected that these adaptive changes in the a]-receptor system should have their neurophysiological correlate in the responsiveness of individual neurons to aa-adrenergic stimulation. Therefore the present study was aimed at gaining insight into al-adrenoceptor function in the hippocampus. To this end the effect of the al-selective agonist, phenylephrine, and the putative neurotransmitter, norepinephfine, on spontaneous discharges of the CA1 layer neurons was studied in the brain slices from rats treated with antidepressant drugs. So far, antidepressant drug-induced alterations in the responsiveness of hippocampal neurons to stimulation of /3-adrenoceptors, serotonin and dopamine receptors have been described (Anwyl and Rowan, 1984; Rowan and Anwyl, 1985; Smia/owski and Bijak, 1987; Bijak and Smia/owski, 1988). The present study shows that different antidepressants evoke supersensitivity of the eqadrenoceptor system in the hippocampus. Therefore electrophysiological evaluation of the reactivity of hippocampal neurons to different neurotransmitters in vitro is proposed as a sensitive method for screening the potential antidepressant activity of drugs.
ing antidepressant drug (10 m g / k g p.o.). The animals were killed by decapitation 48 h after the last dose of the drug. The brain was removed and the hippocampus was dissected. Transverse hippocampal slices, approximately 400 /~m thick, were cut and transferred to an incubation chamber. After at least 1 h a single slice was placed in an experimental chamber (0.5 ml in volume) on a nylon mesh and continuously superfused, at a rate of 1 m l / m i n , with an oxygenated solution consisting of (mM) NaC1 124, KC1 5, CaC12 2.4, MgSO 4 1.3, KHEPO 4 1.25, NaHCO 3 26, glucose 10. The temperature was maintained at 34 o C, the pH at 7.4. Extracellular recordings of the spontaneous activity of cells in the CA1 layer were made by using a tungsten microelectrode (Clark Electromedical Instr. 12 MS2). Neurons were selected on the basis of the characteristics of their spontaneous activity. Units displaying a low firing rate (mean = 6.96 + 0.33 Hz, n = 300) and multiple irregular action potential of long duration were chosen. These neurons are related to complex-spike cells, which may represent pyramidal neurons (Berger et al., 1983; Pang and Rose, 1987). An example of the spontaneous activity, photographed directly from the oscilloscope screen, is shown in fig. 1. The frequency of spontaneous discharge was calculated as the number of spikes per minute, and was simultaneously integrated at 10 s intervals and recorded on a chart recorder. The substances used were dissolved in incubation medium and 0.1 ml was added to the perfusion line just before it entered the experimental chamber after at least 10 min of a stable basal firing rate. The final concentration of the substance in the chamber was about 5 times lower
2. Materials and methods
Male Wistar rats (200-250 g), housed under controlled 12 h light-dark conditions (light on between 7-19 h) and with free access to food and water, were used. The animals received water or antidepressant drug (10 m g / k g p.o.) twice daily for 14 consecutive days. In the acute experiment the rats received a single dose of the correspond-
I Fig. 1. An example of the spontaneous activity of CA1 layer neurons photographed directly from the oscilloscope screen. Vertical bar: 20 FV; horizontal bar: 5 ms.
185
than its initial one. In the experiments in which the effect of the antagonistic drug was investigated, the slices were perfused with the drug-containing solution for at least 20 min. The reaction to the given substance is expressed as a percentage of the mean firing rate during the control period, measured at 1 rain intervals after drug administration. The percent depression (effect below 80% of the control firing rate) and excitation (effect above 120% of the control) in one experimental series were pooled to give a mean change in the neuronal firing rate after administration of the substance. The mean percentage changes in neuronal discharge were compared by using a one-way analysis of variance and a two-tailed Student's t-test. A Chi square test was used to compare the number of units reacting with excitation and inhibition in different experimental groups. The drugs used were: 1-phenylephrine (Boehringer Ingelheim), D L - n o r e p i n e p h r i n e HC1 (Sigma), methoxamine (Wellcome), prazosin (Pfizer), propranolol (Polfa), phentolamine HC1 (Ciba-Geigy); imipramine HC1 (Polfa), mianserin HC1 (Organon), ( +)-oxaprotiline, ( - ) - o x a p r o t i line (Ciba-Geigy).
NE
NE Fig. 2. The effect of norepinephrine (NE, 16 # M ) on the spontaneous firing rate of CA1 neurons in hippocampal slices. Vertical bar: 20 i m p u l s e s / 1 0 s; horizontal bar: 5 rain. TABLE 1 The effect of norepinephrine (NE, 16 ttM) on the spontaneous firing rate of hippocampal CA1 neurons and the influence of acute (1 x ) and prolonged treatment with imipramine (IMI) and mianserin (MIA) on the norepinephrine-evoked reaction. n = n u m b e r of units tested, a Statistical significance vs. control, Chi-square test, P < 0.05. Substance tested
Pretreat-
n
Type of response (%n)
ment NE N E + propranolol 1#M
3. R e s u l t s
In hippocampal slices prepared from nontreated rats norepinephrine (NE, 16 #M) elicited an increase in the spontaneous discharge rate in the majority of the CA1 layer neurons studied, inhibitory reactions were observed in only a few units (fig. 2). As shown in table 1, the NE-mediated excitatory response was blocked by propranolol. N E evoked mainly a decrease in the neuronal
Ph
Inhibition Excitation
-
23
26
74
-
14
86
-
-
9
-
13
-
NE NE
IMI 1 x IMI 1 4 x
16 17
44 18
50 82
NE
MIA 1 x MIA 1 4 ×
21 23
48 78
50 17
NE+phentolamine 10 v M N E + prazosin 0.1 # M
NE
100
a
77
a
Ph
Fig. 3. Examples of inhibitory responses of hippocampal neurons to phenylephrine (Ph, 20 #M). Vertical bar: 10 i m p u l s e s / 1 0 s; horizontal bar: 5 min.
