Noradrenergic modulation of neuronal responses to n-methyl-d -aspartate in the vestibular nuclei: An electrophysiological and immunohistochemical study

Neuroscience 265 (2014) 172–183

NORADRENERGIC MODULATION OF NEURONAL RESPONSES TO N-METHYL-D-ASPARTATE IN THE VESTIBULAR NUCLEI: AN ELECTROPHYSIOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY Key words: NMDA receptors, noradrenaline, complex, firing rate, microiontophoresis.

M. BARRESI, * C. GRASSO, F. LICATA AND G. LI VOLSI Department of Biomedical Sciences, Section of Physiology, University of Catania, Italy

Abstract—Excitatory responses evoked by N-methyl-Daspartate (NMDA) in the vestibular nuclei (VN) of the rat were studied in vivo during microiontophoretic application of noradrenaline (NA) and/or its agonists and antagonists. Ejection of NA-modified excitatory responses mediated by NMDA receptors (NMDAR) in all neurons tested; the effect was enhancement in 59% of cases and depression in the remaining 41%. Enhancements prevailed in all VN with the exception of the lateral vestibular nucleus, where both effects were recorded in an equal number of cases. The enhancing action of NA on NMDAR-mediated responses was mimicked by the noradrenergic beta-receptor agonist isoproterenol, the beta1 specific agonist denopamine and the alpha2 agonist clonidine. These effects were blocked respectively by the generic beta-receptor antagonist timolol, the beta1 antagonist atenolol and the alpha2 antagonist yohimbine. In contrast, application of the alpha1 receptor agonist cirazoline and the specific alpha1 antagonist prazosin respectively mimicked and partially antagonized the depression of NMDAR-mediated excitations induced by NA. Double-labeling immunohistochemical techniques demonstrated broad colocalization of NMDAR (specifically NR1 and NR2 subunits) with noradrenergic receptors (alpha1, alpha2 and beta1) in many VN neurons; only minor differences were found between nuclei. These results indicate that NA can produce generalized modulation of NMDAR-mediated excitatory neurotransmission in VN, which may in turn modify synaptic plasticity within the nuclei. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

vestibular

INTRODUCTION The vestibular nuclei (VN) constitute a sensorimotor complex involved in the control of posture (Pompeiano, 1972; Sarkisian, 2000), eye movements (Pompeiano, 1972; Ito, 1991; Sarkisian, 2000) and motor learning (Broussard and Kassardjian, 2004). Primary vestibular afferents to the VN are mostly glutamatergic (Zhang et al., 2011). The important role played by glutamate in these nuclei is confirmed by the high concentration of the neurotransmitter (Li et al., 1996) and its receptors (De Waele et al., 1994; Vidal et al., 1996), found throughout the whole complex. A significant and selective noradrenergic projection from the locus coeruleus is also delivered to VN (Schuerger and Balaban, 1993, 1999). Modulation of glutamatergic neurotransmission by noradrenaline (NA) has been described in various central structures such as the hippocampus (Stanton et al., 1989; Segal et al., 1991), prefrontal cortex (Ji et al., 2008a,b), amygdala (Ferry et al., 1997) and cerebellar cortex (Pompeiano, 2006). We found in a previous study of VN that GLU-evoked excitatory effects on secondary vestibular neurons are modulated by NA (Barresi et al., 2009). Ionotropic AMPA and N-methyl-D-aspartate receptors (NMDAR) are widely colocalized in VN (Chen et al., 2000). The NMDAR participate in various learning mechanisms throughout the CNS (Malenka and Nicoll, 1993; Dineley et al., 2001; Antic et al., 2010). Specifically, in the VN, a structure characterized by intrinsic plasticity (Gittis and du Lac, 2006), NMDAR are related to long-term potentiation and depression (Scarduzio et al., 2012). We set out to determine whether NA application could modulate NMDAR-mediated responses in VN, and which types of noradrenergic receptor may be involved. We also explored the extent to which NMDAR are co-localized with noradrenergic receptors in single neurons of the VN. Noradrenergic effects, mediated by various receptors, and implicated in learning have been described (Cahill and McGaugh, 1996; Gibbs et al., 2010; McIntyre et al., 2012). A possible involvement of NA in mechanisms of synaptic plasticity in VN by a modulation of NMDARmediated responses is discussed.

*Corresponding author. Address: Department of Biomedical Sciences, Section of Physiology, University of Catania, Viale Andrea Doria 6, I-95125 Catania, Italy. Tel: +39-95-7384221; fax: +39-957384217. E-mail addresses: [email protected] (M. Barresi), [email protected] (C. Grasso), fl[email protected] (F. Licata), [email protected] (G. Li Volsi). Abbreviations: ANOVA, analysis of variance; ATE, atenolol; CIRA, cirazoline hydrochloride; CLO, clonidine hydrochloride; DENO, denopamine; ISO, L-isoproterenol hydrochloride; LVN, lateral vestibular nucleus; MBA, mean background activity; MVN, medial vestibular nucleus; NA, noradrenaline; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptors; PBS, phosphate-buffered saline; PRA, prazosin hydrochloride; SD, standard deviation; SIOC, semiquantitative indicator of co-localization; SpVN, spinal vestibular nucleus; SVN, superior vestibular nucleus; VN, vestibular nuclei; YO, yohimbine hydrochloride. http://dx.doi.org/10.1016/j.neuroscience.2014.01.054 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 172

