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Hippocampal theta rhythm after local administration of procaine or amphetamine into the ventral tegmental area in fear conditioned rats
Neuroscience Letters 589 (2015) 132–137
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Research article
Hippocampal theta rhythm after local administration of procaine or amphetamine into the ventral tegmental area in fear conditioned rats Paweł Matulewicz ∗ , Jolanta Orzeł-Gryglewska, Łukasz Braszka, Piotr Zawistowski, Edyta Jurkowlaniec Department of Animal and Human Physiology, University of Gda´ nsk, 59 Wita Stwosza St., Gda´ nsk 80-308, Poland
h i g h l i g h t s • VTA injection of procaine suppressed avoidance response in fear conditioned animals. • VTA inactivation affects hippocampal theta rhythm linked with fear conditioned immobility. • Administration of amphetamine has no effect on the behavior and hippocampal LFP.
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Article history: Received 7 November 2014 Received in revised form 17 January 2015 Accepted 19 January 2015 Available online 21 January 2015 Keywords: Hippocampal theta rhythm Ventral tegmental area Freezing Foot-shock Avoidance reaction
a b s t r a c t The ventral tegmental area (VTA) is thought to be an important component in the mesocorticolimbic system involved in the regulation of theta rhythm in the hippocampus. In this study we investigate the effect of pharmacological inactivation (local procaine infusion) or activation (local amphetamine infusion) of the VTA on theta rhythm parameters during task specific behavior in fear conditioned, freely moving rats. Animals were implanted with bilateral recording electrodes into the dorsal hippocampus (CA1) and bilateral injection cannulas into the VTA. Behavioral activities and hippocampal local field potentials (LFP) were recorded throughout the experiment, in pre- and post-injection conditions. We found that intra-VTA injection of procaine temporarily suppressed fear conditioned avoidance response (escape from the foot-shock arena) and also influenced hippocampal theta rhythm parameters during immobility linked with arousal and/or attention. Procaine infusion decreased the signal power (Pmax ) of theta rhythm during immobility behavior, in comparison to the control group (water infusion), whereas administration of amphetamine had no effect on the behavior and hippocampal LFP. Our results indicate that temporal inactivation of neuronal activity in the VTA affects hippocampal theta rhythm linked with attentional immobility and suppresses avoidance response in fear conditioned animals. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The theta activity is the one of the most regular rhythms of the brain and can be easily recorded in rats, rabbits, cats, and other small mammals. Depending on behavioral conditions and species, theta frequency can range from 3 to 12 Hz [1,2]. There are two types of hippocampal theta rhythm in freely moving rats, distinguished on the basis of differences in the characteristics of EEG frequency and the type of accompanying behavioral activity. Type I (translation movement-theta) occurs in awake animals in a frequency range between 6 and 12 Hz (or 8–14 Hz). In the experimental con-
∗ Corresponding author. Tel.: +48 58 523 6132. E-mail address: [email protected] (P. Matulewicz). http://dx.doi.org/10.1016/j.neulet.2015.01.049 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
ditions episodes of type I theta can be recorded in rats during spontaneous locomotion or induced locomotor responses [3–7]. This type is thought to be related with the serotonergic system activity [8]. Type II (attentional-theta) is recorded in awake animals during immobility, as well as during paradoxical sleep episodes, and its frequency range is between 3 and 9 Hz (or 4–8 Hz). This type is thought to be related with the activity of cholinergic system [1,2,9]. In awake animals, the theta rhythm is also recorded during freezing behavior induced by an aversive conditioning procedure [10,11]. The two components of theta, present during translational movements or arousal, are not mutually incompatible and their main frequency ranges partially overlap. The theta rhythm synchronization system is composed of different brain structures forming together the brainstemdiencephalo-septohippocampal system [12–15]. The midbrain
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ventral tegmental area (VTA), composed mainly of dopaminergic cells forming the A10 group [16–18], is also believed to be involved in the processes of theta regulation or modulation. Our previous studies have shown that unilateral ibotenate lesions of the VTA in freely moving rats, temporary pharmacological inactivation or permanent lesion by electrocoagulation of the VTA in urethanized rats is capable of reducing the power of hippocampal theta activity [19,20]. On the other hand, electrical stimulation of the VTA induces a regular theta rhythm in hippocampal EEG [21]. Local regulatory mechanisms within the VTA were assessed with a microinjection of amphetamine, a dopaminergic agonist of indirect activity, which enhances dopamine release from presynaptic endings and blocks its reuptake and metabolism [22,23]. Intra-VTA amphetamine administration in uretanized rats elicited theta rhythm [24]. Available evidence suggests a role of VTA-hippocampus pathways in mediating locomotor activity, learning and hippocampal-dependent memory processes [25,26]. Lisman and Otmakhova [27] also suggest that the functional VTAhippocampal loop is involved in detection of novelty and in incorporation of novel information depending on the hippocampal processing. The present study aimed to examine whether temporal pharmacological inactivation (procaine) or activation (amphetamine) of the VTA would affect the hippocampal theta rhythm induced during task specific behavior (attentional immobility) as well as avoidance response in fear conditioned rats. 2. Materials and method 2.1. Animals All experiments were performed on male, adult (4–6 months old) Wistar rats (345 ± 16.7 g body weight), carried out in compliance with the guidelines of the European Communities Council Directive (2010/63/UE) and approved by the Local Ethics Committee in Gdansk. Animals were kept in separate cages (after surgery), in conditions of regular light–dark cycles (12 h day/12 h night), constant temperature (22◦ C), and with access to food (standard pellets) and water ad libitum. 