Interaction of glutamatergic and adrenergic inputs of cortical neurons during conditioning

Pergamon

PII:

Neuroscience Vol. 76, No. 3, pp. 877–890, 1997 Copyright ? 1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/97 $17.00+0.00 S0306-4522(96)00329-6

INTERACTION OF GLUTAMATERGIC AND ADRENERGIC INPUTS OF CORTICAL NEURONS DURING CONDITIONING V. M. STOROZHUK,* A. V. SANZHAROVSKY and V. V. SACHENKO Department of Physiology of Higher Nervous Activity, Bogomoletz Institute of Physiology, Bogomoletz Street 4, Kiev 252024, Ukraine Abstract––Background and evoked activities of sensorimotor cortex neurons have been examined on learning cats with conditioned placing reaction before, during and after iontophoretic application of synaptically active drugs. It was shown that glutamate exerted not only a direct excitatory effect on the cortical neurons during its application, but also developed modulatory influences on background and evoked impulse activity after cessation of application in the subsequent 10–20 min. Adrenergic influences on the activity of neocortical neurons evoked by application of adrenomimetic drugs were complex and consisted of at least two different types. Noradrenaline depressed background and particularly evoked activity of many neurons through â1-adrenoreceptors. At the same time, activation of â2-adrenoreceptors was accompanied by facilitation of background and evoked activity during application and 10–20 min after its cessation, as was shown in experiments with alupent. Co-application of glutamate and alupent improved facilitation of impulse response evoked by conditioned stimuli. It was concluded that â1- and â2-adrenergic inputs to neocortical neurons are involved in plasticity changes of glutamate inputs of some cortical neurons. Copyright ? 1996 IBRO. Published by Elsevier Science Ltd. Key words: conditioning, learning, plasticity, cortical neurons, interaction of glutamatergic and adrenomimetic transmission in neocortex, iontophoretic application.

Although attention of reserchers for many years have been focused on such models of learning as long-term potentiation (LTP), currently the investigation of cortex neuronal activity during conditioned reflexes seems to be of a more perspective trend in the study of neurophysiological mechanisms of learning. The three following factors favour such investigations: (i) conditioned reflexes are real and natural models of learning; (ii) beginning with the pioneer work of Jasper et al.,15 numerous data have been accumulated about the activity of cortical neurons during conditioning; (iii) microiontophoretic application of synaptically active substances to cortical neurons,18–20,23,25,28,30,39,43,44,47 including experiments on awake animals,16,17,21,35,37,38 gives the possibility of affecting different synaptic inputs to neurons and of determining the interaction of synaptic processes upon learning. It is known from experiments with LTP on hippocampal5,6,9,31–33 and cortical3,4,39,40 slices that the main role in plasticity consists of changes in glutamatergic synapses and their modulation by some synaptically active substances. This was the reason why we decided to investigate the action of glutamate on impulse responses of cortical neurons during conditioning and

their changes under the influence of neuromodulators. The aim of our investigation was to appraise the influences of glutamate itself on glutamatergic inputs during repeated neuronal responses to conditioned stimuli and changes in these reactions during application of adrenomimetics.

*To whom correspondence should be addressed. Abbreviations: LTP, long-term potentiation; NA, noradrenaline; NMDA, N-methyl--aspartate. 877

EXPERIMENTAL PROCEDURES

Experiments were carried out on 14 adult male cats weighing 3–3.5 kg with elaborated instrumental conditioned placing reaction. A sound click (2 ms duration and 60 dB intensity) was used as a conditioned stimulus. Coupling of the click and touch to dorsal surface of the anterior left paw was reinforced by food reward after realization of the conditioned movement. The animal was taught to carry out the placing reaction in response to click without touch. Cessation of the reinforcement led to extinction of the conditioned placing reaction. Trained cats were anaesthetized with nembutal (40 mg/kg, i.p.) and operated on. An opening 10 mm in diameter was made in the skull over the sensorimotor cortex in the right hemisphere. The dura mater was removed and a stainless steel cylinder was placed into the opening and fixed with dental acrylic. Several days later, during experiments, a special directing cannula was inserted into the cylinder. The cannula was linked to a micromanipulator containing a multi-barrelled micropipette. The micropipette was used for extracellular recording of impulse activity and application of synaptically active drugs. One of the barrels was filled with 4.0 M NaCl and had a resistance of 5–10 MÙ. The other barrels were filled with the synaptically active drugs to be tested: glutamate (0.5 M, pH 7.4); 0.2 M ketamine hydrochloride (pH 5.5); noradrenaline hydrotartar (0.5 M); ephedrine hydrochloride