M. Barresi et al. / Neuroscience 265 (2014) 172–183

EXPERIMENTAL PROCEDURES Electrophysiological and double-labeling tochemical techniques were employed.

immunohis

Electrophysiology and microiontophoresis Experiments were performed on 18 Wistar rats deeply anesthetized with urethane (1.5 g/kg i.p.). Acquisition and care of laboratory animals conformed to the European Communities Council Directive (86/609/EEC), guidelines in the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) and with Italian law. The experimental protocol was approved by the IACUC (International Animal Care and Use Committee) of the University of Catania. Toe pinch reflex, heart rate and respiratory rate were used to monitor anesthetic levels. Supplementary doses of anesthetic were administered when necessary. Body temperature was maintained between 37 and 38 °C with a heating pad, and a gel of agar-agar (2%) covered the exposed tissue to prevent desiccation. Small holes were drilled in the skull and a five-barrel glass microelectrode was introduced at coordinates corresponding to the VN (Paxinos & Watson, 1997). The final point of each penetration was marked by ejecting Pontamine Sky Blue (Sigma, Milano, Italy) from the recording electrode (negative current pulse of 20 lA for 20 min). At the end of the experiment, the brain was removed and fixed in 10% formalin. Electrode tracks and recording sites were identified in 60 lm coronal sections through the VN that were stained with Neutral Red. Five-barrel glass microelectrodes were used to record single unitary activity of secondary vestibular neurons and to apply drugs with microiontophoresis. The recording barrel (resistance 6–10 MX) was filled with a 4% solution of Pontamine Sky Blue in 3 M NaCl. Action potentials acquired, analyzed and stored using a personal computer (interface: Cambridge Electronic Design 1401, software: Spike2) were checked for their unitary nature and processed if they remained unmodified in amplitude during the test and had a signal to noise ratio of at least 3:1. Three barrels of the micropipette were used for microiontophoresis and contained N-methyl-D-aspartate (NMDA, Sigma, Milano, Italy, 100 mM, pH 8.0) and two of the following: norepinephrine hydrogen tartrate (NA, Sigma, Milano, Italy, 100 mM, pH 4.0–5.0), cirazoline hydrochloride (CIRA, Tocris Bioscience, Milano, Italy, 50 mM, pH 4.5–5.0), prazosin hydrochloride (PRA, Tocris Bioscience, Milano, Italy, 5 mM, pH 4.5–5.0), clonidine hydrochloride (CLO, Tocris Bioscience, Milano, Italy, 50 mM, pH 4.5–5.0), yohimbine hydrochloride (YO, Sigma, Milano, Italy, 20 mM, pH 4.5–5.0), L-isoproterenol hydrochloride (ISO, Sigma, Milano, Italy, 20 mM, pH 4.5–5.0), timolol maleate (TIM, Tocris Bioscience, Milano, Italy, 20 mM, pH 4.5–5.0) denopamine (DENO, Sigma, Milano, Italy, 50 mM, pH 4.5–5.0) and atenolol (ATE, Sigma, Milano, Italy, 10 mM, pH 4.5–5.0). All drugs were dissolved in water with the exception of DENO, which was dissolved in dimethyl sulfoxide. The

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microiontophoretic system (Neurophore BH-2, Harvard Apparatus, Holliston, MA, USA) balanced currents automatically through a barrel filled with 3 M NaCl to neutralize any voltage shift due to the applied currents. Retaining currents (2–10 nA) of appropriate polarity were constantly applied to prevent drug leakage. NMDA was applied with brief (30 s) negative current pulses (intensity up to 80 nA), while NA agonists and antagonists were ejected with longer-lasting currents (up to 20 min, 1– 20 nA). Whenever a single unit was isolated, applications of NMDA (30 s pulses) were routinely followed by three (or more) applications performed during continuous application of NA or one of its agonists at low doses that did not by themselves modify the firing rate of the tested neuron. NMDA was then pulsed for at least 5 min after cessation of NA ejection to ascertain recovery. In some cases the sequence of applications was repeated during simultaneous application of NA and an antagonist specific for a specific type of noradrenergic receptor. The retention– ejection cycle of NMDA applications was determined in each neuron by the duration of the response and the recovery after the NMDA response. A short-lasting, highdose application of NA was finally tested to ascertain the effect of the amine on the background firing rate. The firing rate of each recorded unit was calculated and integrated over 1 s bins for analysis and 5 s bins for display. The mean firing rate, calculated over a sequence of 180 values (3 min) before any drug application, was defined as the mean background activity (MBA). If the standard deviation (SD) of this parameter exceeded 50% of the MBA, the unit was excluded from analyses. Following a drug ejection, a neuron was defined as responsive if its MBA was differed by at least 2 SD from the MBA for at least 20 s. The parameters used to quantify the response were the magnitude (M) and the contrast (C). M indicated the absolute intensity of the effect, and was defined as the difference between the number of spikes recorded during the response and the number recorded during a period with the same duration immediately preceding drug ejection. C (in %) was the ratio between these two values and indicated the signalto-noise value. The effects of NA agonists on the responses to NMDA application were expressed as modifications of the M and C values induced by NMDA applied to the same neuron. As a rule at least three single responses to microiontophoretic applications of NMDA alone (control) were compared with at least three responses to NMDA obtained during continuous application of an NA agonist. Modification of an NMDA response was regarded as significant if at least one of the mean values of the parameters used (M, C) differed significantly from the mean values recorded during the preliminary control (two-tailed Student’s t-test and Mann–Whitney U test for non-parametric data). The trend of the two parameters M and C could only differ significantly if the NA agonist influenced the background firing rate. In fact, in a few cases, NA application modified the background activity even at very low doses of 1–2 nA or 0 (no retention current).