186 TABL E 2 The effect of phenylephrine and methoxamine on the spontaneous firing rate of hippocampal CA1 neurons, n = number of units tested; a p < 0.05 t-test vs. phenylephrine 0.2 # M ; mean, the mean percent decrease in the neuronal firing rate following administration of phenylephrine as compared to the pre-drug baseline control, b p < 0.001. Substance tested (dose #M)
n
Type of response (%n) Inhibition
Excitation
No effect
1.37 3.76 a -
14 14 13
27 36 62
2.37 a
10
10 80
--
--
M e a n _ S.E.M. Phenylephrine (0.2) + prazosin (0.01) + prazosin (1.0)
37 14 8
59 50 25
59.09 69.38 -
Phenylephrine (20) + prazosin (1.0)
13 10
92 10
46.89
Methoxamine (100)
6
100
54.41
-
firing rate in the presence of this fl-receptor antagonist. Conversely, application of phentolamine and prazosin abolished the inhibitory reaction to NE. The specific al-receptor agonist, phenylephrine, generally diminished the neuronal firing rate in hippocampal slices (figs. 3,4). The inhibitory effect of phenylephrine was dose-dependent and was significantly attenuated by the specific al-receptor antagonist, prazosin (table 2). Another th-receptor agonist, methoxamine, produced a decrease in the frequency of neuronal discharges in all the units tested. However, methoxamine had a lower potency than phenylephrine in eliciting the inhibi-
-
3.37
b
tory effect. The latency of the inhibitory reaction evoked by selective stimulation of the al-receptor was longer than the latency of the NE-evoked excitation or inhibition (figs. 2,3,4). Unlike the short-lasting effects of NE, the effect of phenylephrine was frequently of long duration, and the firing rate often did not return to the pre-drug level after a 30 rain washout period. The application of phenylephrine induced inhibition followed by excitation in some units or only elevation of the firing rate (table 2). Interestingly, the excitatory effects were usually disproportionately high in relation to the low dose of phenylephrine.
TABLE 3 The influence of acute (1 × ) and prolonged (14 x ) treatment with antidepressant drugs: imipramine (IMI), mianserin (MIA), ( + ) and ( - ) - o x a p r o t i l i n e (OXA) on the responsiveness of hippocampal neurons to phenylephrine (0.2 /~M). N = number of animals; n = number of units tested. Pretreatment
N
n
Type of response (%n) Inhibition
Excitation
N o effect
Control
8
37
59
14
27
IMI 1 x IMI 1 4 x
5 7
27 28
59 61
15 21
26 18
MIA I x MIA 14x
5 10
23 38
57 82
30 5
13 13
( + )-OXA 1 × ( + ) - O X A 14 x
4 5
18 22
39 73
22 9
39 18
( - )-OXA 1 x ( - )-OXA 14 x
4 5
18 21
56 85
11 10
33 5
187 Cont.
]
acute
D chronic O
140
A
iili
Ph
MIA
|OC
1
Ph Fig. 4. The effect of subchronic treatment with mianserin (MIA) on the inhibitory response of hippocampal CA1 neurons to phenylephrine (Ph, 0.2 #M). Vertical bar: 10 i m p u l s e s / 1 0 s; horizontal bar: 5 rain.
60,
Cont.
Prolonged administration of imipramine, mianserin, ( + ) and ( - ) enantiomers of oxaprotiline potentiated the inhibitory reaction evoked by phenylephrine (figs. 4,5). Imipramine, (+)- and (-)-oxaprotiline, given acutely, did not change the effect of phenylephrine on the neuronal firing
O
IMI
MIA
o
Fig. 6. The effect of acute and subchronic treatment with imipramine (IMI) and mianserin (MIA) on the excitatory and inhibitory reactions of hippocampal neurons to norepinephrine (NE, 16 /LM). The results are expressed as the percent change ( m e a n + S . E . M . ) in the baseline firing rate following N E administration. 0 p < 0.05 vs. control, t-test.
100, X
o
I o
~L_
50 o
_-:::::_-
N Cont.
IMI
[ ] prezosin
l
MIA
[ ] acute
(+) OXA
( . ) OXA
[ ] chronic
Fig, 5. The effect of acute and subchronic treatment with antidepressant drugs: imipramine (IMI), mianserin (MIA), ( + ) - and ( - ) - o x a p r o t i l i n e (OXA), on the reaction of hippocampal neurons to phenylephrine (Ph, 0.2 /~M). The results are expressed as the percent change ( m e a n + S.E.M.) in the baseline firing rate following Ph administration. Prazosin (0.01 /xM) applied to the bathing medium. 0 p < 0.05 vs. control, t-test.