2.2. Stereotaxic implantation The surgery was performed under isoflurane (1–2.5% in oxygen; Aerrane; Baxter, UK) anesthesia (combi-vet system, Rothacher Medical, Switzerland) and butorphanol analgesia (1.5 mg/300 g b.w.; Butomidor; Richter Pharma AG, Austria), Rats were implanted with the use of stereotaxic frame (Kopf, USA) with bilateral hippocampal (AP: −3.7, L: ±2.4, D: −3.1 mm) recording electrodes (125 m diameter, teflon-coated twisted stainless steel wire (SS5T; Science Products, Germany), and bilaterally with standard pedestal guides for infusions (PlasticsOne, USA) to the VTA (AP: −5.1, L: ±0.9, D: −8.1 mm). A silver wire connected to a screw mounted anteriorly to bregma was used as a ground/reference electrode. Electrodes were connected to a 6-pin connector (MillMax, USA) and cemented to the skull with dental acrylic. 2.3. Conditioning procedure A setup for fear conditioning and EEG recordings in freely moving animals was assembled (PC with the SPIKE-2 software (CED, UK), a signal converter unit – MICRO-1410-3 (CED, UK), a commutator (Crist Instruments, USA), signal amplifier (AM Systems, USA) and an electrical stimulator (model A320, WPI, USA) combined with two Plexiglas cages (40 × 40 × 40 cm) connected with a 100 cm long corridor). The floor of one of the cage was made of metal grid (rods
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equally spaced every 1.2 cm) connected to a discontinuous shock source. Conditioning procedures were performed after one week of recovery and consisted of three phases: I – arena familiarization before conditioning (1–2 days); II – conditioning phase I: when rats were put in the shock box and were exposed to 6–8 pairings (separated by 2-min blocks) of an acoustic stimulus (tone of 70 dB, duration 1 s) and a 1-s electrical foot-shock 7 s later (duration 0.1 s pulse rate 0.50–0.60 mA). The tone was associated with the subsequent aversive stimulus, causing freezing, which lasted from the sound stimulus until the electric shock; III – conditioning phase II: when the gate to the corridor was manually opened by the experimentator 5 s after the tone (but before the foot-shock), and rats were allowed to escape from the aversive stimulus box to the neutral box. This procedure allowed us to repeatedly induce both: attentional-theta rhythm episode during the immobility after the acoustic tone (when the rats were waiting for the gate to be open) and active avoidance response (escape to the neutral box to avoid the foot-shock) (Fig. 1(I)). 2.4. Experimental protocol Acquisition of hippocampal local field potentials (LFP – through a JFET preamplifier, filtered 0.1–1000 Hz, amplified × 1000, and digitized at 4 kHz), as well as animals’ behavior data (video camera recording), was carried out through the whole duration of the experiment. In baseline conditions, animals spent 20 min in the neutral box, and then were placed in the aversive stimulus box for 1 min, followed by presentation of the acoustic tone (pre-injection control of immobility and avoidance response). The animals which performed (I) clear and stable immobility after the tone presentation, as well as (II) active locomotion after opening of the gate (escape) were then bilaterally infused into the VTA (0.5 l volume/side, infusion lasted for 1 min) with 20% solution of procaine (N = 5), 10 g amphetamine (N = 6) or water (drug vehicle, N = 6). After the drug infusion rats were placed in the neutral box. Then every 4 min (during the first 20 min after procaine or water infusion) or 9 min (after amphetamine infusion) till the end of the experiment (60 min after the infusion) the rats were placed in the foot-shock box for 1 min, followed by the presentation of acoustic tone and gate opening (and foot-shock in case of no escape from the shock box) (Fig. 1(I)). Each animal received intracerebral infusion of drugs in a randomised order, with at least 3 days gap between each experiment. After completion of the experiment, electrolytic small lesions (an anodal current of 100 A/15 s) were performed through the hippocampal electrodes to mark the tips locations, and an intraVTA injection of alcian blue dye was made to assess the location of the injection cannulas tips. Brain sections were cut using frozen tissue technique and the positions of the recording electrodes and injection cannulas were verified. 2.5. Statistical analysis Signal analysis was performed off-line with SPIKE 2 and GraphPad Prism 6 (GraphPad Software, USA) software, on artifact-free fragments of hippocampal LFP. Fast Fourier transformation (FFT) was calculated for 5-s samples, chosen from the hippocampal LFP recordings obtained during immobility induced by presentation of the acoustic tone in the foot-shock box, in pre- and post-injection conditions. The maximal peak power (FFT peak magnitude, Pmax ) was analyzed in five frequency bands (0.1–3, 3–6, 6–9, 9–12 and 12–15 Hz). To eliminate inter-subject variability, Pmax was expressed as a percentage of the pre-injection baseline value (from 5 s attentional immobility after the tone presentation) taken as 100%, for each frequency
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Fig. 1. (I) A diagram of procedures undertaken on each animal and experimental protocol with the accompanying behavior of rats in the foot-shock box (in pre-and postinjection conditions). (II) Hippocampal local field potential with corresponding FFT power spectra in a representative rat during the experiment. A – neutral box: quiet awake with no locomotor activity. B – neutral box: active locomotion. C – foot-shock box: quiet awake with no locomotor activity. D – foot-shock box: bS – quiet awake with no locomotion before the sound (S) stimulus (1 s, dotted line); aS – immobility after the stimulus, just before the gate opening (arrow).
band separately (Pmax ) and submitted to Two-way ANOVA analysis with groups as factors (control and experimental) and time (after drug microinjection). The within-group differences of Pmax between pre- and post-injection conditions, as well as possible differences between procaine and amphetamine effect on the latency of the escape reaction in comparison to the to the control water group were statistically analyzed with One-way analysis of variance (ANOVA).
