Accepted Manuscript Title: NMDA-glutamatergic activation of the ventral tegmental area induces hippocampal theta rhythm in anesthetized rats Author: Paweł Matulewicz Jolanta Orzeł-Gryglewska Magda Ku´smierczak Edyta Jurkowlaniec PII: DOI: Reference:
S0361-9230(14)00098-7 http://dx.doi.org/doi:10.1016/j.brainresbull.2014.06.001 BRB 8758
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
9-10-2013 20-5-2014 2-6-2014
Please cite this article as: P. Matulewicz, J. Orzel-Gryglewska, M. Ku´smierczak, E. Jurkowlaniec, NMDA-glutamatergic activation of the ventral tegmental area induces hippocampal theta rhythm in anesthetized rats, Brain Research Bulletin (2014), http://dx.doi.org/10.1016/j.brainresbull.2014.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
NMDA-glutamatergic activation of the ventral tegmental area induces hippocampal theta rhythm in anesthetized rats
Paweł Matulewicz, Jolanta Orzeł-Gryglewska*, Magda Kuśmierczak, Edyta Jurkowlaniec
ip t
Department of Animal and Human Physiology, University of Gdańsk, Wita Stwosza 59,
______________
Ac
ce pt
ed
M
an
* Correspondence: E-mail: [email protected]
us
cr
80-308 Gdańsk, Poland
Page 1 of 32
Abstract Glutamate afferents reaching the ventral tegmental area (VTA) affect dopamine (DA) cells in this structure probably mainly via NMDA receptors. VTA appears to be one of the structures involved in regulation of hippocampal theta rhythm, and this work aimed at assessing the role
ip t
of glutamatergic activation of the VTA in the theta regulation. Male Wistar rats (n=17) were divided into groups, each receiving intra-VTA microinjection (0.5 l) of either solvent (water),
cr
glutamatergic NMDA agonist (0.2 g) or antagonist (MK-801, 3.0 g). Changes in local field
us
potential were assessed on the basis of peak power (Pmax) and corresponding peak frequency (Fmax) for the delta (0.5-3 Hz) and theta (3-6 Hz) bands. NMDA microinjection evoked long-
an
lasting hippocampal theta. The rhythm appeared with a latency of ca. 12 min post-injection and
M
lasted for over 30 min; Pmax in this band was significantly increased for 50 min, while simultaneously Pmax in the delta band remained lower than in control conditions. Theta Fmax and
ed
delta Fmax were increased in almost entire post-injection period (by 0.3-0.5 Hz and 0.3-0.7 Hz respectively). MK-801 depressed the sensory-evoked theta: tail pinch could not induce theta for
ce pt
30 min after the injection; Pmax significantly decreased in the theta band and at the same time it increased in the delta band. Theta Fmax decreased 10 and 20 min post injection (by 0.4-0.5 Hz) and delta Fmax decreased in almost entire post injection period (by 0.3-0.7 Hz). NMDA
Ac
injection generates theta rhythm probably through stimulation of dopaminergic activity within the VTA.
Keywords: glutamate, MK-801, NMDA, VTA, hippocampus, theta rhythm
1
Page 2 of 32
1. Background Hippocampal theta rhythm (also known as the rhythmic slow activity, RSA) is a highly synchronous electrical signal recorded in the mammalian brain, especially in rodents, and is characterised with high voltage and low frequency, i.e. 3-12 Hz. Its appearance is associated with voluntary locomotor activities (Oddie and Bland, 1998; Vanderwolf, 1969) as well as
ip t
episodes of REM sleep, (Kemp and Kaada, 1975; Montgomery et al., 2008; Whishaw and Vanderwolf, 1973) and the types I (6-12 Hz) and II (3-9 Hz) of theta are distinguished accordingly (Kramis et al., 1975).