188 rate; however, a single dose of mianserin enhanced the reaction to a~-receptor stimulation. Repeated administration of mianserin potentiated the NE-induced inhibitory reaction, both increasing the potency of the effect and the number of units reacting with inhibition (table 1, fig. 6). A similar change in the reaction to NE, i.e. from excitation, which prevailed in control slices, to inhibition, was observed after a single dose of mianserin; however, this effect was not statistically significant. The excitation elicited by NE was significantly diminished in the hippocampal slices prepared from animals treated either acutely or chronically with mianserin. A single dose of imipramine did not change the responsiveness of CA1 neurons to NE, while prolonged treatment with the antidepressant attenuated the NE-evoked excitatory effect (table 1, fig. 6).
4. Discussion
The data reported above on the excitatory and inhibitory action of norepinephrine (NE) on the spontaneous discharge rate of CA1 neurons and the blockade of the latter effects by propranolol and phentolamine or prazosin, respectively, are in line with the electrophysiological action of the monoamine in the hippocampal formation. It has been shown that exogenously applied norepinephrine interacts with both a- and fladrenoceptors in the hippocampus, producing a decrease and increase, respectively, in neuronal excitability in the CA1 layer (Mueller et al., 1981; 1982a,b; Madison and Nicoll, 1986). Several data suggest that, unlike other brain areas where stimulation of al-adrenoceptors brings about an excitatory reaction, application of selective al-adrenoceptor agonist in the hippocampus results in inhibition of neuronal firing and synaptic responses (Mueller et al., 1981; Madison and Nicoll, 1986; Pang and Rose, 1987; Mynlieff and Dunwiddie, 1988). The data presented provide further electrophysiological evidence for a al-adrenoceptor-mediated attenuation of spontaneous discharges of CA1
neurons. The specific al-adrenoceptor agonists, phenylephrine and methoxamine, diminished the firing rate of hippocampal neurons. Moreover, the phenylephrine-induced inhibition was in most cases blocked by the selective, competitive a 1adrenoceptor antagonist, prazosin. The long-lasting effect of phenylephrine observed is in line with the reported prolonged action of this substance in the brain (Rogawski and Aghajanian, 1980). The potent action of phenylephrine on the spontaneous firing rate of hippocampal neurons parallels the high efficacy of this substance in stimulating the breakdown of inositol phospholipids in the hippocampal formation (Johnson and Minneman, 1985; Fowler et al., 1986). Although inhibition was the predominant action of phenylephrine on the CA1 neuronal discharges, in few cases an excitatory reaction was also observed. This effect was not studied further; however, it is suggested that inhibition of inhibitory interneurons in the hippocampus may lead to excitation of pyramidal cells (Jahr and Nicoll, 1982; Collins et al., 1984). Moreover, it has been implied that phenylephrine may evoke an excitatory effect via non-specific activation of fl-adrenoceptors (Mynlieff and Dunwiddie, 1988). The present results show that imipramine, mianserin, ( + ) - and (-)-oxaprotiline, when administered subchronically, enhance the reaction of hippocampal neurons to phenylephrine. The acute application of imipramine, ( + ) - and ( - ) oxaprotiline has no such sensitizing effect. However, even a single dose of mianserin potentiated the phenylephrine-induced inhibition. Similarly, acute administration of mianserin affects the density of serotonin receptors in the brain (Helmeste, 1986). On the basis of the binding data and biochemical studies it is postulated that potentiation of the electrophysiological effect of phenylephrine could be due to the antidepressant-evoked increase in the number a n d / o r affinity of al-adrenoceptors, or result from enhancement of the second messenger system coupled to the receptor (Menkes et al., 1983; Vetulani et al., 1984; Hong et al., 1986; Newman et al., 1987; Stockmeier et al., 1987). Mianserin was effective not only in augmenting the phenylephrine-induced depression in neuronal
189 firing but also in potentiating the NE-evoked inhibitory reaction, whereas imipramine, although the most potent of the drugs tested in enhancing the action of phenylephrine, did not change the inhibitory reaction to NE. Although the depression of neuronal firing evoked by NE in the hippocampus is most probably due to activation of both a a- and a2-adrenoceptors (Mueller et al., 1981; Madison and Nicoll, 1986) and a l t h o uhg it has been demonstrated that antidepressant treatment enhances a a- and attenuates a2-adrenoceptor function (McNeal and Cimbolic; Lipinski et al., 1987), changes in the a-adrenoceptor-related response were not observed when NE was used. However, mianserin, a potent a2-antagonist, probably does not produce a significant down-regulation of a2-receptors, this might permit the manifestation of'an enhanced a-adrenoceptor-related reaction to NE. The attenuation of the excitatory response to NE in hippocampal slices prepared from animals treated with imipramine and mianserin is in line with the decrease in the number and function of fl-adrenoceptors observed after prolonged administration of antidepressants (Maggi et al., 1980; Mishra et al., 1980; Anwyl and Rowan, 1984). The antidepressants tested had different pharmacological profiles in the acute experiment, yet all of them affect the brain noradrenergic system. The tricyclic antidepressant imipramine, is a nonselective inhibitor of the NE and serotonin uptake, the tetracyclic, (+)-oxaprotiline, is more selective as it inhibits the NE uptake only, while the atypical antidepressants, the tetracyclics, mianserin and (-)-oxaprotiline, are not uptake blockers in vivo. All the antidepressant drugs tested have adrenolytic activities, probably a prerequisite for their sensitizing action on the a 1adrenoceptor system in the hippocampus (Waldmeier et al., 1981; see Fuller and Wong, 1985; McNeal and Cimbolic, 1986). While the uptake-blocking effect of imipramine, which leads to an increase in availability of NE at the postsynaptic receptor, is thought to be responsible for the down-regulation of r-receptors, the direct blockade of al-adrenoceptors by the antidepressant could be responsible for the augmented response to eta-stimulation.