3. Results Histological verification showed that tips of the recording electrodes were localized in the dorsal hippocampus (i.e., CA1 region) in sections between 3.6 and 4.2 mm posterior to bregma. The tips of injection cannulas were located in the anterior part of the VTA, from 4.9 to 5.2 mm posterior to bregma according to the Rat Brain Atlas of Paxinos and Watson [28].
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Fig. 2. (A) The effect of procaine, amphetamine or water microinjection into the VTA on the latency of avoidance reaction (escape from the foot-shock cage) in pre- (control) and post-injection conditions (if a particular rat’s response time (escape) was longer than 60 s or the animal had not escaped at all, the latency was scored as 60 s). Explanations: control – pre-injection conditions, arrow – end of the injection; asterisks, significant changes after procaine (*p < 0.05; ***p < 0.001, mean ± SE, post hoc Bonferroni test). (B) The effect of procaine, amphetamine or water microinjection into the VTA on the Pmax during 5 s immobility after the tone presentation, expressed as a percentage of the pre-injection baseline value taken as 100% (%), for 3–6 Hz and other frequency bands (C: 0.1–3; D: 6–9, E: 9–12 and F: 12–15 Hz) separately. Explanations: control – pre-injection conditions, arrow – end of the injection; asterisks, significant changes after procaine (*p < 0.05; **p < 0.01; ***p < 0.001) or amphetamine (##p < 0.01: #p < 0.05) microinjection (mean ± SE, post hoc Fisher’s LSD test).
Hippocampal LFP recordings showed that in all analyzed rats peak power in the theta frequency band (3–12 Hz) was present during active exploration of the experimental arena as well as during immobility after the tone presentation (linked with arousal and/or attention of an animal – Fig. 1(II)). 3.1. The effect of VTA injections on avoidance response The effect of intra-VTA procaine injection on the avoidance reaction (in comparison to pre-injection conditions) was manifested in significant slowdown, or even lack of, avoidance response (latency longer than 60 s), in the first 2/3 sessions of acoustic tone presentation in the foot-shock box (Fig. 2(A)). One-way ANOVA with Bonferroni’s multiple comparisons test revealed a significant difference in the latency of escape reaction for the procaine group (F(17.72) = 5.574, p < 0.0001) in the 5th and 10th min after the infusion, but no effect for the amphetamine (F(13.65) = 0.8556, p = 0.6019) in comparison to the control water group. 3.2. Pmax of hippocampal LFP in theta frequency band (3–6, 6–9 and 9–12 Hz) after procaine administration In the 3–6 Hz frequency band, two-way ANOVA (group effect F(1.196) = 83.50, p < 0.0001; time effect F(8.196) = 0.73, p = 0.6689;
and interaction F(8.196) = 2.96, p = 0.0038) revealed a significant decrease in Pmax value during the tone induced immobility in the foot-shock box from 5th till 60th min post injection, except for the 50th min (p = 0.1182) (Fig. 2(B)). In the 6–9 Hz frequency, two-way ANOVA (group effect F(1.204) = 1.99, p = 0.1602; time effect F(8.204) = 1.06, p = 0.3946; and interaction F(8.204) = 1.41, p = 0.1933) revealed no significant change in Pmax value, except for the 5th min post injection (p = 0.0121) (Fig. 2(D)). In the 9–12 Hz frequency band, two-way ANOVA (group effect F(1.195) = 13.48, p = 0.0003; time effect F(8.195) = 0.44, p = 0.8961; and interaction F(8.195) = 3.26, p = 0.0016) revealed significant decrease in Pmax value in the 5th (p = 0.0012), 10th (p = 0.0018) and 40th (p = 0.0022) min post-injection (Fig. 2(E)), all in comparison to the control water group (post hoc Fisher’s LSD test). 3.3. Pmax of hippocampal LFP in theta frequency band (3–6, 6–9 and 9–12 Hz) after amphetamine administration Local amphetamine infusion had no influence on Pmax value in 3–6, 6–9 and 9–12 Hz frequency bands during immobility after acoustic signal presentation. Statistical data (Two-way ANOVA) are as follows: 3–6 Hz: group effect F(1.153) = 0.05, p = 0.8251; time effect F(6.153) = 1.22, p = 0.3004; and interaction
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F(6.153) = 0.24, p = 0.9610), Fig. 2(B); 6–9 Hz: group effect F(1.162) = 1.49, p = 0.2239; time effect F(6.162) = 0.66, p = 0.6807; and interaction F(6.162) = 0.46, p = 0.8344), Fig. 2(D); 9–12 Hz group: F(1.146) = 1.20, p = 0.2747; time effect F(6.146) = 0.36, p = 0.9035; and interaction F(6.146) = 0.31, p = 0.9287), Fig. 2(E); all in comparison to the control water group. We performed additional analysis to verify whether the observed decrease in theta power after procaine infusion is specific to tone induced immobility or appeared in relation to overall suppression of signal power in theta frequency band. We found that hippocampal theta during the exploration of the foot-shock box (with translational movements) in the pre-exposure conditions (1min LFP recording just before acoustic signal presentation) was not affected by administration of either procaine or amphetamine (data not shown). Two-way ANOVA (with post hoc Fisher’s LSD test) revealed no major change in Pmax value during the tone induced immobility in the 0.1–3 Hz (except for the 10th min after the procaine injection (p = 0.0049) and for the 30th min after the amphetamine injection (p = 0.0097), when significant changes of Pmax occurred, Fig. 2(C)) and 12–15 Hz frequency band after drugs injection, in comparison to the control water group (Fig. 2(E)).
