HIPPOCAMPO CEREBELLAR THETA BAND PHASE SYNCHRONY IN RABBITS

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1 Neuroscience 165 (2010) HIPPOCAMPO CEREBELLAR THETA BAND PHASE SYNCHRONY IN RABBITS J. WIKGREN,* 1 M. S. NOKIA 1 AND M. PENTTONEN Department of Psychology, University of Jyväskylä, P.O. Box 35, FIN 40014, Jyväskylä, Finland Abstract Hippocampal functioning, in the form of theta band oscillation, has been shown to modulate and predict cerebellar learning of which rabbit eyeblink conditioning is perhaps the most well-known example. The contribution of hippocampal neural activity to cerebellar learning is only possible if there is a functional connection between the two structures. Here, in the context of trace eyeblink conditioning, we show (1) that, in addition to the hippocampus, prominent theta oscillation also occurs in the cerebellum, and (2) that cerebellar theta oscillation is synchronized with that in the hippocampus. Further, the degree of phase synchrony (PS) increased both as a response to the conditioning stimuli and as a function of the relative power of hippocampal theta oscillation. However, the degree of PS did not change as a function of either training or learning nor did it predict learning rate as the hippocampal theta ratio did. Nevertheless, theta band synchronization might reflect the formation of transient neural assemblies between the hippocampus and the cerebellum. These findings help us understand how hippocampal function can affect eyeblink conditioning, during which the critical plasticity occurs in the cerebellum. Future studies should examine cerebellar unit activity in relation to hippocampal theta oscillations in order to discover the detailed mechanisms of theta-paced neural activity IBRO. Published by Elsevier Ltd. All rights reserved. Keywords: hippocampus, cerebellum, theta, oscillation, eyeblink conditioning, phase synchrony. Common to all biologically meaningful learning is the engagement of multiple distinct phases and sub-processes that are governed by different, sometimes widely distributed, brain structures. Rabbit eyeblink conditioning (Gormezano et al., 1962), where a neutral conditioned stimulus (CS, e.g. a tone) is repeatedly paired with a reflex-eliciting unconditioned stimulus (US, e.g. a corneal airpuff) provides an example of a model system of learning which could be modified to tap into different aspects of the learning process. In this paradigm, the primary memory trace of the motor conditioned response (CR) is thought to be 1 J. Wikgren and M. Nokia both contributed equally. *Corresponding author. Tel: ; fax: address: jan.wikgren@jyu.fi (J. Wikgren). Abbreviations: ANOVA, analysis of variance; CS, conditioned stimulus; CR, conditioned response; EMG, electromyogram; FFT, fast fourier transform; HPC, hippocampus; HVI, cerebellar cortical area HVI; LFP, local-field potential; LTP/LTD, long-term potentiation/depression; mpfc, medial prefrontal cortex; PS, phase synchrony; US, unconditioned stimulus /10 $ - see front matter 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi: /j.neuroscience stored within the cerebellar deep nuclei and/or the cerebellar cortical area HVI (Thompson and Steinmetz, 2009; De Zeeuw and Yeo, 2005), whereas various cerebral areas can modulate the learning process. For instance, when the cognitive demands of the task increase, hippocampal contribution becomes critical (Beylin et al., 2001; Moyer et al., 1990; Woodruff-Pak and Disterhoft, 2008). Hippocampal function, especially in the form of thetaband oscillation ( 6 Hz; for a review see, e.g., Bland, 1986 or Buzsáki, 2002), has been linked to a host of cognitive processes, most notably to learning and memory (Buzsáki, 2005; Hasselmo, 2005; Kahana, 2006). The relative power of the hippocampal theta activity (theta ratio) recorded before learning correlates strongly with the learning rate during subsequent eyeblink conditioning in rabbits, as shown both using the simple delay paradigm and using the trace paradigm, during which learning is hippocampally mediated (Berry and Thompson, 1978; Berry and Seager, 2001; Griffin et al., 2004; Nokia et al., 2008, 2009; Nokia and Wikgren, 2009; Seager et al., 2002). Moreover, blocking hippocampal theta oscillation with, for example, scopolamine injections before eyeblink conditioning retards behavioral learning and virtually abolishes any learningrelated unit responses in the hippocampus (Salvatierra and Berry, 1989). Previous research begs the questions, how do the hippocampus and the cerebellum interact, and what is the special role of the theta oscillation? In order for the hippocampus to modulate memory trace formation in the cerebellum we have to assume interaction between these areas. One indication of interaction between two brain areas is synchronized oscillation (e.g. Fries, 2005; Singer, 1999). By synchronizing and desynchronizing their activity, brain structures can select which inputs arrive and