Similarly, it may be assumed that mianserin increases the concentration of endogenous NE in the synaptic cleft by blocking a2-presynaptic receptors, leading to r-receptor down-regulation and a reduction in fl-adrenergic excitatory activity in the hippocampus. In contrast, antagonism of a 1adrenoceptors is a possible mechanism for the mianserin-induced supersensitivity to phenylephrine. The same mode of action can be proposed for both enantiomers of oxaprotiline, which also have adrenolytic activity (Waldmeier et al., 1982). (-)-Oxaprotiline, which is ineffective in potentiating some behaviorally assessed functions related to the al-adrenoceptor system (Maj, 1984), evoked sensitization in the present electrophysiological model. However, in other studies the effects of both the ( + ) and ( - ) enantiomers of oxaprotiline were similar, which indicates that the inhibition of uptake is not essential for at least part of the antidepressant evoked actions (Kopanski et al., 1983; Maj and Wedzony, 1987; Mogilnicka et al., 1987). The sensitizing effect of mianserin, which is devoid of uptake-blocking activity in vivo, on the phenylephrine- and NE-induced inhibition demonstrated in the presented study corroborates the latter assumption. Antidepressant-evoked changes in different receptor systems may depend on complex interactions in the brain. Several authors describe the involvement of the serotonergic system in the regulation of adrenergic transmission (Sulser, 1984; Racagni et al., 1986), thus the sensitizing effect of the antidepressants studied on the al-adrenoceptor-related response in the hippocampus may be due partly to antidepressant-induced changes in the serotonergic system (Rowan and Anwyl, 1985). The latter possibility is supported by the established serotonergic involvement in the regulation of aa-adrenoceptors in the hippocampus (Consolo et al., 1985). The present results demonstrate that, irrespective of their different acute effects, the long-term administration of the antidepressants tested leads to supersensitivity of the al-adrenoceptor system in the hippocampus. These data are in line with behavioral evidence of antidepressant-evoked central etl-supersensitivity (Maj, 1984).
19o
Acknowledgement This work was supported by the Polish Academy of Sciences. Research Project No. 06-02.I.
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biochemical changes induced by prolonged antidepressant administration, Clin. Neuropharmacol. 9, 110. Rehavi, M., O. Ramot, B. Yavetz and M. Sokolovsky, 1980, Amitriptyline: long-term treatment elevates a-adrenergic and muscarinic receptor binding in mouse brain, Brain Res. 194, 443. Rogawski, M.A. and C.K. Aghajanian, 1980, Activation of lateral geniculate neurons by norepinephrine: mediation by an a-adrenergic receptor, Brain Res. 182, 345. Rowan, M.J. and R. Anwyl, 1985, The effect of prolonged treatment with tricyclic antidepressants on the actions of 5-hydroxytryptamine in the hippocampal slice of the rat, Neurol~harmacology 24, 131. Smialowski, A. and M. Bijak, 1987, Repeated treatment with imipramine enhances the excitatory response of hippocampal neurons to dopamine, Neuroscience 23, 1021. Stockmeier, C.A., S.W. McLeskey, J.A. Blendy, N.R. Armstrong and K.J. KeUar, 1987, Electroconvulsive shock but not antidepressant drugs increases al-adrenoceptor binding sites in rat brain, European J. Pharmacol. 139, 259. Sugrue, M.F., 1983, Do antidepressants possess a common mechanism of action?, Biochem. Pharmacol. 32, 1811. Sulser, F., 1984, Regulation and function of noradrenaline receptor systems in brain. Psychopharmacological aspects, Neuropharmacology 23, 255. Vetulani, J., L. Antkiewicz-Michaluk and A. Rokosz-Pelc, 1984, Chronic administration of antidepressant drugs increases the density of cortical [3H]prazosin binding sites in the rat, Brain Res. 310, 360. Waldmeier, P.C., P.A. Baumann, K. Hanser, L. Maire and A. Stomi, 1982, Oxaprotiline, a noradrenaline uptake inhibitor with an active and an inactive enantiomer, Biochem. Pharmacol. 31, 2169.