4. Discussion In this study we observed that temporary pharmacological inactivation of the VTA, by local procaine administration, resulted in suppression of fear conditioned avoidance response and clear decrease in the power of the theta rhythm (Pmax ) during the conditioned fear response (immobility linked with arousal and/or attention). No such effects occurred after VTA activation by local amphetamine infusion. As documented in the literature, the predominant brain electrical activity in the hippocampus during active exploration is the theta rhythm [1,6]. This finding was corroborated also by Machado et al. [29], in their investigations on the relationship between hippocampal rhythms and characterization of recent and remote aversive memories in rats, based on the theta/delta frequency bands ratio. They showed that peak power in the theta frequency band (4–10 Hz) was present during active exploration as well as during freezing reaction. Seidenbecher et al. [42] also indicated that elicitation of conditioned freezing behavior in mice is associated with type II theta activity. Similar effect was also observed in our study (Fig. 1(II)). Studies in animals indicate strong functional link between dopaminergic system and memory processes in the hippocampus. Dopamine acting at hippocampal synapses is a necessary precursor not only of long-term potentiation (LTP), a prime cellular model of learning and memory [30,31], but also of the behavioral persistence of long-term memories [32,33]. Importantly, as shown in models of episodic memory, dopamine-dependent facilitation of neural plasticity is evident even after a single event [34]. It has been reported that the hippocampus and VTA form a neural loop in which dopamine released from the VTA enhances hippocampal LTP [26] and is critical for consolidation and acquisition of novel information [35]. The loss of VTA dopaminergic neurons impairs memory acquisition in the Morris water maze task and prevents rats from transferring information to the next test day [36]. In addition to connections with the hippocampus, the VTA also sends dopaminergic projections to the prefrontal cortex (PFC) [37,38] and may alter neural activity in the PFC and neural transmission between the hippocampus and PFC, which is necessary for LTP to occur in the hippocampal–PFC pathway during memory processing [39]. Apart from mediating LTP induction in the hippocampus and PFC, the VTA also affects hippocampal theta activity. Our studies on VTA lesions
also strongly indicates that this brain area is an important part of the theta synchronizing system [19,20] and the projection, through which the VTA enhances theta rhythm, might be indirect and incorporated into the main route of theta generation which involves the septum and diagonal band of Broca [21,40]. It should be also noted that dopamine release in the hippocampus is itself modulated by hippocampal activity: outputs from the hippocampus facilitate dopaminergic signaling in the midbrain, which in turn can enhance hippocampal plasticity via dopamine release [26]. The report from Moaddab et al. [41], in which authors observed no significant effect on general locomotor activity after lidocaine reversible inactivation of the VTA, gives us strong evidence that the prolonged latency of the avoidance response observed in our study is not caused by post-injection impairment of motor function. In our experiments, procaine was infused to the VTA only in those animals in which the previously learned behavior linked with tone induced immobility and avoidance response (escape from the foot-shock box) was undisturbed during control trials performed 5–10 min prior to each infusion. Therefore, we assumed that the observed prolonged escape latency after procaine infusion can be probably more related to the motivational processes rather than to disturbed memory recall (animals were allowed to recall previously learned behavior just before each infusion). As showed by Seidenbecher et al. [42] the retrieval and/or expression of conditioned fear may be functionally related to theta synchronization in amygdalohippocampal pathways, therefore it should be also considered that temporal inactivation of the VTA might also disrupted this processes, what in turn affected avoidance response of animal. It can be assumed that intra-VTA procaine infusion led to temporal decrease in dopaminergic mesolimbic transmission, which in turn led to dysregulation of hippocampal activity by direct or indirect pathway. The infusion could have also influenced cortical, amygdalohippocampal pathways and limbic loops that are involved in the motivation, attention and memory processes. 5. Conclusion Overall, our findings indicate that the VTA inactivation leads to a disruption in the hippocampal theta rhythm during fear conditioned immobility linked with arousal and/or attention, which occurs in parallel with suppression of avoidance response. Pharmacological activation of the VTA (amphetamine) had no effect on theta rhythm or avoidance reaction parameters. Conflicts of interest The authors declare no conflicts of interest. Acknowledgments This research was supported from the Polish Ministry of Science and Higher Education (Iuventus Plus grant, IP2011 034371). The Authors would like to thank Magda Ku´smierczak, Ph.D., for linguistic revision and for inspiring comments on an earlier version of the manuscript. References [1] C.H. Vanderwolf, Hippocampal electrical activity and voluntary movement in the rat, Electroencephalogr. Clin. Neurophysiol. 26 (1969) 407–418. [2] R. Kramis, C.H. Vanderwolf, B.H. Bland, Two types of hippocampal rhythmical slow activity in both the rabbit and the rat: relations to behavior and effects of atropine, diethyl ether, urethane and pentobarbital, Exp. Neurol. 49 (1975) 58–85.