24 6 18 32 9 89

Glutamate Ketamine Ephedrine Propranolol Alupent

14 — 8 13 6

Increase 4 3 7 7 —

Decrease 6 3 3 12 3

No change 24 2 — 6 9

n 12 — — 1 5

Increase

n, number of neurons investigated. Significant data are underlined.

n

Drug

On application

Background activity

7 2 — 2 —

Decrease

After 10 min

5 — — 3 4

No change 21 6 9 19 9 64

n 11 1 1 12 9

Increase

4 2 6 2 —

Decrease

On application

6 3 2 5 —

No change

21 3 — 6 8

n

11 — — 2 7

Increase

Evoked activity

5 2 — — —

Decrease

After 10 min

Table 1. Early and late effects of application of synaptically active substances on the background and evoked impulse activities of sensorimotor cortex neurons

5 1 — 4 1

No change

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Fig. 1. (A)

(1 M); alupent (metaproterenol sulphate; 2 mM); propranolol (obzidan; 3 mM). Ejection currents varied between 20 and 50 nA; retaining currents were 10–20 nA. Background and evoked impulse activities in isolated neurons were recorded in response to unconditioned and conditioned stimuli before, during and after cessation of iontophoretic application of drugs. The iontophoretic application began 2 s prior to each realization and lasted 5 s. The electromyogram response from the biceps muscle was used as an indicator of the beginning of the placing reaction. This muscle is the first to be involved in placing movement.36 The neuronal impulse activity, electromyogram, initiation of movement and time of stimulus presentation were connected on-line to a computer. Analysis of neuronal impulse activity was performed by means of averaged histograms (50-ms bin) of two types: peristimulus histograms and histograms from the same realizations were constructed relative to the onset of the placing movement. Parts of the histograms exceeding the background by the mean & 2S.D. range were evaluated as significantly evoked responses. The paired Wilcoxon test was used to compare reactions before and after iontophoretic application of

synaptically active drugs. Differences at the 0.05 level were considered significant. RESULTS

Sixty-four of 89 recorded cells responded to conditioned sound stimuli with a change in impulse activity. The effects of synaptically active substances were tested during application and 10 min after cessation of application. Twenty minutes after application the impulse activity returned to its initial level. The number of neurons studied in each series of experiments and the character of changes in impulse background and evoked activity during application of corresponding drugs are shown in Table 1. Separate action of glutamate and adrenomimetics The experiments with glutamate application showed that the intensity of impulse reaction of many

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Fig. 1. (B) Fig. 1. Effects of glutamate on background and evoked impulse activities of sensorimotor cortex neurons. Changes of reactions of ‘‘sensory’’ (A) and ‘‘motor’’ (B) neurons are shown. Averaged peristimulus histograms (1, 3, 5) and histograms of the same realization constructed by the onset of the placing movement (2, 4, 6) are displayed. (1, 2) Control. (3, 4) During iontophoretic application. (5, 6) Ten minutes after cessation of application. N, number of trials. The vertical broken line indicates conditioned sound (left) or the onset of placing reaction (right). The horizontal line is the mean of the background activity. The broken horizontal line indicates the mean & 2S.D. level. Iontophoretic application began 2 s before the stimulus and lasted 5 s.

neurons (14 of 20 neurons investigated) increased not only during this procedure but also 5–10 min later. At the same time, the background impulse activity of these neurons increased by 1.5- to 2.0-fold (Fig. 1). The part of the histogram in Fig. 1A1 obtained before presentation of the sound stimulus shows the level of initial background activity. The same part of the histogram in Fig. 1A3 shows an increase of impulse activity during iontophoretic glutamate application, which began 2 s before the sound stimulus. This is a reaction of the neuron to iontophoretic application and we cannot estimate the natural background activity. The initial part of the histogram in