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Elsevier RIP 50872
Antidepressant drugs potentiate the al-adrenoceptor effect in hippoemnp~ slices M a r i a Bijak * Polish Academy of Sciences, Institute of Pharmacology, 12 Smetna, 31-343 Krakow, Poland
Received 25 August 1988, revised MS received 28 March 1989, accepted 25 April 1989
The effect of prolonged treatment with antidepressant drugs on the phenylephrine- and norepinephrine (NE)-evoked reaction in hippocampal slices was examined by extracellular recording of the spontaneous activity of CA1 layer neurons. The eq-adrenoceptor agonists, phenylephrine and methoxarnine, depressed the neuronal discharges of most of the units tested, while NE evoked both excitatory and inhibitory effects which were blocked by propranolol and phentolamine or prazosin, respectively. Imipramine, mianserin, (+)- and (-)-oxaprotiline administered subchronically (10 mg/kg p.o., twice daily for 14 days, withdrawal 48 h), potentiated the inhibitory reaction to phenylephrine. Mianserin was the only drug tested in the acute dose to effectively augment the reaction to al-adrenoceptor stimulation. Prolonged administration of mianserin and imipramine attenuated the excitatory effect to NE, which probably reflects r-receptor down-regulation; however, only mianserin, but not imipramine, enhanced the NE-induced inhibition. The observed potentiation of the al-adrenoceptor-related inhibitory reaction to phenylephrine produced by antidepressant drugs may reflect the development of the al-adrenergic system supersensitivity in the hippocampus. Antidepressants; al-Adrenoceptors; Phenylephrine; Hippocampal slice
1. Introduction
Experimental evidence suggests that alterations in the central noradrenergic system is one of the most consistent effects of treatment with antidepressant drugs. While a decrease in both the function and number of a 2- and fl-adrenoceptors after repeated administration of antidepressant drugs and electroconvulsive shock has been reported by several laboratories, it is still a matter of controversy as to whether there are changes in central %-adrenoceptors after these treatments (see Sugrue, 1983; McNeal and Cimbolic, 1986; Lipin-
* To whom all correspondence should be addressed at present address (till 31 January 1990): Max-Planck-Inst. f'firPsychiatrie, Department of Neurophysiology, Am Klopferspitz 18A, 8033 Planegg-Martinsried, F.R.G.
ski et al., 1987). Some biochemical, electrophysiological and behavioral studies have demonstrated an increase in the number and function of a 1adrenoceptors after antidepressant therapy (Rehavi et al., 1980; Menkes and Aghajanian, 1981; Menkes et al., 1983; Vetulani et al., 1984; Maj, 1984; Hong et al., 1986; Mogilnicka et al., 1987; Stockmeier et al., 1987), but there is also considerable experimental evidence that does not confirm the antidepressant-evoked alterations in the a 1adrenoceptor system (Heal 1984; Stockmeier et al., 1987). It has been suggested that the hippocampal formation, being a part of the limbic brain, is involved in the regulation of emotional and motivational functions, processes that are affected in depression (Angst and Dobler-Mikola, 1984; Post, 1986). Therefore the hippocampus m a y be an important anatomical substratum for the action of
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
184
antidepressant drugs. This suggestion is supported by reports concerning alterations in the hippocampal fl-adrenergic, dopaminergic, serotonergic, cholinergic and GABA-ergic systems after antidepressant treatment (Lerer et al., 1983; Anwyl and Rowan, 1984; Lloyd et al., 1986; Newman et al., 1987; Smia/owski and Bijak, 1987; Bijak and Smia/owski, 1988). Moreover, there is direct behavioral evidence for al-adrenoceptor supersensitivity in the hippocampus of rats treated with antidepressant drugs (Plaznik et al., 1984; Plaznik and Kostowski, 1985). It is expected that these adaptive changes in the a]-receptor system should have their neurophysiological correlate in the responsiveness of individual neurons to aa-adrenergic stimulation. Therefore the present study was aimed at gaining insight into al-adrenoceptor function in the hippocampus. To this end the effect of the al-selective agonist, phenylephrine, and the putative neurotransmitter, norepinephfine, on spontaneous discharges of the CA1 layer neurons was studied in the brain slices from rats treated with antidepressant drugs. So far, antidepressant drug-induced alterations in the responsiveness of hippocampal neurons to stimulation of /3-adrenoceptors, serotonin and dopamine receptors have been described (Anwyl and Rowan, 1984; Rowan and Anwyl, 1985; Smia/owski and Bijak, 1987; Bijak and Smia/owski, 1988). The present study shows that different antidepressants evoke supersensitivity of the eqadrenoceptor system in the hippocampus. Therefore electrophysiological evaluation of the reactivity of hippocampal neurons to different neurotransmitters in vitro is proposed as a sensitive method for screening the potential antidepressant activity of drugs.
ing antidepressant drug (10 m g / k g p.o.). The animals were killed by decapitation 48 h after the last dose of the drug. The brain was removed and the hippocampus was dissected. Transverse hippocampal slices, approximately 400 /~m thick, were cut and transferred to an incubation chamber. After at least 1 h a single slice was placed in an experimental chamber (0.5 ml in volume) on a nylon mesh and continuously superfused, at a rate of 1 m l / m i n , with an oxygenated solution consisting of (mM) NaC1 124, KC1 5, CaC12 2.4, MgSO 4 1.3, KHEPO 4 1.25, NaHCO 3 26, glucose 10. The temperature was maintained at 34 o C, the pH at 7.4. Extracellular recordings of the spontaneous activity of cells in the CA1 layer were made by using a tungsten microelectrode (Clark Electromedical Instr. 12 MS2). Neurons were selected on the basis of the characteristics of their spontaneous activity. Units displaying a low firing rate (mean = 6.96 + 0.33 Hz, n = 300) and multiple irregular action potential of long duration were chosen. These neurons are related to complex-spike cells, which may represent pyramidal neurons (Berger et al., 1983; Pang and Rose, 1987). An example of the spontaneous activity, photographed directly from the oscilloscope screen, is shown in fig. 1. The frequency of spontaneous discharge was calculated as the number of spikes per minute, and was simultaneously integrated at 10 s intervals and recorded on a chart recorder. The substances used were dissolved in incubation medium and 0.1 ml was added to the perfusion line just before it entered the experimental chamber after at least 10 min of a stable basal firing rate. The final concentration of the substance in the chamber was about 5 times lower
2. Materials and methods
Male Wistar rats (200-250 g), housed under controlled 12 h light-dark conditions (light on between 7-19 h) and with free access to food and water, were used. The animals received water or antidepressant drug (10 m g / k g p.o.) twice daily for 14 consecutive days. In the acute experiment the rats received a single dose of the correspond-
I Fig. 1. An example of the spontaneous activity of CA1 layer neurons photographed directly from the oscilloscope screen. Vertical bar: 20 FV; horizontal bar: 5 ms.