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Research article
Hippocampal theta rhythm after local administration of procaine or amphetamine into the ventral tegmental area in fear conditioned rats Paweł Matulewicz ∗ , Jolanta Orzeł-Gryglewska, Łukasz Braszka, Piotr Zawistowski, Edyta Jurkowlaniec Department of Animal and Human Physiology, University of Gda´ nsk, 59 Wita Stwosza St., Gda´ nsk 80-308, Poland
h i g h l i g h t s • VTA injection of procaine suppressed avoidance response in fear conditioned animals. • VTA inactivation affects hippocampal theta rhythm linked with fear conditioned immobility. • Administration of amphetamine has no effect on the behavior and hippocampal LFP.
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Article history: Received 7 November 2014 Received in revised form 17 January 2015 Accepted 19 January 2015 Available online 21 January 2015 Keywords: Hippocampal theta rhythm Ventral tegmental area Freezing Foot-shock Avoidance reaction
a b s t r a c t The ventral tegmental area (VTA) is thought to be an important component in the mesocorticolimbic system involved in the regulation of theta rhythm in the hippocampus. In this study we investigate the effect of pharmacological inactivation (local procaine infusion) or activation (local amphetamine infusion) of the VTA on theta rhythm parameters during task specific behavior in fear conditioned, freely moving rats. Animals were implanted with bilateral recording electrodes into the dorsal hippocampus (CA1) and bilateral injection cannulas into the VTA. Behavioral activities and hippocampal local field potentials (LFP) were recorded throughout the experiment, in pre- and post-injection conditions. We found that intra-VTA injection of procaine temporarily suppressed fear conditioned avoidance response (escape from the foot-shock arena) and also influenced hippocampal theta rhythm parameters during immobility linked with arousal and/or attention. Procaine infusion decreased the signal power (Pmax ) of theta rhythm during immobility behavior, in comparison to the control group (water infusion), whereas administration of amphetamine had no effect on the behavior and hippocampal LFP. Our results indicate that temporal inactivation of neuronal activity in the VTA affects hippocampal theta rhythm linked with attentional immobility and suppresses avoidance response in fear conditioned animals. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The theta activity is the one of the most regular rhythms of the brain and can be easily recorded in rats, rabbits, cats, and other small mammals. Depending on behavioral conditions and species, theta frequency can range from 3 to 12 Hz [1,2]. There are two types of hippocampal theta rhythm in freely moving rats, distinguished on the basis of differences in the characteristics of EEG frequency and the type of accompanying behavioral activity. Type I (translation movement-theta) occurs in awake animals in a frequency range between 6 and 12 Hz (or 8–14 Hz). In the experimental con-
∗ Corresponding author. Tel.: +48 58 523 6132. E-mail address: [email protected] (P. Matulewicz). http://dx.doi.org/10.1016/j.neulet.2015.01.049 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
ditions episodes of type I theta can be recorded in rats during spontaneous locomotion or induced locomotor responses [3–7]. This type is thought to be related with the serotonergic system activity [8]. Type II (attentional-theta) is recorded in awake animals during immobility, as well as during paradoxical sleep episodes, and its frequency range is between 3 and 9 Hz (or 4–8 Hz). This type is thought to be related with the activity of cholinergic system [1,2,9]. In awake animals, the theta rhythm is also recorded during freezing behavior induced by an aversive conditioning procedure [10,11]. The two components of theta, present during translational movements or arousal, are not mutually incompatible and their main frequency ranges partially overlap. The theta rhythm synchronization system is composed of different brain structures forming together the brainstemdiencephalo-septohippocampal system [12–15]. The midbrain
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ventral tegmental area (VTA), composed mainly of dopaminergic cells forming the A10 group [16–18], is also believed to be involved in the processes of theta regulation or modulation. Our previous studies have shown that unilateral ibotenate lesions of the VTA in freely moving rats, temporary pharmacological inactivation or permanent lesion by electrocoagulation of the VTA in urethanized rats is capable of reducing the power of hippocampal theta activity [19,20]. On the other hand, electrical stimulation of the VTA induces a regular theta rhythm in hippocampal EEG [21]. Local regulatory mechanisms within the VTA were assessed with a microinjection of amphetamine, a dopaminergic agonist of indirect activity, which enhances dopamine release from presynaptic endings and blocks its reuptake and metabolism [22,23]. Intra-VTA amphetamine administration in uretanized rats elicited theta rhythm [24]. Available evidence suggests a role of VTA-hippocampus pathways in mediating locomotor activity, learning and hippocampal-dependent memory processes [25,26]. Lisman and Otmakhova [27] also suggest that the functional VTAhippocampal loop is involved in detection of novelty and in incorporation of novel information depending on the hippocampal processing. The present study aimed to examine whether temporal pharmacological inactivation (procaine) or activation (amphetamine) of the VTA would affect the hippocampal theta rhythm induced during task specific behavior (attentional immobility) as well as avoidance response in fear conditioned rats. 2. Materials and method 2.1. Animals All experiments were performed on male, adult (4–6 months old) Wistar rats (345 ± 16.7 g body weight), carried out in compliance with the guidelines of the European Communities Council Directive (2010/63/UE) and approved by the Local Ethics Committee in Gdansk. Animals were kept in separate cages (after surgery), in conditions of regular light–dark cycles (12 h day/12 h night), constant temperature (22◦ C), and with access to food (standard pellets) and water ad libitum. 