Fig. 1A5 shows the background activity 10 min after cessation of glutamate application. It should be noted that the initial level of background activity was also increased two-fold from this initial level. The moderate impulse response evoked by sound increased during glutamate aplication (Fig. 1A3), but a more intensive reaction to sound was preserved even 10 min after cessation of application. Fig. 1A2, A4 and A6 show changes in impulse responses of the same neuron with respect to initiation of conditioned movement. It is known from experiments performed on the somatic cortex of monkey8 that pyramidal tract neurons develop impulse responses to a stimulus

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Fig. 2. Effects of ketamine on evoked and background activities of cortical neurons. (1, 2) Control. (3, 4) During iontophoretic application. (5, 6) Decrease in background and evoked impulse activities 10 min after application. Designations as in Fig. 1.

which precede conditioned movement by 100 ms or more. The cortical neurons are sensory if their impulse reaction begins simultaneously or no longer than 60 ms before the initiation of movement. This was also found in experiments performed on cats.35 The initial impulse reaction started simultaneously with the initiation of movement, indicating that these neurons are sensory (Fig. 1A2). In control experiments after glutamate application neurons show an impulse response 150 ms before the initiation of conditioned movement, which can be estimated as a motor response. In Fig. 1B impulse neuronal reactions originally outstripped the initiation of movement. Such an outstripping increased during application of glutamate and after its abolition. These neurons are ‘‘motor’’ neurons. In controls, the background activity increased more than two-fold after cessation of application. Application of ketamine, a non-selective antagonist of glutamate receptors, did not change background or evoked activity during 10 successive

applications. However, 3–5 min after cessation of application the background and evoked activities decreased significantly (Fig. 2). These delayed changes are probably linked with the slow development of the blocking action of ketamine on glutamate receptors. It is obvious that the depressing action of ketamine has some latent period. We concluded that glutamate, apart from its direct excitatory effect on glutamate receptors of cortical nerve cells, can modulate glutamate inputs of neurons. This effect persisted 5–20 min after cessation of application. Application of noradrenaline (NA) or the adrenomimetic ephedrine, which is more convenient to use in experiments, depressed the background and evoked activities of many cortical neurons that were observed earlier.37 Examples of the depressing effect can be seen in Fig. 3. The responses to sound stimulus are not significant (Fig. 3A3, B3). On the contrary, use of propranolol, a non-selective antagonist of â-adrenoreceptors, facilitated impulse

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Fig. 3. Depressing effects of ephedrine on neuronal impulse activity during conditioning. (1, 2) Two ‘‘motor’’ neurons with early (A) and late (B) control responses to conditioned sound. (3, 4) During iontophoretic application of ephedrine. Designations as in Fig. 1.

activity; background and especially evoked activity increased significantly (Fig. 4). There are reasons to believe that under natural conditions in awake animals a persisting tonic depressing effect of noradrenergic inputs on the background and evoked impulse activities of the somatosensory cortex neurons takes place. This depression must increase with stressor factors. The noradrenergic depression of impulse activity is realized through â1-adrenoreceptors. However, adrenergic effects on cortical neurons are more complex, since alupent (metaproterenol), an agonist of adrenaline selectively activating â2-adrenoreceptors,2 evoked facilitation of impulse responses of cortical neurons to conditioned stimuli. The background impulse activity of some neurons also increased.