cr
The structures participating in induction and regulation of theta rhythm include the brainstem, medial septum and hippocampal formation, and they constitute „the theta rhythm
us
synchronization system” (Bland and Oddie, 1998; Vertes, 1981, 1982; Vertes and Kocsis, 1997; Vertes et al., 2004; Vinogradova, 1995). Our research indicates that this brainstem-
an
diencephalo-septohippocampal system also includes the midbrain ventral tegmental area (VTA). We found that unilateral cytotoxic lesion of the VTA caused bilateral decrease in
M
neocortical and hippocampal signal power during such behavioural reactions that are easily elicited by stimulation of the VTA and are usually accompanied by hippocampal theta rhythm (e.g. during exploratory sniffing) (Jurkowlaniec et al., 2003). Furthermore, procaine injections
ed
into the VTA temporarily blocked the appearance of sensory stimulated theta rhythm in urethane anesthetized rats, whereas lesions of this area considerably changed synchronization
ce pt
of electrical activity of the hippocampus: peak power in 3-6 and 6-9 Hz bands decreased immediately after the lesion, and a few days later the decrease in 3-6 Hz was still present but in 6-9 and 9-12 Hz bands an increase in signal peak power was observed (Orzeł-Gryglewska et al., 2006). On the other hand, activation of the VTA triggered regular theta band activity in
Ac
the hippocampus. This was achieved by electrical stimulation or local microinjections of bicuculline (GABAA antagonist) (Orzeł-Gryglewska et al., 2010; 2012). Microinjection of phaclofen (GABAB antagonist), flupenthixol (D2 receptor antagonist if applied at low doses) or amphetamine (increases the DA release) into the VTA also induced theta rhythm in the hippocampal signal; however, the changes were less clear after these agents (OrzełGryglewska et al., 2010; 2013). The VTA is composed mainly (almost 70%) of the A10 dopaminergic (DA) cells group (Dahlstrom and Fuxe, 1964; Oades and Halliday, 1987) with widespread projections to the forebrain structures, and GABA cells, representing approximately 25% of the total population 2
Page 3 of 32
of neurons in this area (Adell and Artigas, 2004; Kalivas, 1993; Oades and Halliday, 1987; Swanson, 1982). A large part of the GABA cells are interneurons but some of them send GABAergic projections to the prefrontal cortex (Carr and Sesack, 2000a), nucleus accumbens (Van Bockstaele and Pickel, 1995), and dorsal raphe nucleus (Kirouac et al., 2004). Inhibitory GABAergic afferents reaching the VTA from the nucleus accumbens, ventral pallidum and pedunculopontine nucleus (Charara et al., 1996; Waalas and Fonmun, 1980), as well as the
ip t
GABAergic interneurons within the VTA, exert powerful regulatory control over the activity of the perikarya of the dopamine neurons (Kalivas, 1993; Stinus et al., 1982).
cr
Glutamatergic afferents that innervate DA cells within the VTA play a significant role in the regulation of cell activity and are crucial for the functioning of this area (Geisler, Wise, 2008).
us
Induction of the burst firing of VTA dopamine neurons depends largely on glutamatergic inputs (reviewed in Overton, Clarck, 1997; White 1996) and local infusions of glutamate
an
receptor agonists induce burst firing of DA neurons in vivo (Chergui et al., 1993; Johnson et al., 1992). The critical modulatory functions of DA in the nervous system often involve regulation of or by glutamate (Sesack et al., 2003). The DA neurons in the whole midbrain
M
(A10 and A9) receive glutamatergic afferents mainly from the medial prefrontal cortex, but also from the lateral and medial preoptic area, lateral and medial hypothalamus, habenula,
ed
central gray, dorsal raphe, pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus, etc. (Blaha et al., 1996; Geisler et al., 2007; Geisler and Wise, 2008; Omelchenko and Sesack, 2005; for review see: Kalivas, 1993).
ce pt
DA neurons express ionotropic NMDA and non-NMDA as well as metabotropic glutamate receptors (Meltzer et al., 1997). Blockers of the glutamate NMDA and AMPA receptors injected into the VTA were shown to decrease dopamine release in the VTA target structures, i.e. the nucleus accumbens (Karreman et al., 1996; Westerink et al., 1996) and the prefrontal
Ac
cortex (Enrico et al., 1998; Takahata and Moghaddam, 1998; Westerink et al., 1998), which suggests that the glutamatergic system exerts a tonic excitatory influence over DA neurons (Meltzer et al., 1997). NMDA receptors are present in the VTA on both DA and non-DA cells (Seutin et al., 1990; Wang and French, 1993b; 1995). Tonic activation of NMDA receptors caused spontaneous burst discharge of midbrain DA neurons in rats (Chergui et al., 1993). The excitatory effect of glutamate on A10 DA neurons appears to be preferentially mediated by NMDA rather than non-NMDA receptors (Gronier, 2008; Meltzer et al., 1997; Wang and French, 1993a). Activity of the metabotropic glutamate receptors seems to be negligible in the VTA (Adell and Artigas, 2004). 3
Page 4 of 32
Glutamatergic synapses localised on VTA DA neurons can undergo both long-term potentiation and long-term depression to regulate their synaptic strength and consequently the dopaminergic output (Bonci and Malenka, 1999; Chergui et al., 1994; Overton and Clark, 1997; Overton et al., 1999). DA neurons in the mesocortical system are under greater tonic control of AMPA and NMDA receptors than are the DA cells which innervate the nucleus accumbens (Takahata and Moghaddam, 1998; 2000). Dizocilpine (MK-801) was found to
ip t
have different effects on the mesolimbic and mesocortical dopamine systems (Mathe et al., 1999), which shows heterogeneity of the glutamatergic control over the dopaminergic
cr
pathways. Hence, the effect of glutamate on dopaminergic activity within the VTA in the process of theta rhythm generation is difficult to predict.