2.2. Stereotaxic implantation The surgery was performed under isoflurane (1–2.5% in oxygen; Aerrane; Baxter, UK) anesthesia (combi-vet system, Rothacher Medical, Switzerland) and butorphanol analgesia (1.5 mg/300 g b.w.; Butomidor; Richter Pharma AG, Austria), Rats were implanted with the use of stereotaxic frame (Kopf, USA) with bilateral hippocampal (AP: −3.7, L: ±2.4, D: −3.1 mm) recording electrodes (125 m diameter, teflon-coated twisted stainless steel wire (SS5T; Science Products, Germany), and bilaterally with standard pedestal guides for infusions (PlasticsOne, USA) to the VTA (AP: −5.1, L: ±0.9, D: −8.1 mm). A silver wire connected to a screw mounted anteriorly to bregma was used as a ground/reference electrode. Electrodes were connected to a 6-pin connector (MillMax, USA) and cemented to the skull with dental acrylic. 2.3. Conditioning procedure A setup for fear conditioning and EEG recordings in freely moving animals was assembled (PC with the SPIKE-2 software (CED, UK), a signal converter unit – MICRO-1410-3 (CED, UK), a commutator (Crist Instruments, USA), signal amplifier (AM Systems, USA) and an electrical stimulator (model A320, WPI, USA) combined with two Plexiglas cages (40 × 40 × 40 cm) connected with a 100 cm long corridor). The floor of one of the cage was made of metal grid (rods
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equally spaced every 1.2 cm) connected to a discontinuous shock source. Conditioning procedures were performed after one week of recovery and consisted of three phases: I – arena familiarization before conditioning (1–2 days); II – conditioning phase I: when rats were put in the shock box and were exposed to 6–8 pairings (separated by 2-min blocks) of an acoustic stimulus (tone of 70 dB, duration 1 s) and a 1-s electrical foot-shock 7 s later (duration 0.1 s pulse rate 0.50–0.60 mA). The tone was associated with the subsequent aversive stimulus, causing freezing, which lasted from the sound stimulus until the electric shock; III – conditioning phase II: when the gate to the corridor was manually opened by the experimentator 5 s after the tone (but before the foot-shock), and rats were allowed to escape from the aversive stimulus box to the neutral box. This procedure allowed us to repeatedly induce both: attentional-theta rhythm episode during the immobility after the acoustic tone (when the rats were waiting for the gate to be open) and active avoidance response (escape to the neutral box to avoid the foot-shock) (Fig. 1(I)). 2.4. Experimental protocol Acquisition of hippocampal local field potentials (LFP – through a JFET preamplifier, filtered 0.1–1000 Hz, amplified × 1000, and digitized at 4 kHz), as well as animals’ behavior data (video camera recording), was carried out through the whole duration of the experiment. In baseline conditions, animals spent 20 min in the neutral box, and then were placed in the aversive stimulus box for 1 min, followed by presentation of the acoustic tone (pre-injection control of immobility and avoidance response). The animals which performed (I) clear and stable immobility after the tone presentation, as well as (II) active locomotion after opening of the gate (escape) were then bilaterally infused into the VTA (0.5 l volume/side, infusion lasted for 1 min) with 20% solution of procaine (N = 5), 10 g amphetamine (N = 6) or water (drug vehicle, N = 6). After the drug infusion rats were placed in the neutral box. Then every 4 min (during the first 20 min after procaine or water infusion) or 9 min (after amphetamine infusion) till the end of the experiment (60 min after the infusion) the rats were placed in the foot-shock box for 1 min, followed by the presentation of acoustic tone and gate opening (and foot-shock in case of no escape from the shock box) (Fig. 1(I)). Each animal received intracerebral infusion of drugs in a randomised order, with at least 3 days gap between each experiment. After completion of the experiment, electrolytic small lesions (an anodal current of 100 A/15 s) were performed through the hippocampal electrodes to mark the tips locations, and an intraVTA injection of alcian blue dye was made to assess the location of the injection cannulas tips. Brain sections were cut using frozen tissue technique and the positions of the recording electrodes and injection cannulas were verified. 2.5. Statistical analysis Signal analysis was performed off-line with SPIKE 2 and GraphPad Prism 6 (GraphPad Software, USA) software, on artifact-free fragments of hippocampal LFP. Fast Fourier transformation (FFT) was calculated for 5-s samples, chosen from the hippocampal LFP recordings obtained during immobility induced by presentation of the acoustic tone in the foot-shock box, in pre- and post-injection conditions. The maximal peak power (FFT peak magnitude, Pmax ) was analyzed in five frequency bands (0.1–3, 3–6, 6–9, 9–12 and 12–15 Hz). To eliminate inter-subject variability, Pmax was expressed as a percentage of the pre-injection baseline value (from 5 s attentional immobility after the tone presentation) taken as 100%, for each frequency
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Fig. 1. (I) A diagram of procedures undertaken on each animal and experimental protocol with the accompanying behavior of rats in the foot-shock box (in pre-and postinjection conditions). (II) Hippocampal local field potential with corresponding FFT power spectra in a representative rat during the experiment. A – neutral box: quiet awake with no locomotor activity. B – neutral box: active locomotion. C – foot-shock box: quiet awake with no locomotor activity. D – foot-shock box: bS – quiet awake with no locomotion before the sound (S) stimulus (1 s, dotted line); aS – immobility after the stimulus, just before the gate opening (arrow).
band separately (Pmax ) and submitted to Two-way ANOVA analysis with groups as factors (control and experimental) and time (after drug microinjection). The within-group differences of Pmax between pre- and post-injection conditions, as well as possible differences between procaine and amphetamine effect on the latency of the escape reaction in comparison to the to the control water group were statistically analyzed with One-way analysis of variance (ANOVA).