are sent during a time window of maximal effect, i.e. cause a change in the desired direction in the excitability of the receiving group of neurons (Fries, 2005). We recorded LFPs simultaneously from the rabbit hippocampus, cerebellar cortex, and medial prefrontal cortex (mpfc) during trace eyeblink conditioning and assessed the degree of phase synchrony (PS) (Palva et al., 2005; Lachaux et al., 1998) between these structures. Since synchronous oscillatory activity supposedly indicates co-operation, and because hippocampal theta oscillation has been associated with the learning rate during cerebellum-dependent eyeblink conditioning, we expected to observe (1) theta oscillation also in the cerebellum and (2) PS between the hippocampus and the cerebellum at the theta band. In addition, we aimed to determine whether the degree of PS changes as a function of training and/or learning and

2 J. Wikgren et al. / Neuroscience 165 (2010) whether, like the hippocampal theta ratio does, it predicts learning rate. EXPERIMENTAL PROCEDURES Subjects and surgery The subjects were 27 adult male New Zealand white rabbits (Harlan, Netherlands, BV, Horst, Netherlands) aged 4 months and weighing 2.7 kg at the time of surgery. The rabbits were housed in individual metal cages on the premises of the animal research unit of the University of Jyväskylä. Food and water were freely available, and room temperature and humidity were controlled. All procedures were conducted during the light portion of the 12/12 hour light/dark cycle and implemented in accordance with the European Communities Council Directive (86/609/EEC, uri CELEX: 31986L0609:EN:HTML) on the care and use of animals for research purposes. Every effort to minimize the number of animals and suffering caused to them was undertaken. For a detailed description of the surgery see Nokia et al. (2009). Two or three monopolar recording electrodes were chronically implanted into the right hippocampus 5 mm posterior and 4 6 mm lateral to the bregma and one electrode into the right cerebellar cortex lobule HVI 1 mm anterior and 5 mm lateral to the lambda. In addition, two electrodes were implanted into the left mpfc 4 5 mm anterior and 0.8 mm lateral to the bregma. The electrodes were attached to a pin connector and the whole construction cemented in place with dental acrylic. At least 1 week was allowed for postsurgical recovery. Stimuli and procedure Prior to the experiments, the rabbits were placed (for approximately 20 min) in a Plexiglas restraining box located in a ventilated, electrically insulated, and sound-attenuated conditioning chamber to familiarize them with the experimental situation, and to ensure the functioning of the implanted electrodes. Thereafter, sessions were conducted once per day on consecutive days. The CS was a 2 khz, 85 db, 200 ms tone and the US was a 100 ms corneal airpuff (0.35 bar source pressure, sound pressure level 64 db) delivered through a nozzle (inner diameter 2 mm) placed approximately 1 cm away from the eye. A fan located inside the conditioning chamber behind the rabbit created a steady background noise of approximately 65 db. E-Prime software (Psychology Software Tools Inc., Pittsburgh, PA, USA) was used to control the presentation of stimuli. The rabbits were randomly assigned to unpaired (n 10) and paired (n 17) groups. Both received 10 daily sessions of either trace eyeblink conditioning (paired) or explicitly unpaired training (unpaired). The unpaired sessions consisted of 70 CS-alone and 70 US-alone trials presented in a random order with an intertrial interval averaging out at 20 s (range, s). The conditioning sessions consisted of 80 trials: 60 conditioning, 10 CS-alone, and 10 US-alone trials were presented in a pseudorandom order with an average intertrial interval of 40 s (range, s). During the conditioning trials, CS onset preceded US onset by 700 ms, thus creating a 500 ms trace period. Recordings Eyeblinks were measured using stainless steel wire hooks placed around the upper and lower eyelids for the duration of the training session. To acquire neural measures, a low-noise pre-amplifier (MPA8I, Multi Channel Systems, Reutlingen, Germany) was directly attached to the electrode coupler anchored with dental acrylic to the rabbit s head. A flexible, insulated cable was used to connect the animal to the amplifier (Axon Cyberamp 380, Molecular Devices Corporation, Union City, CA, USA). Both the neural data and the bipolar electromyogram (EMG) were recorded with AxoScope (Molecular Devices Corporation) software and digitized (Digidata 1322A, Molecular Devices Corporation) using a khz sampling rate. Before digitization, the LFPs were band-passfiltered between 0.1 and 4000 Hz, and the EMG was filtered between 30 and 300 Hz. Data analyses Clampfit (Molecular Devices Corporation), MATLAB (The MathWorks Inc., Natick, MA, USA) and SPSS (SPSS Inc., Chicago, IL, USA) were used for the data analyses. Eyeblinks. The EMG