These changes can persist for 10–20 min after cessation of iontophoretic application of the drug (Fig. 5A, B). Interaction between glutamate and adrenomimetics We investigated the separate effects of glutamate and NA on the impulse activity of cortical neurons which are supposed to be effected through glutamate inputs. The aim was to ascertain the complex effects of glutamate and NA on impulse neuronal activity in awake animals during conditioned reflex. Co-application of glutamate and propranolol, a nonselective â-adrenoblocker, two substances which separately facilitate the impulse activity of cortical neurons, prevented an increase of background

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Fig. 4. (A) and (B)

activity and led to an improvement of the ratio between evoked and background impulse activities. Impulse responses became more pronounced as compared to control (Fig. 6). Co-application of glutamate and alupent led to some increase of neuronal impulse activity. However, the effect manifested itself 10 min after application (3 in Fig. 7). These late

changes in neuronal impulse activity were more prominent as compared with control after iontophoretic application of alupent or glutamate alone (5 and 7 in Fig. 7). The selective adrenaline agonist alupent induced more intensive changes in impulse activity of neurons than the non-selective âadrenoblocker propranolol. Co-application of the

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Fig. 4. (C) Fig. 4. Facilitating effect of propranolol on background and evoked activities of different types of cortical neurons: ‘‘sensory’’ (A), ‘‘motor’’ (B) and a neuron activated during reinforcement (C). Designations as in Fig. 1.

two synaptically active substances was accompanied by minimal changes in evoked activity (Fig. 8). Comparison of the changes in the background activity between the two groups of cortical neurons demonstrated that glutamate or alupent when applied alone increased background impulse activity. Their co-application leads initially to a decrease of the background activity, which increased by more than 200% 10 min later. Co-application of alupent and propranolol did not evoke any significant changes in background activity (Fig. 9).

DISCUSSION

We failed to find any direct evidence in the neurohistological literature about the nature of neurotransmitters in the endings of thalamocortical fibres. There are some indirect data which indicate that glutamate and aspartate may be transmitters in thalamocortical fibres in visual, somatosensory and motor cortices.1,23,41,45 It was demonstrated, for example, that excitatory postsynaptic potentials of cortical neurons evoked by stimulation of the ventrolateral thalamic nucleus were insensitive to N-methyl--aspartate (NMDA) receptor antagonists, although they were susceptible to a blockade by a non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. The authors supposed that the thalamic input may differ from that of the local intracortical glutamatergic pathways, but these inputs still have glutamatergic nature.27 It is necessary to note that the latency of excitatory postsynaptic potentials, blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, was 4–10 ms

and they could belong not to thalamic glutamatergic fibres, as suggested by the authors, but to other intracortical neurons, because a significant part of cortical neurons responds to stimulation of the ventrolateral thalamic nucleus with a latency of 2 ms. On the other hand, intracortical connections of layer II and III cortex neurons in the rat were found to be sensitive to NMDA receptor antagonists and/or nonNMDA receptor antagonists.39,40,42 These and many other facts support the idea that the transmitter in the thalamocortical fibres remains unknown. This allows us to assert that the changes observed in our experiments which were evoked by iontophoretic application of glutamate take place in intracortical or corticocortical glutamatergic connections of cortical neurons. The ways by which glutamate, through its action on postsynaptic receptors, may affect signal transduction processes involved in LTP have recently been summarized by Bliss and Collingridge.5 These data can be useful for understaning the plasticity changes in neocortical neurons during conditioning. However, one should bear in mind that during conditioning combination of excitation of two or more neuronal inputs with different synaptic transmitters takes place. It is an interesting fact that the increase of glutamate levels under natural conditions after excitation of intracortical connections may support a new increased level of impulse activity 10–20 min after cessation of glutamatergic excitation. The functional role of adrenomimetics in the neocortex still remains poorly understood. NA has been described as an inhibitory11,12 and an excitatory tansmitter.24,26,29 Some investigators considered NA as a modulator of glutamatergic and GABAergic

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Fig. 5. Modulating effects of the selective â2-adrenoreceptor agonist, alupent, on cortical neurons. (A) Depression of background and facilitation of evoked activity. (B) Facilitation of background and decrease of evoked activity. Designations as in Fig. 1.