us
In the present study we aimed to investigate whether NMDA-mediated glutamatergic transmission within the VTA has any involvement in the regulation of hippocampal electric
an
activity, particularly in the theta band. We used a standard experimental model of sensoryelicited theta rhythm recorded from the hippocampus in urethane-anesthetized rats. This model permits the analysis of the capacity of the hippocampus to generate synchronous
M
activity in strictly controlled conditions and was used in numerous studies (Bland et al., 1995; Kinney et al., 1996; Kirk et al., 1996; Llinas and Alonso, 1992; McNaughton et al., 1995;
ed
Nowacka et al., 2002; Vanderwolf and Baker, 1986). We analysed power, frequency and regularity of hippocampal signal, in the theta and delta bands. Delta rhythm was also analysed, since changes in the delta and theta bands are related: delta becomes
ce pt
desynchronized and supressed when theta rhythm dominates the signal, for example during tail pinch stimulation (Vinogradova, 1995). A preliminary report of a part of the present work was published in an abstract form
Ac
(Matulewicz et al., 2012).
2. Materials and methods 2.1. Animals Experiments were performed on male Wistar rats (n= 17; Tri-City Central Animal Laboratory – Research and Service Centre of the Medical University of Gdansk, Poland) weighing approximately 300 - 350 g. The rats were maintained in a 12-h light-dark cycle (lights on at 6 a.m.), at 22±1ºC and provided with food and water ad libitum. The animals were randomly 4
Page 5 of 32
divided into experimental groups receiving intrategmental injections of either water, NMDA or MK-801. The experiments were evaluated and approved by the Local Ethical Committee of the Medical University of Gdańsk. 2.2. Surgery Animals were anesthetized with urethane (Urethane, Sigma 1.5 g/kg i.p.) and positioned in the
ip t
frame of a stereotaxic apparatus. The depth of anaesthesia was assessed by monitoring respiratory rate (unpublished study) and maintained at such a level that hind-limb withdrawal reflex is not present and theta rhythm can be elicited by sensory, pharmacological or electrical
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stimulation but does not appear spontaneously. Bilateral recording electrodes were implanted
us
in the stratum moleculare of the dorsal blade of the dentate gyrus of the dorsal hippocampus according to the standard stereotaxic procedure. The electrodes were monopolar, made of stainless steel wire, 0.2 mm in diameter, insulated on the entire length except for the flat-cut
an
tip. The following Paxinos and Watson (1998) stereotaxic coordinates (skull levelled) were used for implantation: 3.7 mm posterior to bregma, 2.4 mm lateral to midline and 3.1 mm
M
ventral to skull surface. Stainless steel screws positioned in the frontal area and over the olfactory bulb served as ground and reference electrodes respectively; they were inserted into holes drilled in the skull and additionally secured with dental acrylic. The olfactory bulb
ed
seems to be an appropriate reference for theta because of the low-amplitude and unsynchronized patterns of electrical activity present in this area (Chaput et al., 1992; Kay
ce pt
and Lazzara, 2010). A small hole was drilled over the VTA to allow subsequent insertion of an injection cannula. Stereotaxic coordinates for the VTA implantation were: 5.1 mm posterior to bregma, 0.9 mm lateral to midline and 8.0 mm ventral to skull surface. As an injection cannula, we used a needle (0.4 mm in diameter, with a flat-cut tip) of 10 l Hamilton
Ac
syringe. The syringe was placed in the stereotaxic holder with a microinfusion pump (Kopf Instruments).
2.3. Experimental design Urethane-anesthetized rats were held in the stereotaxic apparatus throughout the experiment, with body temperature maintained at 37 oC. Local field potential (LFP) was recorded from the hippocampal electrodes during the whole experiment with the use of EEG DigiTrack computer system (ELMIKO, Warsaw, Poland; sampling rate: 240 Hz). The anaesthesia was maintained at the depth that allowed theta to be evoked with sensory stimulation but did not permit spontaneous appearance of this rhythm. Sensory stimulation (tail pinch lasting 60 s) 5
Page 6 of 32
was applied 3 - 4 times in the preinjection conditions. The animals which did not yield theta rhythm of an amplitude of at least 400 V during control tail pinch stimulations were discarded. After completion of the control recordings, the needle of the Hamilton syringe (injection cannula) was slowly lowered to just above the VTA. A 10-min period of adjustment after the insertion of the cannula was allowed, after which tail pinch was reapplied and if theta was successfully elicited, the intra-VTA injection procedure was started.