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‘Recovery’ was defined as the interval between the end of NA application and the earliest NMDA response with parameters equaling the mean values (±SD) recorded during the preliminary control. To normalize the effects, the action of a drug on the NMDA responses of a neuron was expressed as a percentage (M%) of the magnitude M and the fraction (Cfrac) of the contrast C, evoked by NMDA applied alone to the same cell. An analysis of variance (ANOVA) test for non-parametric data was applied to compare sets of neurons (M% and Cfrac) recorded in the various nuclei. A paired test (Wilcoxon rank test) was used to analyze the effects of the antagonists in neuronal populations. The effect of an NA agonist on NMDA responses was considered to be antagonized if the parameters were reduced by 50–70% (partial antagonism) or more (complete antagonism). Immunohistochemistry NMDA receptors consist of various subunits (Kutsuwada et al., 1992; Monyer et al., 1992; Yashiro and Philpot, 2008) as are noradrenergic receptors (Hieble, 2007). The latter are differentially distributed throughout the brain (Nicholas et al., 1993a,b; Domyancic and Morilak, 1997). This study focused on NR1 and NR2A subunits. Fourteen adult male Wistar rats weighing 200–240 g were used. The principles followed for the care and use of laboratory animals have been cited above. Rats were deeply anesthetized with chloral hydrate (60–80 mg/kg, i.p.) and perfused intracardially with 200 ml of saline solution (0.1 M, pH 7.4), followed by 300 ml of 4% paraformaldehyde. The brain was removed, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight and cryoprotected by immersion in 30% sucrose in PBS. Frontal sections of the brain (30 lm thick) were cut using a freezing microtome and mounted on gelatin-coated slides. Brain sections were then processed for immunohistochemical runs. Antibodies were used against: NMDA receptor subunits NR1 and NR2A (NR1, 1:50 and NR2A, 1:100; mouse monoclonal antibodies – Invitrogen, Milano, Italy), noradrenergic receptor subunits alpha and beta (alpha1, 1:100, rabbit polyclonal antibody – AB CAM, Milano, Italy; alpha2, 1:50 and beta1, 1:100; rabbit polyclonal antibodies – Santa Cruz Biotechnology, ‘Chemicon’, ‘Jackson Immuno Research’ Heidelberg, Germany). Slices were washed in PBS, permeabilized and preincubated with blocking solution (0.2% Triton X-100, 5% normal goat serum) in PBS for 1 h. The sections were then incubated in mixtures of the primary antibodies (NR1/alpha1, NR1/alpha2, NR1/beta1, NR2A/ alpha1, NR2A/alpha2 and NR2A/beta1) diluted at appropriate concentrations in the same blocking solution overnight at 4 °C. Tissues were washed three times with PBS and incubated for 1 h at room temperature in a mixture of secondary antibodies, goat anti-rabbit IgG conjugated with fluorescein (FITC, 1:100 – Chemicon) and goat anti-mouse IgG conjugated with indocarbocyanine (CY3, 1:100 – Jackson Immuno Research) diluted in a blocking solution (0.2% Triton

X-100, 1% normal goat serum, NGS in PBS) for 1 h at room temperature. After two washes in PBS, the sections were mounted in PBS/glycerol (3:1) and coverslips were applied. In control experiments, one or both primary antibodies were omitted, substituted by an equal volume of NGS. No immunoreactivity was found for the antibodies that were omitted (data not shown). Immunolabeled tissues were examined under a Leica DMRB fluorescence microscope (20 magnification) equipped with a computer-assisted Nikon digital camera. For semi-quantification of immunopositive, double-labeled neurons, they were counted in selected fields (20 objective) of random sections from each nucleus. Randomly selected sections (20 lm thick) of VN were used to evaluate the extent of colocalization of NMDA receptor subunits (NR1 and NR2A) with noradrenergic receptor subunits (alpha1, alpha2 and beta1) in single neurons within a rectangular area of 500  350 lm. In each of the VN, the semi-quantitative indicator of co-localization (SIOC) was the ratio a/b, a being the number of cells immunoreactive for both a NMDA receptor sub-unit (either NR1 or NR2) and a NA receptor (alpha1, alpha2 or beta) and b being the number of cells immunoreactive for a NMDA receptor sub-unit (NR1 or NR2). A univariate test was applied to compare sets of colocalized neurons (NR1/alpha1, NR2/alpha1, NR1/ alpha2, NR2/alpha2, NR1/beta1 and NR2/beta1,) labeled in the various nuclei.