transmission,30,46 or as an important factor in the development of LTP in hippocampal neurons.13,14,31,33,34 The phenomenon of â-adrenergic potentiation10 has been shown and attempts to investigate its mechanisms have been undertaken.7 The long-term excitability changes induced by the nonselective â-adrenergic agonist isopreterenol did not depend upon the calcium concentration of the medium. The authors hypothesized that activation of â-adrenoreceptors may induce an alteration of the hippocampal ‘‘state’’ that can persist for several hours, during which time the induction of other forms of plasticity may be enhanced. Our results allow the possibility of ascertaining the complex effect of NA and adrenaline on the impulse activity of cortical neurons. NA depresses the background

and evoked activities of neurons through â1adrenoreceptors, while adrenaline facilitates this activity through â2-adrenoreceptors. The results depend on the ratio of â1- and â2-receptors on cortical neurons and the ratio of NA and adrenaline concentrations. It is known from neuropharmacology that NA activates á-, â1- and â3-adrenoreceptors. Under natural conditions, â2-adrenoreceptors are activated by adrenaline. In our experiments, these receptors could be activated by ephedrine or the selective â2-agonists metaproterenol and terbutaline.22 Unfortunately, we failed to find any morphological evidence of adrenergic projections to neocortical neurons. We can only suppose that there are adrenergic projections to the cortex which activate

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Fig. 6. Interaction of glutamate and propranolol. (A1, A2) Control. (A3, A4) Increased background and decreased evoked activity during glutamate application. (B1, B2) Decreased and delayed response to sound. (B3, B4) Increase of background activity 10 min after cessation of the co-application of the drugs. Designations as in Fig. 1.

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Fig. 7. Effect of co-application of glutamate and alupent. (1) Control. (2) Co-application. (3) Development of facilitation after co-application. (4) Alupent application. (5) Control after alupent. (6) Glutamate application. (7) Control after glutamate. The vertical broken line indicates the onset of placing reaction in all histograms. Control histograms were obtained 10–20 min after cessation of applications. Other designations as in Fig. 1.

Fig. 8. Effect of joint application of alupent and propranolol. (1) Control. (2) Facilitation evoked by alupent. (3) Control after application of alupent. (4) Action of propranolol. (5) Control after application. (6) Depression of the facilitating action of alupent evoked by propranolol. (7) Control after application. Designations as in Fig. 7.

â2-adrenoreceptors. A general ratio of â1- and â2-adrenoreceptors in rat cortex is, for example, 65:35.28 Our experiments with alupent showed that NA, through â2-adrenoreceptors, can facilitate

impulse responses and background activity in some cortical neurons. These changes are realized through the adenylate cyclase system, cyclic AMP and protein kinase C, and do not depend on the increase of

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interaction of glutamate and alupent changes neuronal responses to conditioned stimuli in a different way to glutamate alone: the responses became more prominent. Hence, this result suggests that glutamate and alupent evoked different processes that interacted during plasticity changes of cortical neurons. It is necessary to note that NA inputs are not able to evoke impulse activity of cortical neurons by themselves: they only modulate the activity of other modality inputs. At the same time, the conditioned stimulus starting the process in the neocortex through thalamocortical inputs is not linked directly with glutamatergic transmission. The increased responses of cortical neurons are a result of facilitation of intracortical interneuronal connections, which are often glutamatergic. Thus, preliminary glutamate modulation of cortical neurons and their modulation by â2-adrenoreceptors manifested themselves by increasing the interneuronal intracortical activity linked with the improvement of glutamatergic connections. CONCLUSIONS

Fig. 9. Changes of background activity of cortical neurons evoked by application of some synaptically active drugs. Every second column shows the level of background impulse activity 10 min after cessation of application.

calcium influx into the cell.7 However, this does not mean that alupent acts in the same way as glutamate, through metabotropic glutamate receptors. The

This study shows that glutamate, apart from its direct excitatory action on cortical neurons, modulates background and evoked impulse activities for at least 10–20 min. NA and adrenaline have different effects on cortical neuronal activity: NA depresses background and, in particular, evoked activity of many neurons through â1adrenoreceptors, while adrenaline facilitates them through â2-adrenoreceptors in the subsequent 10– 20 min after cessation of application. The latter is the reason why co-application of glutamate and alupent improves the impulse response evoked by conditioned stimuli. It is suggested that â1- and â2adrenergic inputs to neocortical neurons participate in plasticity changes of glutamate inputs to some cortical neurons. Acknowledgement—This work was partly supported by PECO Grant No. ERBCIPDCT 940236.

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