ip t
Animals were divided into 3 experimental groups, depending on the drug used for intra-VTA injections (an injection lasted for 3-5 min and was 0.5 µl in volume). Rats from the first group
cr
(n=7) received unilateral microinjections of NMDA (Sigma-Aldrich, Germany) at a dose of 0.2 g/0.5l and in the second group of rats (n=5) MK-801 (Tocris Bioscience, USA) was
us
microinjected at a dose of 3.0 g/0.5l. Hippocampal LFP was recorded continuously for 90 min after the injection and 1-min tail pinch was applied at 9 min intervals throughout the
an
recording time. In cases when spontaneous theta rhythm appeared as a result of the intracerebral injection, no tail-pinch was applied until the end of the theta episode. The third, control group (n=5) received an injection of the drugs solvent (Aqua pro injectione,
M
Polpharma, Starogard Gdański, Poland) and the hippocampal LFP was recorded for 90 min afterwards. Hippocampal signal in the control group was assessed both in spontaneous and
ed
tail-pinch conditions, with animals randomly assigned to one of the two protocols: 1) water injection followed by 1.5-hour recording of spontaneous signal, followed by another water
ce pt
injection and 1.5-hour recording with 60-s tail pinch applied every 9 min; 2) the opposite order: recording with tail pinch after the first water injection, followed by another injection and recording of only the spontaneous signal. 2.4. Data Analysis
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Spectral analysis of hippocampal LFP (band pass filtered 0–30 Hz), ipsi- and contralateral (in relation to the injection side), was performed off line by fast Fourier transformation (FFT) on 10-14 artefact-free 4.1-s epochs chosen from the 60-s tail pinch fragments applied before and after each drug injection (by SPIKE 2, Cambridge, UK software). In the cases of drug-elicited theta rhythm LFP samples were taken every 5 minutes from the post-injection period. In the water group samples were taken from 60-s fragments of spontaneous LFP signal every 5 min and also from the 60-s tail pinch, and the results were separated into two respective control groups.
6
Page 7 of 32
Two parameters of the power spectrum, i.e. the maximal peak power (FFT peak magnitude, Pmax [V2]) and the frequency corresponding to the maximal peak power (FFT peak frequency, Fmax [Hz]), were assessed separately for the following bands: 3.0-6.0 Hz, 6.0-9.0 Hz and 9.0-12.0 Hz (we divided the 3-12 Hz band corresponding to theta activity into 3 subbands to allow more detailed analysis), 0.5-3.0 Hz (delta) and 12.0-15.0 Hz (beta). Pmax and Fmax were submitted to statistical analysis (Two-way ANOVA with groups as factors (control
ip t
and experimental) and time (after drug microinjection) and post hoc Fisher’s LSD test). The time factor in water groups was analysed using one-way ANOVA test (GraphPad Prism 6.02
cr
software). To eliminate inter-subject variability, power was expressed as a percentage of the pre-injection baseline value taken as 100%, for each frequency band separately. The latency
us
and duration of NMDA-induced theta rhythm episodes were measured on the basis of the FFT analysis of the peak power in 4.2-s samples of the signal (previously described in Matulewicz
an
et al., 2010). 2.5. Histology
M
After completion of the experiment, a small electrolytic lesion (anodal current of 100 A/15 s) was performed through the hippocampal electrodes to visualize the localization of their tips.
ed
Rats were then treated with an overdose of anaesthetic and intracardially perfused with 0.9% saline followed by a 10% solution of formalin. The brains were removed from the skulls and placed in 10% formalin. After fixation, brains were cut into 60-m slices using frozen tissue
ce pt
technique, and the position of the hippocampal electrodes and the VTA cannulas was verified. In a representative rat, 2% saline solution of alcian blue 8GHX (Fluka, Switzerland) was injected into the VTA in a volume of 0.5 µl and diffusion area around the injection cannula was
Ac
assessed under a light microscope. 3. Results
3.1. Hippocampal LFP pattern after administration of NMDA into the VTA In all animals (n=7) intra-VTA injection of NMDA induced robust and long lasting episodes of rhythmic slow activity with a mean latency of 11.8±2.5 min and mean duration of 33.3±6.9 min (mean±SE), with no differences between both hippocampi. Only a few very short episodes of irregular activity interrupted the episodes of the rhythm and together they accounted for 4.6% of the entire NMDA-induced theta episode. Figure 1 shows the course of the experiment in representative animals: power spectra and fragments of hippocampal LFP 7
Page 8 of 32
traces before and during tail pinch without NMDA and after NMDA administration (left panel) and before and after water administration (right panel). Part A of the spectrogram shows a 5-min sample of hippocampal LFP before manipulations. A 60-sec fragment of this spontaneous signal served as a baseline for calculating control values for subsequent frequency bands; during this preliminary recording we also tested whether theta rhythm appears as a response to tail pinch. This first part of recording lasted for
ip t
20-40 min. Part B depicts a 5-min LFP fragment encompassing theta rhythm that was elicited with 1-min tail pinch. Microinjection procedure followed and lasted 5 min (interval between
cr
B and C). Part C shows the final seconds of an injection followed by a 60-min post-injection period. After NMDA, activity in the theta band increased and this effect had a latency of ca.