RESULTS Effects of NA on NMDA-evoked responses The activities of 81 VN neurons were studied, 33 of which were located in the lateral vestibular nucleus (LVN), 23 in the medial vestibular nucleus (MVN), 15 in the superior vestibular nucleus (SVN), and 10 in the spinal vestibular nucleus (SpVN). The background firing rate ranged from 1 to 66 spikes/s (mean ± standard error = 18 ± 1 spikes/s; mode = 5–10 spikes/s; median = 14 spikes/ s); no significant difference was observed between nuclei. All cells responded to short-lasting (30 s) ejections of NMDA (current intensities: 2–60 nA) with a significant increase in firing rate that appeared 3–17 s from the beginning of the ejection and lasted 30–115 s after the end of the NMDA application. In 39 VN neurons (16 in LVN, 9 in MVN, 6 in SVN and 8 in SpVN) responses to NMDA application were studied during long-lasting (20–60 min) ejections of NA at low doses (0–20 nA), which generally had little effect on background firing. A complete map of these units is shown in Fig. 1A. In the presence of NA, NMDA-induced excitations were significantly enhanced (p < 0.05) in 59% of the 39 units (8 in LVN, 6 in MVN, 4 in SVN and 5 in SpVN) and depressed (p < 0.05) in 41% (8 in LVN, 3 in MVN, 2 in SVN and 3 in SpVN). M was significantly modified in all units and C was significantly modified in a great majority of cases (85%). The mean values of M% and Cfrac induced by NA on NMDA responses in the neurons are reported for each vestibular nucleus in

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(A)

(B)

200 Decrease

Increase

M%

SVN

LVN

SpVN (5)

SVN (4)

MVN (6)

LVN (8)

SpVN (3)

SVN (2)

LVN (8)

MVN (3)

0

SpVN

MVN

100

P 2.0

(C) 2.0

Decrease

200

Increase

Cfrac

M%

150

100

1.0

50

0

SpVN (5)

SVN (4)

MVN (6)

Mean firing rate (spikes/s)

LVN (8)

40

SpVN (3)

30

SVN (2)

20

MVN (3)

10

LVN (8)

0.0 0

Fig. 1. Effects of noradrenaline (NA) application on the excitatory responses evoked by N-methyl-D-aspartate (NMDA) in the vestibular nuclei (VN). (A) Map showing the sites in vestibular nuclei (VN) where noradrenaline (NA) ejection enhanced (open circles) or depressed (filled circles) the excitatory effects induced by NMDA ejection. The locations of recorded neurons, reconstructed by histological examination of brain slices after each experiment, are projected onto a schematic drawing of a frontal section of the rat brain based on the Paxinos and Watson atlas (1997) for stereotaxic plane P 2.0. LVN, lateral vestibular nucleus; MVN, medial vestibular nucleus; SVN, superior vestibular nucleus; SpVN, spinal vestibular nucleus. (B) Mean magnitude (M%) and contrast (Cfrac) of both enhancements and depressions induced by NA in NMDA responses for each of the vestibular nuclei. The number of units is indicated in parentheses. In both graphs, horizontal lines indicate the reference values (no effect). Mean values lower (filled columns) or higher (open columns) than the reference values indicate decreases or increases of NMDA-evoked excitations, respectively. NA evoked both effects in each VN nucleus, but not in the same percentages. (C) Magnitude of NA effects on the response to NMDA (M%) in each vestibular unit versus the mean background firing rate of the same unit. In the graph the dotted horizontal line indicates the reference values (no effect). Neither the type nor the intensity of NA-induced effects was related to the background firing rate (r2 < 0.1).

Table 1. Enhancing and depressive effects of NA application on NMDA-evoked excitatory responses Nucleus

VC (39) LVN (16) MVN (9) SVN (6) SpVN (8)

Increases

Decreases

M%

Cfrac

n

M%

Cfrac

n

143.18 ± 6.05 152.67 ± 9.76 146.62 ± 13.08 123.83 ± 9.15 139.37 ± 15.25

1.36 ± 0.08 1.52 ± 0.14 1.19 ± 0.06 1.35 ± 0.22 1.30 ± 0.19

23 8 6 4 5

61.47 ± 5.52 65.62 ± 9.70 61.96 ± 8.35 71.19 43.45 ± 4.25

0.78 ± 0.007 0.84 ± 0.14 0.74 ± 0.08 0.71 0.72 ± 0.08

16 8 3 2 3

The table shows the mean values (±standard error) of either enhancements (increases) or depressions (decreases) induced by noradrenaline (NA) application on NMDAinduced responses. The number of units tested in each nucleus is indicated in parentheses. M%, percentage of magnitude; Cfrac, fraction of the contrast; n, number of units; VC, vestibular complex; LVN, lateral vestibular nucleus; MVN, medial vestibular nucleus; SVN, superior vestibular nucleus; SpVN, spinal vestibular nucleus.

Fig. 1B and in Table 1. The NA-induced enhancements of NMDA responses appeared more intense in LVN than in SVN; on the whole no significant difference was found between VN nuclei with regard to the noradrenergic modulation of NMDA responses. The magnitudes (M%) of NA-induced effects on NMDA responses in each neuron (Fig. 1C) were not correlated with the

background firing rate of the same neuron (r2 < 0.1 for both enhancements and depressions). NA application had significant effects on the magnitudes, but not on the durations of NMDA responses. In four units, the long lasting ejection of NA induced an initial depression of NMDA responses, that reverted to an enhancing effect in 15–20 min.