3. Results Histological verification showed that tips of the recording electrodes were localized in the dorsal hippocampus (i.e., CA1 region) in sections between 3.6 and 4.2 mm posterior to bregma. The tips of injection cannulas were located in the anterior part of the VTA, from 4.9 to 5.2 mm posterior to bregma according to the Rat Brain Atlas of Paxinos and Watson [28].
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Fig. 2. (A) The effect of procaine, amphetamine or water microinjection into the VTA on the latency of avoidance reaction (escape from the foot-shock cage) in pre- (control) and post-injection conditions (if a particular rat’s response time (escape) was longer than 60 s or the animal had not escaped at all, the latency was scored as 60 s). Explanations: control – pre-injection conditions, arrow – end of the injection; asterisks, significant changes after procaine (*p < 0.05; ***p < 0.001, mean ± SE, post hoc Bonferroni test). (B) The effect of procaine, amphetamine or water microinjection into the VTA on the Pmax during 5 s immobility after the tone presentation, expressed as a percentage of the pre-injection baseline value taken as 100% (%), for 3–6 Hz and other frequency bands (C: 0.1–3; D: 6–9, E: 9–12 and F: 12–15 Hz) separately. Explanations: control – pre-injection conditions, arrow – end of the injection; asterisks, significant changes after procaine (*p < 0.05; **p < 0.01; ***p < 0.001) or amphetamine (##p < 0.01: #p < 0.05) microinjection (mean ± SE, post hoc Fisher’s LSD test).
Hippocampal LFP recordings showed that in all analyzed rats peak power in the theta frequency band (3–12 Hz) was present during active exploration of the experimental arena as well as during immobility after the tone presentation (linked with arousal and/or attention of an animal – Fig. 1(II)). 3.1. The effect of VTA injections on avoidance response The effect of intra-VTA procaine injection on the avoidance reaction (in comparison to pre-injection conditions) was manifested in significant slowdown, or even lack of, avoidance response (latency longer than 60 s), in the first 2/3 sessions of acoustic tone presentation in the foot-shock box (Fig. 2(A)). One-way ANOVA with Bonferroni’s multiple comparisons test revealed a significant difference in the latency of escape reaction for the procaine group (F(17.72) = 5.574, p < 0.0001) in the 5th and 10th min after the infusion, but no effect for the amphetamine (F(13.65) = 0.8556, p = 0.6019) in comparison to the control water group. 3.2. Pmax of hippocampal LFP in theta frequency band (3–6, 6–9 and 9–12 Hz) after procaine administration In the 3–6 Hz frequency band, two-way ANOVA (group effect F(1.196) = 83.50, p < 0.0001; time effect F(8.196) = 0.73, p = 0.6689;
and interaction F(8.196) = 2.96, p = 0.0038) revealed a significant decrease in Pmax value during the tone induced immobility in the foot-shock box from 5th till 60th min post injection, except for the 50th min (p = 0.1182) (Fig. 2(B)). In the 6–9 Hz frequency, two-way ANOVA (group effect F(1.204) = 1.99, p = 0.1602; time effect F(8.204) = 1.06, p = 0.3946; and interaction F(8.204) = 1.41, p = 0.1933) revealed no significant change in Pmax value, except for the 5th min post injection (p = 0.0121) (Fig. 2(D)). In the 9–12 Hz frequency band, two-way ANOVA (group effect F(1.195) = 13.48, p = 0.0003; time effect F(8.195) = 0.44, p = 0.8961; and interaction F(8.195) = 3.26, p = 0.0016) revealed significant decrease in Pmax value in the 5th (p = 0.0012), 10th (p = 0.0018) and 40th (p = 0.0022) min post-injection (Fig. 2(E)), all in comparison to the control water group (post hoc Fisher’s LSD test). 3.3. Pmax of hippocampal LFP in theta frequency band (3–6, 6–9 and 9–12 Hz) after amphetamine administration Local amphetamine infusion had no influence on Pmax value in 3–6, 6–9 and 9–12 Hz frequency bands during immobility after acoustic signal presentation. Statistical data (Two-way ANOVA) are as follows: 3–6 Hz: group effect F(1.153) = 0.05, p = 0.8251; time effect F(6.153) = 1.22, p = 0.3004; and interaction
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F(6.153) = 0.24, p = 0.9610), Fig. 2(B); 6–9 Hz: group effect F(1.162) = 1.49, p = 0.2239; time effect F(6.162) = 0.66, p = 0.6807; and interaction F(6.162) = 0.46, p = 0.8344), Fig. 2(D); 9–12 Hz group: F(1.146) = 1.20, p = 0.2747; time effect F(6.146) = 0.36, p = 0.9035; and interaction F(6.146) = 0.31, p = 0.9287), Fig. 2(E); all in comparison to the control water group. We performed additional analysis to verify whether the observed decrease in theta power after procaine infusion is specific to tone induced immobility or appeared in relation to overall suppression of signal power in theta frequency band. We found that hippocampal theta during the exploration of the foot-shock box (with translational movements) in the pre-exposure conditions (1min LFP recording just before acoustic signal presentation) was not affected by administration of either procaine or amphetamine (data not shown). Two-way ANOVA (with post hoc Fisher’s LSD test) revealed no major change in Pmax value during the tone induced immobility in the 0.1–3 Hz (except for the 10th min after the procaine injection (p = 0.0049) and for the 30th min after the amphetamine injection (p = 0.0097), when significant changes of Pmax occurred, Fig. 2(C)) and 12–15 Hz frequency band after drugs injection, in comparison to the control water group (Fig. 2(E)).