us
12 min. This change in activity is evident in the LFP traces and it is also expressed as an increase in FFT power in the 3-6 Hz theta band, which is the dominating theta frequency in
an
urethane-anaesthetized rats (Cervera-Ferri et al., 2011; Kiss et al., 2013; Kowalczyk et al., 2014), and as a reduction of power in the delta (0.5–3 Hz) band. Water injection did not cause
M
any changes in LFP as compared with the preliminary spontaneous recording (Fig. 1). 3.1.1. Changes of Pmax and Fmax in 3.0-6.0 Hz band
A two-way ANOVA (group effect F(1,142)=148.70, p
S0361-9230(14)00098-7 http://dx.doi.org/doi:10.1016/j.brainresbull.2014.06.001 BRB 8758
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
9-10-2013 20-5-2014 2-6-2014
Please cite this article as: P. Matulewicz, J. Orzel-Gryglewska, M. Ku´smierczak, E. Jurkowlaniec, NMDA-glutamatergic activation of the ventral tegmental area induces hippocampal theta rhythm in anesthetized rats, Brain Research Bulletin (2014), http://dx.doi.org/10.1016/j.brainresbull.2014.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
NMDA-glutamatergic activation of the ventral tegmental area induces hippocampal theta rhythm in anesthetized rats
Paweł Matulewicz, Jolanta Orzeł-Gryglewska*, Magda Kuśmierczak, Edyta Jurkowlaniec
ip t
Department of Animal and Human Physiology, University of Gdańsk, Wita Stwosza 59,
______________
Ac
ce pt
ed
M
an
* Correspondence: E-mail: [email protected]
us
cr
80-308 Gdańsk, Poland
Page 1 of 32
Abstract Glutamate afferents reaching the ventral tegmental area (VTA) affect dopamine (DA) cells in this structure probably mainly via NMDA receptors. VTA appears to be one of the structures involved in regulation of hippocampal theta rhythm, and this work aimed at assessing the role
ip t
of glutamatergic activation of the VTA in the theta regulation. Male Wistar rats (n=17) were divided into groups, each receiving intra-VTA microinjection (0.5 l) of either solvent (water),
cr
glutamatergic NMDA agonist (0.2 g) or antagonist (MK-801, 3.0 g). Changes in local field
us
potential were assessed on the basis of peak power (Pmax) and corresponding peak frequency (Fmax) for the delta (0.5-3 Hz) and theta (3-6 Hz) bands. NMDA microinjection evoked long-
an
lasting hippocampal theta. The rhythm appeared with a latency of ca. 12 min post-injection and
M
lasted for over 30 min; Pmax in this band was significantly increased for 50 min, while simultaneously Pmax in the delta band remained lower than in control conditions. Theta Fmax and
ed
delta Fmax were increased in almost entire post-injection period (by 0.3-0.5 Hz and 0.3-0.7 Hz respectively). MK-801 depressed the sensory-evoked theta: tail pinch could not induce theta for
ce pt
30 min after the injection; Pmax significantly decreased in the theta band and at the same time it increased in the delta band. Theta Fmax decreased 10 and 20 min post injection (by 0.4-0.5 Hz) and delta Fmax decreased in almost entire post injection period (by 0.3-0.7 Hz). NMDA
Ac
injection generates theta rhythm probably through stimulation of dopaminergic activity within the VTA.
Keywords: glutamate, MK-801, NMDA, VTA, hippocampus, theta rhythm
1
Page 2 of 32
1. Background Hippocampal theta rhythm (also known as the rhythmic slow activity, RSA) is a highly synchronous electrical signal recorded in the mammalian brain, especially in rodents, and is characterised with high voltage and low frequency, i.e. 3-12 Hz. Its appearance is associated with voluntary locomotor activities (Oddie and Bland, 1998; Vanderwolf, 1969) as well as
ip t
episodes of REM sleep, (Kemp and Kaada, 1975; Montgomery et al., 2008; Whishaw and Vanderwolf, 1973) and the types I (6-12 Hz) and II (3-9 Hz) of theta are distinguished accordingly (Kramis et al., 1975).