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After the end of NA application, the recovery time varied over an extended period (5–40 min). The rate meter records in Fig. 2 illustrate examples of opposite effects induced by NA application on the NMDA responses of two VN neurons, both located in LVN. During application of NA, the NMDA response of the LVN neuron was reversibly enhanced, with recovery completed 60 min after the end of NA application. In contrast, responsiveness of the SVN neuron to NMDA was depressed during NA ejection (M%: 37%) and the effect persisted for no more than 2 min after the end of NA application. The effect of NA on the background firing rate was analyzed in a previous work (Licata et al., 1993), and was systematically tested in the current study with injections of short duration (30 s) and high current intensity (80 nA). Consistent with our previous results (Licata et al., 1993), the action of NA on the background firing was inhibitory in most cases and unrelated to the type of influence exerted by NA on NMDA responses.

modulation of NMDA responses, agonists and antagonists of alpha and beta adrenoceptors were tested. PRA, a selective antagonist of alpha1 noradrenergic receptors, was tested in six VN neurons during longlasting NA applications that induced depression of NMDA responses in three neurons and enhancements in the remainder. PRA partially (50–70%) but significantly (t-test, p < 0.05) antagonized the depressive action of NA in two out of three units, whereas it significantly increased the enhancing action of NA in one out of three neurons and was ineffective in the remaining two. In contrast, YO, a selective antagonist of alpha2 noradrenergic receptors, was tested in 14 VN neurons during long-lasting NA application and partially (50–70%) antagonized (paired test, p < 0.05) all NA-induced enhancements of NMDA responses (eight units) and had no effect on the depressive effects of NA (six units). TIM, a non-selective antagonist of beta noradrenergic receptors, was tested in eight VN neurons during longlasting NA applications, and partially (50–70%) antagonized (paired test p < 0.05) enhancements of NMDA responses induced by NA (six units) and was ineffective on the depressive effects induced by NA (two units). Similarly, ATE, a selective antagonist of beta1 noradrenergic receptors, was tested in nine VN neurons during long-lasting NA applications and significantly

Effects of noradrenergic agents on NA-modulated NMDA responses To check the specificity of the recorded effects and identify the noradrenergic receptors responsible for 5 nA

10 nA

15 nA NA NMDA 60nA

A

400

# of spikes

300

200

100 bin 5s 0 1000 s/div

4nA

B

8nA

12nA

3200 s

20nA NA NMDA 5nA

120

# of spikes

80

40

bin 5s 0 400 s/div

800 s

Fig. 2. Rate meter records (5 s bins) illustrating the effects induced by NA application on the excitatory responses evoked by NMDA in two LVN neurons. The horizontal bars above the histograms indicate the duration of ejection of the indicated drugs at the given current (in nanoamps). The level recorded in the absence of any drug application represents the background activity. NMDA-evoked responses, indicated by an increase of the firing rate under the horizontal bars, were significantly enhanced during NA application in the neuron (A) and depressed in the neuron (B). Both effects were reversible and dose-dependent.

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antagonized (paired test, p < 0.05) all enhancements induced by NA on NMDA responses (four units) and was ineffective on the depressive effects (five units).

p < 0.01) reduced the excitatory effects induced by NMDA in eight units and was ineffective in the remaining two. In two units it was found that the depressive action of CIRA on NMDA responses was significantly (t-test, p < 0.05) antagonized by PRA. The mean level of depression of NMDA responses evoked by CIRA was not significantly different from that evoked by NA. Fig. 3A illustrates an example of CIRA depression of NMDA responses and the strong, long-lasting antagonistic effect of PRA in an SVN neuron. A selective alpha2 receptor agonist, CLO, was applied to 11 VN neurons and enhanced the NMDA-induced

Effects of noradrenergic alpha and beta receptor agonists on NMDA responses A summary of the effects induced on NMDA responses by long-lasting application of various NA receptor agonists is reported in Table 2. CIRA, a selective alpha1 receptor agonist, was tested during NMDA application in 10 neurons, located throughout the VN. CIRA ejection significantly (t-test,

Table 2. Enhancing and depressive effects of noradrenergic agonist on NMDA-induced responses All neurons

Increases

CIRA (10) CLO (11) ISO (9) DENO (9)

Decreases

Not responsive

M%

Cfrac

n

M%

Cfrac

n

n

– 142.76 ± 16.86 216.59 ± 64.99 187 ± 18

– 1.57 ± 0.23 1.49 ± 0.09 1.71 ± 0.16

– 5 4 8

57.69 ± 8.74 – – –

0.78 ± 0.14 – – –

8 – – –

2 6 3 1

The table shows the mean values (± standard error) of either enhancements (increases) or depressions (decreases) induced by application of noradrenergic agonist on NMDA-induced responses. CIRA, cirazoline; CLO, clonidine; ISO, isoproterenol; DENO, denopamine. The number of units tested for each drug is indicated in parentheses. n is the number of units showing enhancement, depression or no modification of NMDA-evoked responses. M%, percentage of magnitude; Cfrac, fraction of the contrast.