185
than its initial one. In the experiments in which the effect of the antagonistic drug was investigated, the slices were perfused with the drug-containing solution for at least 20 min. The reaction to the given substance is expressed as a percentage of the mean firing rate during the control period, measured at 1 rain intervals after drug administration. The percent depression (effect below 80% of the control firing rate) and excitation (effect above 120% of the control) in one experimental series were pooled to give a mean change in the neuronal firing rate after administration of the substance. The mean percentage changes in neuronal discharge were compared by using a one-way analysis of variance and a two-tailed Student's t-test. A Chi square test was used to compare the number of units reacting with excitation and inhibition in different experimental groups. The drugs used were: 1-phenylephrine (Boehringer Ingelheim), D L - n o r e p i n e p h r i n e HC1 (Sigma), methoxamine (Wellcome), prazosin (Pfizer), propranolol (Polfa), phentolamine HC1 (Ciba-Geigy); imipramine HC1 (Polfa), mianserin HC1 (Organon), ( +)-oxaprotiline, ( - ) - o x a p r o t i line (Ciba-Geigy).
NE
NE Fig. 2. The effect of norepinephrine (NE, 16 # M ) on the spontaneous firing rate of CA1 neurons in hippocampal slices. Vertical bar: 20 i m p u l s e s / 1 0 s; horizontal bar: 5 rain. TABLE 1 The effect of norepinephrine (NE, 16 ttM) on the spontaneous firing rate of hippocampal CA1 neurons and the influence of acute (1 x ) and prolonged treatment with imipramine (IMI) and mianserin (MIA) on the norepinephrine-evoked reaction. n = n u m b e r of units tested, a Statistical significance vs. control, Chi-square test, P < 0.05. Substance tested
Pretreat-
n
Type of response (%n)
ment NE N E + propranolol 1#M
3. R e s u l t s
In hippocampal slices prepared from nontreated rats norepinephrine (NE, 16 #M) elicited an increase in the spontaneous discharge rate in the majority of the CA1 layer neurons studied, inhibitory reactions were observed in only a few units (fig. 2). As shown in table 1, the NE-mediated excitatory response was blocked by propranolol. N E evoked mainly a decrease in the neuronal
Ph
Inhibition Excitation
-
23
26
74
-
14
86
-
-
9
-
13
-
NE NE
IMI 1 x IMI 1 4 x
16 17
44 18
50 82
NE
MIA 1 x MIA 1 4 ×
21 23
48 78
50 17
NE+phentolamine 10 v M N E + prazosin 0.1 # M
NE
100
a
77
a
Ph
Fig. 3. Examples of inhibitory responses of hippocampal neurons to phenylephrine (Ph, 20 #M). Vertical bar: 10 i m p u l s e s / 1 0 s; horizontal bar: 5 min.
186 TABL E 2 The effect of phenylephrine and methoxamine on the spontaneous firing rate of hippocampal CA1 neurons, n = number of units tested; a p < 0.05 t-test vs. phenylephrine 0.2 # M ; mean, the mean percent decrease in the neuronal firing rate following administration of phenylephrine as compared to the pre-drug baseline control, b p < 0.001. Substance tested (dose #M)
n
Type of response (%n) Inhibition
Excitation
No effect
1.37 3.76 a -
14 14 13
27 36 62
2.37 a
10
10 80
--
--
M e a n _ S.E.M. Phenylephrine (0.2) + prazosin (0.01) + prazosin (1.0)
37 14 8
59 50 25
59.09 69.38 -
Phenylephrine (20) + prazosin (1.0)
13 10
92 10
46.89
Methoxamine (100)
6
100
54.41
-
firing rate in the presence of this fl-receptor antagonist. Conversely, application of phentolamine and prazosin abolished the inhibitory reaction to NE. The specific al-receptor agonist, phenylephrine, generally diminished the neuronal firing rate in hippocampal slices (figs. 3,4). The inhibitory effect of phenylephrine was dose-dependent and was significantly attenuated by the specific al-receptor antagonist, prazosin (table 2). Another th-receptor agonist, methoxamine, produced a decrease in the frequency of neuronal discharges in all the units tested. However, methoxamine had a lower potency than phenylephrine in eliciting the inhibi-
-
3.37
b
tory effect. The latency of the inhibitory reaction evoked by selective stimulation of the al-receptor was longer than the latency of the NE-evoked excitation or inhibition (figs. 2,3,4). Unlike the short-lasting effects of NE, the effect of phenylephrine was frequently of long duration, and the firing rate often did not return to the pre-drug level after a 30 rain washout period. The application of phenylephrine induced inhibition followed by excitation in some units or only elevation of the firing rate (table 2). Interestingly, the excitatory effects were usually disproportionately high in relation to the low dose of phenylephrine.