cr
The structures participating in induction and regulation of theta rhythm include the brainstem, medial septum and hippocampal formation, and they constitute „the theta rhythm
us
synchronization system” (Bland and Oddie, 1998; Vertes, 1981, 1982; Vertes and Kocsis, 1997; Vertes et al., 2004; Vinogradova, 1995). Our research indicates that this brainstem-
an
diencephalo-septohippocampal system also includes the midbrain ventral tegmental area (VTA). We found that unilateral cytotoxic lesion of the VTA caused bilateral decrease in
M
neocortical and hippocampal signal power during such behavioural reactions that are easily elicited by stimulation of the VTA and are usually accompanied by hippocampal theta rhythm (e.g. during exploratory sniffing) (Jurkowlaniec et al., 2003). Furthermore, procaine injections
ed
into the VTA temporarily blocked the appearance of sensory stimulated theta rhythm in urethane anesthetized rats, whereas lesions of this area considerably changed synchronization
ce pt
of electrical activity of the hippocampus: peak power in 3-6 and 6-9 Hz bands decreased immediately after the lesion, and a few days later the decrease in 3-6 Hz was still present but in 6-9 and 9-12 Hz bands an increase in signal peak power was observed (Orzeł-Gryglewska et al., 2006). On the other hand, activation of the VTA triggered regular theta band activity in
Ac
the hippocampus. This was achieved by electrical stimulation or local microinjections of bicuculline (GABAA antagonist) (Orzeł-Gryglewska et al., 2010; 2012). Microinjection of phaclofen (GABAB antagonist), flupenthixol (D2 receptor antagonist if applied at low doses) or amphetamine (increases the DA release) into the VTA also induced theta rhythm in the hippocampal signal; however, the changes were less clear after these agents (OrzełGryglewska et al., 2010; 2013). The VTA is composed mainly (almost 70%) of the A10 dopaminergic (DA) cells group (Dahlstrom and Fuxe, 1964; Oades and Halliday, 1987) with widespread projections to the forebrain structures, and GABA cells, representing approximately 25% of the total population 2
Page 3 of 32
of neurons in this area (Adell and Artigas, 2004; Kalivas, 1993; Oades and Halliday, 1987; Swanson, 1982). A large part of the GABA cells are interneurons but some of them send GABAergic projections to the prefrontal cortex (Carr and Sesack, 2000a), nucleus accumbens (Van Bockstaele and Pickel, 1995), and dorsal raphe nucleus (Kirouac et al., 2004). Inhibitory GABAergic afferents reaching the VTA from the nucleus accumbens, ventral pallidum and pedunculopontine nucleus (Charara et al., 1996; Waalas and Fonmun, 1980), as well as the
ip t
GABAergic interneurons within the VTA, exert powerful regulatory control over the activity of the perikarya of the dopamine neurons (Kalivas, 1993; Stinus et al., 1982).
cr
Glutamatergic afferents that innervate DA cells within the VTA play a significant role in the regulation of cell activity and are crucial for the functioning of this area (Geisler, Wise, 2008).
us
Induction of the burst firing of VTA dopamine neurons depends largely on glutamatergic inputs (reviewed in Overton, Clarck, 1997; White 1996) and local infusions of glutamate
an
receptor agonists induce burst firing of DA neurons in vivo (Chergui et al., 1993; Johnson et al., 1992). The critical modulatory functions of DA in the nervous system often involve regulation of or by glutamate (Sesack et al., 2003). The DA neurons in the whole midbrain
M
(A10 and A9) receive glutamatergic afferents mainly from the medial prefrontal cortex, but also from the lateral and medial preoptic area, lateral and medial hypothalamus, habenula,
ed
central gray, dorsal raphe, pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus, etc. (Blaha et al., 1996; Geisler et al., 2007; Geisler and Wise, 2008; Omelchenko and Sesack, 2005; for review see: Kalivas, 1993).
ce pt
DA neurons express ionotropic NMDA and non-NMDA as well as metabotropic glutamate receptors (Meltzer et al., 1997). Blockers of the glutamate NMDA and AMPA receptors injected into the VTA were shown to decrease dopamine release in the VTA target structures, i.e. the nucleus accumbens (Karreman et al., 1996; Westerink et al., 1996) and the prefrontal
Ac
cortex (Enrico et al., 1998; Takahata and Moghaddam, 1998; Westerink et al., 1998), which suggests that the glutamatergic system exerts a tonic excitatory influence over DA neurons (Meltzer et al., 1997). NMDA receptors are present in the VTA on both DA and non-DA cells (Seutin et al., 1990; Wang and French, 1993b; 1995). Tonic activation of NMDA receptors caused spontaneous burst discharge of midbrain DA neurons in rats (Chergui et al., 1993). The excitatory effect of glutamate on A10 DA neurons appears to be preferentially mediated by NMDA rather than non-NMDA receptors (Gronier, 2008; Meltzer et al., 1997; Wang and French, 1993a). Activity of the metabotropic glutamate receptors seems to be negligible in the VTA (Adell and Artigas, 2004). 3
Page 4 of 32
Glutamatergic synapses localised on VTA DA neurons can undergo both long-term potentiation and long-term depression to regulate their synaptic strength and consequently the dopaminergic output (Bonci and Malenka, 1999; Chergui et al., 1994; Overton and Clark, 1997; Overton et al., 1999). DA neurons in the mesocortical system are under greater tonic control of AMPA and NMDA receptors than are the DA cells which innervate the nucleus accumbens (Takahata and Moghaddam, 1998; 2000). Dizocilpine (MK-801) was found to
ip t
have different effects on the mesolimbic and mesocortical dopamine systems (Mathe et al., 1999), which shows heterogeneity of the glutamatergic control over the dopaminergic
cr
pathways. Hence, the effect of glutamate on dopaminergic activity within the VTA in the process of theta rhythm generation is difficult to predict.