2 nA

4 nA PRA

A

CIRA 1 nA 400

NMDA 5nA

# of spikes

300

200

100

bin 5s

0 200 s/div

4nA 6nA

B

8nA YO

9nA

CLO NMDA 10nA

200

# of spikes

150

100

50

bin 5 s

0 200 s/div

Fig. 3. Examples of effects of alpha receptor noradrenergic agonists on NMDA-evoked excitatory effects. The horizontal bars above the histograms indicate the duration of ejection of the indicated drugs at the given current (in nanoamps). The level recorded in the absence of any drug application represents the background activity. (A) The noradrenergic alpha1 receptor agonist cirazoline (CIRA) induces reversible depression of NMDAevoked excitations in a neuron of the superior vestibular nucleus (SVN). This effect is totally antagonized by application of the alpha1 receptor antagonist prazosine (PRA). (B) The noradrenergic alpha2 receptor agonist clonidine (CLO) induces reversible enhancement of NMDA-evoked excitations in a neuron in the medial vestibular nucleus (MVN). This effect is antagonized by application of the alpha2 receptor antagonist yohimbine (YO).

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excitations in five of them, but was ineffective in the remaining six (Table 2). YO, tested in two neurons, partially antagonized the effects of CLO. Mean values of CLO- and NA-evoked enhancements of NMDA responses were not significantly different. Fig. 3B illustrates the firing rate of an MVN neuron whose NMDA responses were enhanced by CLO application. This effect was antagonized by YO. ISO, a generic beta receptor agonist was applied to nine VN neurons. It enhanced NMDA-induced excitations in four neurons, and the values of M% were significantly higher (ANOVA p < 0.05) than those induced by NA ejection (Table 2). The beta receptor antagonist TIM was applied to two units; it antagonized the enhancing action of ISO on NMDA-evoked excitations. Fig. 4A illustrates the marked enhancement of NMDA responses that was induced by ISO and antagonized by TIM in an MVN neuron. Application of DENO, a specific beta1 receptor agonist, also enhanced the excitatory effects induced by NMDA in eight out of nine tested neurons. These effects were significantly stronger (ANOVA p < 0.05) than those induced by NA (Table 2) and were antagonized by ATE (three units tested). Fig. 4B shows the enhancement of the NMDA responses in an LVN neuron that was induced by DENO application, and the effective blockade of the effect reversibly evoked by ATE.

160

Colocalization of NMDA and noradrenergic receptors Immunohistochemical analyses gave evidence of extensive colocalization of NMDA receptors (NR1 or NR2 subunits) with noradrenergic receptors (alpha1, alpha2 and beta1) throughout the VN, double labeling being strongly present at neuronal somata and their neighboring. The mean number of neurons and SIOC values with each of the noradrenergic receptors tested are summarized in Table 3 for each nucleus. In this study, the boundaries of VN were defined with the aid of an atlas of the rat brain (Paxinos and Watson, 1997). The co-localization of NMDARs with alpha1 noradrenergic receptors was high and quite homogeneous in all VN nuclei. In contrast, alpha2 receptors had a lower SIOC with NMDARs in the MVN than in the remaining nuclei, and a very high index in SpVN. This nucleus was also characterized by the highest SIOC of NMDARs with beta1 receptors, which were widely scattered across all nuclei. Overall, the SIOC of NMDARs with alpha1 receptors ranged from 84% to 96%, with alpha2 receptors from 72% to 97% and beta1 receptors from 76% to 94%. Fig. 5 shows examples of co-localization of NMDAR subunits (NR1 or NR2) with noradrenergic alpha1, alpha2 and beta1 receptors in VN.

2nA

A

4nA TIM ISO 1nA NMDA 30nA

# of spikes

120

80

40

bin 5s 0 200 s/div

5nA

B

10nA ATE DENO 5nA NMDA 40nA

200

# of spikes

150

100

50 bin 5s 0 400 s/div

Fig. 4. Examples of effects of beta receptor noradrenergic agonists on NMDA-evoked excitatory effects. The horizontal bars above the histograms indicate the duration of ejection of the indicated drugs at the given current (in nanoamps). The level recorded in the absence of any drug application represents the background activity. (A) The noradrenergic beta receptor generic agonist isoproterenol (ISO) induces reversible enhancement of NMDA-evoked excitations in a neuron of the medial vestibular nucleus (MVN). This effect is partially antagonized by application of the generic beta receptor antagonist timolol (TIM). (B) The noradrenergic beta1 receptor agonist denopamine (DENO) induces marked, reversible enhancement of NMDA-evoked excitation in a neuron in the lateral vestibular nucleus (LVN). This effect is antagonized by application of the beta1 receptor antagonist atenolol (ATE).

M. Barresi et al. / Neuroscience 265 (2014) 172–183 Table 3. Co-localization of noradrenergic and NMDA neurons in vestibular nuclei LVN

MVN

SVN

SpVN

NR1/alpha1 I.C. NR2/alpha1 I.C.

7.6 ± 0.4 90% 7.1 ± 0.4 86%

9.6 ± 1.0 84% 6.1 ± 0.3 84%

8.2 ± 0.8 86% 6.5 ± 0.6 96%

8.6 ± 0.9 89% 7.5 ± 0.8 89%

NR1/alpha2 I.C. NR2/alpha2 I.C.

8.0 ± 0.5 85% 6.9 ± 0.4 88%

15.1 ± 1.7 80% 5.9 ± 0.6 72%

4.9 ± 0.7 93% 6.1 ± 1.0 83%

9.1 ± 1.5 97% 4.2 ± 0.4 97%

NR1/beta1 I.C. NR2/beta1 I.C.