4. Discussion In this study we observed that temporary pharmacological inactivation of the VTA, by local procaine administration, resulted in suppression of fear conditioned avoidance response and clear decrease in the power of the theta rhythm (Pmax ) during the conditioned fear response (immobility linked with arousal and/or attention). No such effects occurred after VTA activation by local amphetamine infusion. As documented in the literature, the predominant brain electrical activity in the hippocampus during active exploration is the theta rhythm [1,6]. This finding was corroborated also by Machado et al. [29], in their investigations on the relationship between hippocampal rhythms and characterization of recent and remote aversive memories in rats, based on the theta/delta frequency bands ratio. They showed that peak power in the theta frequency band (4–10 Hz) was present during active exploration as well as during freezing reaction. Seidenbecher et al. [42] also indicated that elicitation of conditioned freezing behavior in mice is associated with type II theta activity. Similar effect was also observed in our study (Fig. 1(II)). Studies in animals indicate strong functional link between dopaminergic system and memory processes in the hippocampus. Dopamine acting at hippocampal synapses is a necessary precursor not only of long-term potentiation (LTP), a prime cellular model of learning and memory [30,31], but also of the behavioral persistence of long-term memories [32,33]. Importantly, as shown in models of episodic memory, dopamine-dependent facilitation of neural plasticity is evident even after a single event [34]. It has been reported that the hippocampus and VTA form a neural loop in which dopamine released from the VTA enhances hippocampal LTP [26] and is critical for consolidation and acquisition of novel information [35]. The loss of VTA dopaminergic neurons impairs memory acquisition in the Morris water maze task and prevents rats from transferring information to the next test day [36]. In addition to connections with the hippocampus, the VTA also sends dopaminergic projections to the prefrontal cortex (PFC) [37,38] and may alter neural activity in the PFC and neural transmission between the hippocampus and PFC, which is necessary for LTP to occur in the hippocampal–PFC pathway during memory processing [39]. Apart from mediating LTP induction in the hippocampus and PFC, the VTA also affects hippocampal theta activity. Our studies on VTA lesions
also strongly indicates that this brain area is an important part of the theta synchronizing system [19,20] and the projection, through which the VTA enhances theta rhythm, might be indirect and incorporated into the main route of theta generation which involves the septum and diagonal band of Broca [21,40]. It should be also noted that dopamine release in the hippocampus is itself modulated by hippocampal activity: outputs from the hippocampus facilitate dopaminergic signaling in the midbrain, which in turn can enhance hippocampal plasticity via dopamine release [26]. The report from Moaddab et al. [41], in which authors observed no significant effect on general locomotor activity after lidocaine reversible inactivation of the VTA, gives us strong evidence that the prolonged latency of the avoidance response observed in our study is not caused by post-injection impairment of motor function. In our experiments, procaine was infused to the VTA only in those animals in which the previously learned behavior linked with tone induced immobility and avoidance response (escape from the foot-shock box) was undisturbed during control trials performed 5–10 min prior to each infusion. Therefore, we assumed that the observed prolonged escape latency after procaine infusion can be probably more related to the motivational processes rather than to disturbed memory recall (animals were allowed to recall previously learned behavior just before each infusion). As showed by Seidenbecher et al. [42] the retrieval and/or expression of conditioned fear may be functionally related to theta synchronization in amygdalohippocampal pathways, therefore it should be also considered that temporal inactivation of the VTA might also disrupted this processes, what in turn affected avoidance response of animal. It can be assumed that intra-VTA procaine infusion led to temporal decrease in dopaminergic mesolimbic transmission, which in turn led to dysregulation of hippocampal activity by direct or indirect pathway. The infusion could have also influenced cortical, amygdalohippocampal pathways and limbic loops that are involved in the motivation, attention and memory processes. 5. Conclusion Overall, our findings indicate that the VTA inactivation leads to a disruption in the hippocampal theta rhythm during fear conditioned immobility linked with arousal and/or attention, which occurs in parallel with suppression of avoidance response. Pharmacological activation of the VTA (amphetamine) had no effect on theta rhythm or avoidance reaction parameters. Conflicts of interest The authors declare no conflicts of interest. Acknowledgments This research was supported from the Polish Ministry of Science and Higher Education (Iuventus Plus grant, IP2011 034371). The Authors would like to thank Magda Ku´smierczak, Ph.D., for linguistic revision and for inspiring comments on an earlier version of the manuscript. References [1] C.H. Vanderwolf, Hippocampal electrical activity and voluntary movement in the rat, Electroencephalogr. Clin. Neurophysiol. 26 (1969) 407–418. [2] R. Kramis, C.H. Vanderwolf, B.H. Bland, Two types of hippocampal rhythmical slow activity in both the rabbit and the rat: relations to behavior and effects of atropine, diethyl ether, urethane and pentobarbital, Exp. Neurol. 49 (1975) 58–85.
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