us
In the present study we aimed to investigate whether NMDA-mediated glutamatergic transmission within the VTA has any involvement in the regulation of hippocampal electric
an
activity, particularly in the theta band. We used a standard experimental model of sensoryelicited theta rhythm recorded from the hippocampus in urethane-anesthetized rats. This model permits the analysis of the capacity of the hippocampus to generate synchronous
M
activity in strictly controlled conditions and was used in numerous studies (Bland et al., 1995; Kinney et al., 1996; Kirk et al., 1996; Llinas and Alonso, 1992; McNaughton et al., 1995;
ed
Nowacka et al., 2002; Vanderwolf and Baker, 1986). We analysed power, frequency and regularity of hippocampal signal, in the theta and delta bands. Delta rhythm was also analysed, since changes in the delta and theta bands are related: delta becomes
ce pt
desynchronized and supressed when theta rhythm dominates the signal, for example during tail pinch stimulation (Vinogradova, 1995). A preliminary report of a part of the present work was published in an abstract form
Ac
(Matulewicz et al., 2012).
2. Materials and methods 2.1. Animals Experiments were performed on male Wistar rats (n= 17; Tri-City Central Animal Laboratory – Research and Service Centre of the Medical University of Gdansk, Poland) weighing approximately 300 - 350 g. The rats were maintained in a 12-h light-dark cycle (lights on at 6 a.m.), at 22±1ºC and provided with food and water ad libitum. The animals were randomly 4
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divided into experimental groups receiving intrategmental injections of either water, NMDA or MK-801. The experiments were evaluated and approved by the Local Ethical Committee of the Medical University of Gdańsk. 2.2. Surgery Animals were anesthetized with urethane (Urethane, Sigma 1.5 g/kg i.p.) and positioned in the
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frame of a stereotaxic apparatus. The depth of anaesthesia was assessed by monitoring respiratory rate (unpublished study) and maintained at such a level that hind-limb withdrawal reflex is not present and theta rhythm can be elicited by sensory, pharmacological or electrical
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stimulation but does not appear spontaneously. Bilateral recording electrodes were implanted
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in the stratum moleculare of the dorsal blade of the dentate gyrus of the dorsal hippocampus according to the standard stereotaxic procedure. The electrodes were monopolar, made of stainless steel wire, 0.2 mm in diameter, insulated on the entire length except for the flat-cut
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tip. The following Paxinos and Watson (1998) stereotaxic coordinates (skull levelled) were used for implantation: 3.7 mm posterior to bregma, 2.4 mm lateral to midline and 3.1 mm
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ventral to skull surface. Stainless steel screws positioned in the frontal area and over the olfactory bulb served as ground and reference electrodes respectively; they were inserted into holes drilled in the skull and additionally secured with dental acrylic. The olfactory bulb
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seems to be an appropriate reference for theta because of the low-amplitude and unsynchronized patterns of electrical activity present in this area (Chaput et al., 1992; Kay
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and Lazzara, 2010). A small hole was drilled over the VTA to allow subsequent insertion of an injection cannula. Stereotaxic coordinates for the VTA implantation were: 5.1 mm posterior to bregma, 0.9 mm lateral to midline and 8.0 mm ventral to skull surface. As an injection cannula, we used a needle (0.4 mm in diameter, with a flat-cut tip) of 10 l Hamilton
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syringe. The syringe was placed in the stereotaxic holder with a microinfusion pump (Kopf Instruments).
2.3. Experimental design Urethane-anesthetized rats were held in the stereotaxic apparatus throughout the experiment, with body temperature maintained at 37 oC. Local field potential (LFP) was recorded from the hippocampal electrodes during the whole experiment with the use of EEG DigiTrack computer system (ELMIKO, Warsaw, Poland; sampling rate: 240 Hz). The anaesthesia was maintained at the depth that allowed theta to be evoked with sensory stimulation but did not permit spontaneous appearance of this rhythm. Sensory stimulation (tail pinch lasting 60 s) 5
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was applied 3 - 4 times in the preinjection conditions. The animals which did not yield theta rhythm of an amplitude of at least 400 V during control tail pinch stimulations were discarded. After completion of the control recordings, the needle of the Hamilton syringe (injection cannula) was slowly lowered to just above the VTA. A 10-min period of adjustment after the insertion of the cannula was allowed, after which tail pinch was reapplied and if theta was successfully elicited, the intra-VTA injection procedure was started.