9.2 ± 0.6 85% 6.6 ± 0.4 88%

6.3 ± 0.4 94% 12.4 ± 1.6 76%

4.9 ± 0.6 85% 7.4 ± 1.1 79%

5.3 ± 0.4 92% 4.6 ± 0.4 94%

Double labeled neurons (a noradrenergic receptor + a NMDA receptor subunit) found in the vestibular complex: LVN, lateral vestibular nucleus; MVN, medial vestibular nucleus; SVN, superior vestibular nucleus; SpVN, spinal vestibular nucleus. In each values indicated are: mean number (±standard error) of double labeled neurons found in a slide and number of double labeled neurons versus NMDA (either NR1 or NR2) labeled ones (semi-quantitative indicator of co-localization, S.I.O.C.).

DISCUSSION These results demonstrate that NA levels can modulate NMDAR-induced excitations in almost all secondary vestibular neurons, but that this modulation is not unidirectional. NA action on NMDA responses appears to be widespread, but not uniform. In fact, various types of noradrenergic receptors are involved and the modulation of NMDAR-mediated responses is not the same in all the nuclei. Glutamate is the main neurotransmitter used by primary vestibular fibers (Lewis et al., 1989; Sasa et al., 2001; Zhang et al., 2011) and plays an essential role in the VN, where high levels are found (Li et al., 1996). As a broad noradrenergic projection is delivered to the VN (Schuerger and Balaban, 1993, 1999), the hypothesis that NA may influence glutamatergic neurotransmission is plausible. In fact we had observed an NA-dependent modulation of glutamate-evoked excitation (Barresi et al., 2009). Furthermore, the diffuse co-localization of AMPA and NMDA receptors described in the VN (Chen et al., 2000) suggests that NA might also modulate the function of NMDARs and thus exerting some control on the efficacy of both receptor types. NMDARs play a role in excitatory transmission, contributing to synaptic integration and learning (Malenka and Nicoll, 1993; Dineley et al., 2001; Antic et al., 2010). The noradrenergic system has also a significant influence on the same mechanisms (McGaugh, 2004; Van Stegeren, 2008; Tully and Bolshakov, 2010). Noradrenergic control of the efficacy of NMDARs appears to be crucial in the nervous system; NA deficiency even impairs NMDA-mediated neurotransmission and exacerbates some early cognitive symptoms of Alzheimer’s disease (Hammerschmidt et al., 2013).

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In VN, NMDARs are also involved in glutamatergic neurotransmission (Kinney et al., 1994; Takahashi et al., 1994; Tyurin, 2009) and specifically in mechanisms of plasticity (Scarduzio et al., 2012). It is known that noradrenergic mechanisms play a role in adaptation of vestibulo-ocular and vestibulo-spinal reflexes by an indirect action exerted on the cerebellar cortex (Pompeiano, 2006). Our results demonstrate that a direct modulation of NA on the NMDAR-mediated responses of secondary vestibular neurons exists, but not in what possible adaptive mechanism it is inserted. NA enhances NMDAR-mediated excitation in a significant majority of cells and has a depressive action on about half of the LVN neurons tested. In contrast, NA application induced depression more frequently than enhancement of excitations induced by glutamate application (Barresi et al., 2009). The influence of NA on NMDAR-mediated responses could therefore be viewed as a component of a wider control exerted by NA in the short and long term on glutamatergic transmission to secondary vestibular neurons. The classical concept of the actions of NMDARs is based on their post-synaptic location. However, in recent years identification of a growing number of presynaptic NMDARs, even if they are mostly confined to brain structures involved in the control of autonomic and limbic functions, has suggested their involvement in a variety of roles (Duguid and Smart, 2009). The location of these receptors cannot be identified with our experimental set-up. However, the presence of presynaptic NMDA receptors in the VN cannot be excluded; their presence would imply a greater and more differentiated functional significance for the modulation exerted by NA. Our data were recorded under anesthesia induced by urethane, that is widely used in electrophysiology and thought to minimally interfere with neurophysiological processes and synaptic transmission. In our experiments it was important to preserve the kinetic properties of neurons (rise and decay time of potentials). Any action of the anesthetic on the release mechanisms was not critical because we tested drugs (NMDA and NA agonists and antagonists) for their effect on the firing, applying them by iontophoresis and therefore directly. Tian et al. (2012) demonstrated that urethane does not alter the kinetic properties of hippocampal CA1 neurons, but induces a depression of the excitability, related to a reduction of presynaptic glutamatergic release. In the light of these findings we cannot exclude that even in the VN urethane can induce a decrease of neuronal background firing. However, the systematic variation of the background firing rate would regard the whole neuronal pool, in no way modifying results concerning the responsiveness to drugs applied by iontophoresis. Our results indicate that in VN, the enhancements of NMDAR-induced excitations were mediated by noradrenergic beta receptors with a minor contribution by alpha2 receptors. Noradrenergic beta receptors are known to be potent regulators of synaptic plasticity. Beta receptors, even if not those located in VN, mediate

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50μm

50mm

50μm

50μm

50μm

50μm

NR1

α1

NR1/α1

NR2A

α1

NR2A/α1

NR1

α2

NR1/α2

NR2A

α2

NR2A/α2

NR1

β1

NR1/β1

NR2A

β1

NR2A/β1

Fig. 5. Co-localization of NMDA receptors with noradrenergic receptors (alpha1, alpha2 and beta1) in single neurons of vestibular nuclei (VN). Immunolabeled sections (fluorescence microscope, 20 magnification) show immunoreactivity for NR1 or NR2A sub-units of NMDA receptors (column 1, red) and either alpha or beta noradrenergic receptor (column 2, green) in single VN neurons. Double immunofluorescence was detected in single neurons throughout all the vestibular nuclei (column 3, orange).