TABLE 3 The influence of acute (1 × ) and prolonged (14 x ) treatment with antidepressant drugs: imipramine (IMI), mianserin (MIA), ( + ) and ( - ) - o x a p r o t i l i n e (OXA) on the responsiveness of hippocampal neurons to phenylephrine (0.2 /~M). N = number of animals; n = number of units tested. Pretreatment
N
n
Type of response (%n) Inhibition
Excitation
N o effect
Control
8
37
59
14
27
IMI 1 x IMI 1 4 x
5 7
27 28
59 61
15 21
26 18
MIA I x MIA 14x
5 10
23 38
57 82
30 5
13 13
( + )-OXA 1 × ( + ) - O X A 14 x
4 5
18 22
39 73
22 9
39 18
( - )-OXA 1 x ( - )-OXA 14 x
4 5
18 21
56 85
11 10
33 5
187 Cont.
]
acute
D chronic O
140
A
iili
Ph
MIA
|OC
1
Ph Fig. 4. The effect of subchronic treatment with mianserin (MIA) on the inhibitory response of hippocampal CA1 neurons to phenylephrine (Ph, 0.2 #M). Vertical bar: 10 i m p u l s e s / 1 0 s; horizontal bar: 5 rain.
60,
Cont.
Prolonged administration of imipramine, mianserin, ( + ) and ( - ) enantiomers of oxaprotiline potentiated the inhibitory reaction evoked by phenylephrine (figs. 4,5). Imipramine, (+)- and (-)-oxaprotiline, given acutely, did not change the effect of phenylephrine on the neuronal firing
O
IMI
MIA
o
Fig. 6. The effect of acute and subchronic treatment with imipramine (IMI) and mianserin (MIA) on the excitatory and inhibitory reactions of hippocampal neurons to norepinephrine (NE, 16 /LM). The results are expressed as the percent change ( m e a n + S . E . M . ) in the baseline firing rate following N E administration. 0 p < 0.05 vs. control, t-test.
100, X
o
I o
~L_
50 o
_-:::::_-
N Cont.
IMI
[ ] prezosin
l
MIA
[ ] acute
(+) OXA
( . ) OXA
[ ] chronic
Fig, 5. The effect of acute and subchronic treatment with antidepressant drugs: imipramine (IMI), mianserin (MIA), ( + ) - and ( - ) - o x a p r o t i l i n e (OXA), on the reaction of hippocampal neurons to phenylephrine (Ph, 0.2 /~M). The results are expressed as the percent change ( m e a n + S.E.M.) in the baseline firing rate following Ph administration. Prazosin (0.01 /xM) applied to the bathing medium. 0 p < 0.05 vs. control, t-test.
188 rate; however, a single dose of mianserin enhanced the reaction to a~-receptor stimulation. Repeated administration of mianserin potentiated the NE-induced inhibitory reaction, both increasing the potency of the effect and the number of units reacting with inhibition (table 1, fig. 6). A similar change in the reaction to NE, i.e. from excitation, which prevailed in control slices, to inhibition, was observed after a single dose of mianserin; however, this effect was not statistically significant. The excitation elicited by NE was significantly diminished in the hippocampal slices prepared from animals treated either acutely or chronically with mianserin. A single dose of imipramine did not change the responsiveness of CA1 neurons to NE, while prolonged treatment with the antidepressant attenuated the NE-evoked excitatory effect (table 1, fig. 6).
4. Discussion
The data reported above on the excitatory and inhibitory action of norepinephrine (NE) on the spontaneous discharge rate of CA1 neurons and the blockade of the latter effects by propranolol and phentolamine or prazosin, respectively, are in line with the electrophysiological action of the monoamine in the hippocampal formation. It has been shown that exogenously applied norepinephrine interacts with both a- and fladrenoceptors in the hippocampus, producing a decrease and increase, respectively, in neuronal excitability in the CA1 layer (Mueller et al., 1981; 1982a,b; Madison and Nicoll, 1986). Several data suggest that, unlike other brain areas where stimulation of al-adrenoceptors brings about an excitatory reaction, application of selective al-adrenoceptor agonist in the hippocampus results in inhibition of neuronal firing and synaptic responses (Mueller et al., 1981; Madison and Nicoll, 1986; Pang and Rose, 1987; Mynlieff and Dunwiddie, 1988). The data presented provide further electrophysiological evidence for a al-adrenoceptor-mediated attenuation of spontaneous discharges of CA1
neurons. The specific al-adrenoceptor agonists, phenylephrine and methoxamine, diminished the firing rate of hippocampal neurons. Moreover, the phenylephrine-induced inhibition was in most cases blocked by the selective, competitive a 1adrenoceptor antagonist, prazosin. The long-lasting effect of phenylephrine observed is in line with the reported prolonged action of this substance in the brain (Rogawski and Aghajanian, 1980). The potent action of phenylephrine on the spontaneous firing rate of hippocampal neurons parallels the high efficacy of this substance in stimulating the breakdown of inositol phospholipids in the hippocampal formation (Johnson and Minneman, 1985; Fowler et al., 1986). Although inhibition was the predominant action of phenylephrine on the CA1 neuronal discharges, in few cases an excitatory reaction was also observed. This effect was not studied further; however, it is suggested that inhibition of inhibitory interneurons in the hippocampus may lead to excitation of pyramidal cells (Jahr and Nicoll, 1982; Collins et al., 1984). Moreover, it has been implied that phenylephrine may evoke an excitatory effect via non-specific activation of fl-adrenoceptors (Mynlieff and Dunwiddie, 1988). The present results show that imipramine, mianserin, ( + ) - and (-)-oxaprotiline, when administered subchronically, enhance the reaction of hippocampal neurons to phenylephrine. The