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Animals were divided into 3 experimental groups, depending on the drug used for intra-VTA injections (an injection lasted for 3-5 min and was 0.5 µl in volume). Rats from the first group
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(n=7) received unilateral microinjections of NMDA (Sigma-Aldrich, Germany) at a dose of 0.2 g/0.5l and in the second group of rats (n=5) MK-801 (Tocris Bioscience, USA) was
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microinjected at a dose of 3.0 g/0.5l. Hippocampal LFP was recorded continuously for 90 min after the injection and 1-min tail pinch was applied at 9 min intervals throughout the
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recording time. In cases when spontaneous theta rhythm appeared as a result of the intracerebral injection, no tail-pinch was applied until the end of the theta episode. The third, control group (n=5) received an injection of the drugs solvent (Aqua pro injectione,
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Polpharma, Starogard Gdański, Poland) and the hippocampal LFP was recorded for 90 min afterwards. Hippocampal signal in the control group was assessed both in spontaneous and
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tail-pinch conditions, with animals randomly assigned to one of the two protocols: 1) water injection followed by 1.5-hour recording of spontaneous signal, followed by another water
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injection and 1.5-hour recording with 60-s tail pinch applied every 9 min; 2) the opposite order: recording with tail pinch after the first water injection, followed by another injection and recording of only the spontaneous signal. 2.4. Data Analysis
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Spectral analysis of hippocampal LFP (band pass filtered 0–30 Hz), ipsi- and contralateral (in relation to the injection side), was performed off line by fast Fourier transformation (FFT) on 10-14 artefact-free 4.1-s epochs chosen from the 60-s tail pinch fragments applied before and after each drug injection (by SPIKE 2, Cambridge, UK software). In the cases of drug-elicited theta rhythm LFP samples were taken every 5 minutes from the post-injection period. In the water group samples were taken from 60-s fragments of spontaneous LFP signal every 5 min and also from the 60-s tail pinch, and the results were separated into two respective control groups.
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Two parameters of the power spectrum, i.e. the maximal peak power (FFT peak magnitude, Pmax [V2]) and the frequency corresponding to the maximal peak power (FFT peak frequency, Fmax [Hz]), were assessed separately for the following bands: 3.0-6.0 Hz, 6.0-9.0 Hz and 9.0-12.0 Hz (we divided the 3-12 Hz band corresponding to theta activity into 3 subbands to allow more detailed analysis), 0.5-3.0 Hz (delta) and 12.0-15.0 Hz (beta). Pmax and Fmax were submitted to statistical analysis (Two-way ANOVA with groups as factors (control
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and experimental) and time (after drug microinjection) and post hoc Fisher’s LSD test). The time factor in water groups was analysed using one-way ANOVA test (GraphPad Prism 6.02
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software). To eliminate inter-subject variability, power was expressed as a percentage of the pre-injection baseline value taken as 100%, for each frequency band separately. The latency
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and duration of NMDA-induced theta rhythm episodes were measured on the basis of the FFT analysis of the peak power in 4.2-s samples of the signal (previously described in Matulewicz
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et al., 2010). 2.5. Histology
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After completion of the experiment, a small electrolytic lesion (anodal current of 100 A/15 s) was performed through the hippocampal electrodes to visualize the localization of their tips.
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Rats were then treated with an overdose of anaesthetic and intracardially perfused with 0.9% saline followed by a 10% solution of formalin. The brains were removed from the skulls and placed in 10% formalin. After fixation, brains were cut into 60-m slices using frozen tissue
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technique, and the position of the hippocampal electrodes and the VTA cannulas was verified. In a representative rat, 2% saline solution of alcian blue 8GHX (Fluka, Switzerland) was injected into the VTA in a volume of 0.5 µl and diffusion area around the injection cannula was
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assessed under a light microscope. 3. Results
3.1. Hippocampal LFP pattern after administration of NMDA into the VTA In all animals (n=7) intra-VTA injection of NMDA induced robust and long lasting episodes of rhythmic slow activity with a mean latency of 11.8±2.5 min and mean duration of 33.3±6.9 min (mean±SE), with no differences between both hippocampi. Only a few very short episodes of irregular activity interrupted the episodes of the rhythm and together they accounted for 4.6% of the entire NMDA-induced theta episode. Figure 1 shows the course of the experiment in representative animals: power spectra and fragments of hippocampal LFP 7
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traces before and during tail pinch without NMDA and after NMDA administration (left panel) and before and after water administration (right panel). Part A of the spectrogram shows a 5-min sample of hippocampal LFP before manipulations. A 60-sec fragment of this spontaneous signal served as a baseline for calculating control values for subsequent frequency bands; during this preliminary recording we also tested whether theta rhythm appears as a response to tail pinch. This first part of recording lasted for
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20-40 min. Part B depicts a 5-min LFP fragment encompassing theta rhythm that was elicited with 1-min tail pinch. Microinjection procedure followed and lasted 5 min (interval between
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B and C). Part C shows the final seconds of an injection followed by a 60-min post-injection period. After NMDA, activity in the theta band increased and this effect had a latency of ca.
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12 min. This change in activity is evident in the LFP traces and it is also expressed as an increase in FFT power in the 3-6 Hz theta band, which is the dominating theta frequency in
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urethane-anaesthetized rats (Cervera-Ferri et al., 2011; Kiss et al., 2013; Kowalczyk et al., 2014), and as a reduction of power in the delta (0.5–3 Hz) band. Water injection did not cause
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any changes in LFP as compared with the preliminary spontaneous recording (Fig. 1). 3.1.1. Changes of Pmax and Fmax in 3.0-6.0 Hz band
A two-way ANOVA (group effect F(1,142)=148.70, p