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NA-induced adaptive mechanisms on vestibular reflexes (Pompeiano, 2006). The same receptors mediate enhancement of NMDAR responsiveness, inducing LTP, in various neural structures as the amygdale (Gean et al., 1992; Huang et al., 1993), hippocampus (Raman et al., 1996) and prefrontal cortex (Ji et al., 2008a,b). Beta receptors also enhance NMDAR-mediated responses indirectly by inducing the release of homocysteic acid from glial cells (Do et al., 1997). In the VN, alpha2 receptors had a selective but important role in the enhancement exerted by NA on NMDAR-mediated excitation. These effects were specific as they were antagonized by yohimbine and mimicked, at least in some units, by clonidine. An involvement of alpha2 receptors was described in the control of short-term and long-term associative plasticity of parallel fiber synapses (Carey and Regehr, 2009), in the same network controlling vestibular reflexes. Alpha2 noradrenergic receptors however generally induce depression of NMDAR-mediated responses, as described in the prefrontal cortex (Law-Tho et al., 1993; Liu et al., 2006; Ji et al., 2008a,b) and the behaviorallyrelated effects mediated by alpha2 receptors on NMDAR responses appear to be depressive (Harkin et al., 2001). Few enhancing effects mediated by these receptors were recorded in the medulla (Zhang et al., 1998), where, in any case, the effect was depressive in a huge majority of cells. On the other hand, an alpha2-induced enhancement of NMDAR neurotransmission could be consistent with the observation that in the prefrontal cortex, activation of alpha2 noradrenergic receptors improves working memory performance via complex membrane mechanisms and an enhancement of temporal integration (Carr et al., 2007). In some VN units, mostly located in LVN, we recorded depressions of NMDAR responses induced by NA application and mediated by alpha1 noradrenergic receptors. These results are more in line with data reported in the literature. In fact, activation of alpha1 noradrenergic receptors in the prefrontal cortex induces LTD by modulating NMDAR-mediated excitation (Liu et al., 2006; Marzo et al., 2010). On the whole our results indicate that in the VN, alpha1, alpha2 and beta receptors are all involved in the control of NMDAR-mediated responses. Immunohistochemical data provide no evidence for segregation either in the distribution of alpha1 and beta noradrenergic receptors or in the location of NMDARs throughout all VN. However, the same data and the electrophysiological results suggest an uneven distribution of alpha2 noradrenergic receptors in VN. Our results show that NA application always influences the NMDA response in all the VN but that the effect may be of opposite sign. Given that noradrenergic receptors appear fairly evenly distributed in all nuclei, the differences could be due to the different types of neurons and/or to the different locations (pre-or postsynaptic) of the receptors. As an example, the two types of effect evoked by NA on NMDAR-mediated responses might affect two different populations of neurons (e.g. projection vs. commissural units or ascending vs.

descending projection), but a functional identification of the units would be necessary to validate this hypothesis. It is possible that increasing the sample might have given evidence of differences between the nuclei, but the fact remains that these differences must anyway brought back to the functional characteristics of neurons. Our experimental set-up and extracellular recordings of the firing rate, however, do not allow to speculate on the specific characteristics of single neuronal units. In this phase of the research, given that both types of responses were recorded in all of the nuclei, and that we were not in the experimental conditions to verify the functional specificity of neurons, we considered useless to further broaden the sampling. An alternative hypothesis is that all the noradrenergic receptors mediating opposite effects act on the same neurons. In fact, alpha1, beta and, to a lesser extent, alpha2 receptors were all widely present in the VN, and each type was broadly colocalized with NMDA receptors. Given the higher affinity of NA for alpha receptors than for beta receptors, the concentration of NA could be critical in determining the type of modulation exerted on NMDAR function. In four neurons, we observed a reversal of the effect of NA on NMDA- induced excitations from depressive into enhancing after a long lasting application of NA, which would be consistent with this hypothesis.

CONCLUSIONS These results demonstrate that NA modulates the excitatory effects mediated by activation of NMDA receptors in secondary vestibular neurons. Both alpha and beta noradrenergic receptors are involved in these effects, which include enhancement and depression. As adrenoreceptors are widely distributed throughout the VN, one can conclude that all physiological mechanisms involving NMDA receptors in the vestibular complex are modulated by local concentrations of NA. Acknowledgments—This work was supported by a grant from the Catania University (Italy). The authors are grateful to Prof. David Tracey for proofreading the manuscript, Prof. Rosario Giuffrida and Dr. Rosalia Pellitteri for their valuable assistance. This research was supported by a grant from the Universita` degli Studi di Catania (Italy).

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(Accepted 29 January 2014) (Available online 7 February 2014)