acute application of imipramine, ( + ) - and ( - ) oxaprotiline has no such sensitizing effect. However, even a single dose of mianserin potentiated the phenylephrine-induced inhibition. Similarly, acute administration of mianserin affects the density of serotonin receptors in the brain (Helmeste, 1986). On the basis of the binding data and biochemical studies it is postulated that potentiation of the electrophysiological effect of phenylephrine could be due to the antidepressant-evoked increase in the number a n d / o r affinity of al-adrenoceptors, or result from enhancement of the second messenger system coupled to the receptor (Menkes et al., 1983; Vetulani et al., 1984; Hong et al., 1986; Newman et al., 1987; Stockmeier et al., 1987). Mianserin was effective not only in augmenting the phenylephrine-induced depression in neuronal
189 firing but also in potentiating the NE-evoked inhibitory reaction, whereas imipramine, although the most potent of the drugs tested in enhancing the action of phenylephrine, did not change the inhibitory reaction to NE. Although the depression of neuronal firing evoked by NE in the hippocampus is most probably due to activation of both a a- and a2-adrenoceptors (Mueller et al., 1981; Madison and Nicoll, 1986) and a l t h o uhg it has been demonstrated that antidepressant treatment enhances a a- and attenuates a2-adrenoceptor function (McNeal and Cimbolic; Lipinski et al., 1987), changes in the a-adrenoceptor-related response were not observed when NE was used. However, mianserin, a potent a2-antagonist, probably does not produce a significant down-regulation of a2-receptors, this might permit the manifestation of'an enhanced a-adrenoceptor-related reaction to NE. The attenuation of the excitatory response to NE in hippocampal slices prepared from animals treated with imipramine and mianserin is in line with the decrease in the number and function of fl-adrenoceptors observed after prolonged administration of antidepressants (Maggi et al., 1980; Mishra et al., 1980; Anwyl and Rowan, 1984). The antidepressants tested had different pharmacological profiles in the acute experiment, yet all of them affect the brain noradrenergic system. The tricyclic antidepressant imipramine, is a nonselective inhibitor of the NE and serotonin uptake, the tetracyclic, (+)-oxaprotiline, is more selective as it inhibits the NE uptake only, while the atypical antidepressants, the tetracyclics, mianserin and (-)-oxaprotiline, are not uptake blockers in vivo. All the antidepressant drugs tested have adrenolytic activities, probably a prerequisite for their sensitizing action on the a 1adrenoceptor system in the hippocampus (Waldmeier et al., 1981; see Fuller and Wong, 1985; McNeal and Cimbolic, 1986). While the uptake-blocking effect of imipramine, which leads to an increase in availability of NE at the postsynaptic receptor, is thought to be responsible for the down-regulation of r-receptors, the direct blockade of al-adrenoceptors by the antidepressant could be responsible for the augmented response to eta-stimulation.
Similarly, it may be assumed that mianserin increases the concentration of endogenous NE in the synaptic cleft by blocking a2-presynaptic receptors, leading to r-receptor down-regulation and a reduction in fl-adrenergic excitatory activity in the hippocampus. In contrast, antagonism of a 1adrenoceptors is a possible mechanism for the mianserin-induced supersensitivity to phenylephrine. The same mode of action can be proposed for both enantiomers of oxaprotiline, which also have adrenolytic activity (Waldmeier et al., 1982). (-)-Oxaprotiline, which is ineffective in potentiating some behaviorally assessed functions related to the al-adrenoceptor system (Maj, 1984), evoked sensitization in the present electrophysiological model. However, in other studies the effects of both the ( + ) and ( - ) enantiomers of oxaprotiline were similar, which indicates that the inhibition of uptake is not essential for at least part of the antidepressant evoked actions (Kopanski et al., 1983; Maj and Wedzony, 1987; Mogilnicka et al., 1987). The sensitizing effect of mianserin, which is devoid of uptake-blocking activity in vivo, on the phenylephrine- and NE-induced inhibition demonstrated in the presented study corroborates the latter assumption. Antidepressant-evoked changes in different receptor systems may depend on complex interactions in the brain. Several authors describe the involvement of the serotonergic system in the regulation of adrenergic transmission (Sulser, 1984; Racagni et al., 1986), thus the sensitizing effect of the antidepressants studied on the al-adrenoceptor-related response in the hippocampus may be due partly to antidepressant-induced changes in the serotonergic system (Rowan and Anwyl, 1985). The latter possibility is supported by the established serotonergic involvement in the regulation of aa-adrenoceptors in the hippocampus (Consolo et al., 1985). The present results demonstrate that, irrespective of their different acute effects, the long-term administration of the antidepressants tested leads to supersensitivity of the al-adrenoceptor system in the hippocampus. These data are in line with behavioral evidence of antidepressant-evoked central etl-supersensitivity (Maj, 1984).
19o
Acknowledgement This work was supported by the Polish Academy of Sciences. Research Project No. 06-02.I.
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