signal was further high-pass filtered over 100 Hz and Hilbert-transformed. Following this, an envelope curve following the peaks of the signal was calculated using the real and imaginary parts of the Hilbert transformation. Baseline EMG activity was calculated for each animal and session as the mean of the maximum EMG amplitude during a 500 ms prestimulus period (MAXpre). In addition, the mean of the standard deviation of the EMG activity during the 500 ms pre-stimulus period (SDpre) was determined. Eyeblinks were defined as EMG activity exceeding a threshold of [MAXpre 4 SDpre]. Blinks occurring during the 500-ms period following the offset of the tone (trace period) were counted as CRs. The learning rate was quantified as the number of trials needed to reach the 5th CR and the learning criterion. Phase synchrony calculations. To obtain comparable data from the control and experimental groups, only CS-alone and US-alone trials were included in the analyses. Phase synchrony (PS) between two signals was calculated as described by Palva et al. (2005). First, the LFP signals were band-pass filtered (delta 2 Hz, theta 6 Hz, alpha 12 Hz, beta 20 Hz, and gamma 60 Hz). Next, the filtered signals were transformed into a complex form using the Hilbert transform. Following this, the amplitudes of the signals were normalized to 1 (one) by dividing each data point by its absolute value. Then, the phase difference of the two signals in comparison was calculated by multiplying the first signal with the complex conjugate of the second signal (each data point of each trial). Finally, the PS was derived by averaging the phase difference matrix over trials, taking the absolute value. In order to standardize the PS values, 100 surrogate datasets per each real PS were created by shuffling the real trials. The PS was then determined for each surrogate dataset and the mean and the 95th percentile of the surrogate PSs derived. The mean of the surrogate PS values was subtracted from the real PS and the outcome divided point-by-point by the difference between the 95th percentile and the mean of the surrogate PSs ([realps surrps]/ [surr95th percentile surrps]) yielding a standardized PS. Thus standardized PS values exceeding 1 (one) represent statistically significant synchrony at the level of P.05. The group level statistical significance for the PS values was calculated using binomial statistics. Theta ratio. The FFT was calculated from the 1-s pre-stimulus period data, with a resolution of 0.5 Hz. The theta ratio was then calculated using (theta/[delta theta]). The delta and theta frequencies were used as the sole reference for theta, firstly, because in absolute power, delta and theta frequencies are fairly comparable and, secondly, because the absolute power of the higher frequencies (8 Hz ) is considerably lower than that of theta. To compare PS during periods of a high versus low hippocampal theta ratio, the tone-alone (10) and airpuff-alone (10) trials from each session (10) and each animal were sorted according to the hippocampal theta ratio calculated from the pre-stimulus period. Then, the trials with the lowest and highest theta ratio were selected to form sets of 10 trials per animal, pre-stimulus theta ratio level and stimulus type.

3 1540 J. Wikgren et al. / Neuroscience 165 (2010) Fig. 1. Recording electrodes were placed in the dorsal hippocampus, the cerebellar cortex lobule HVI, and in the medial prefrontal cortex (mpfc). All of the rabbits had at least one electrode in the hippocampal fissure or in the dentate gyrus/hilus and in the cerebellar cortex (A). In addition, 23 subjects had a recording electrode also in the mpfc (B). The open triangles refer to the subjects in the unpaired group and the filled circles to the subjects in the paired group. Statistical analyses. A paired samples t-test was used to compare pre- and post-stimulus PS values. Analysis of variance (ANOVA) for repeated measures was used for the assessment of training and group effects. Pearson correlation coefficient was used when examining the connection between PS and learning rate. Histology After the experiments, the rabbits were anesthetized with an i.m. injection of ketamine xylazine cocktail and then overdosed with an i.v. injection of pentobarbital (Mebunat vet, Orion-Yhtymä Oyj, Espoo, Finland). A detailed description of the perfusion and histology procedure can be found in Nokia et al. (2009). The electrode-tip locations were determined from the stained slides with the help of a microscope and stereotaxic atlases (Bures et al., 1967; Lavond and Steinmetz, 2003). Histology RESULTS All 27 subjects had recording electrodes both in the hippocampus and in the cerebellar cortex lobule HVI (Fig. 1), and 23 also had an electrode in the mpfc (Fig. 1B). was low (Fig. 3B, D), the cerebellar theta oscillation was also weak. Phase synchrony is specific to hippocampo cerebellar theta band oscillation To assess the degree of PS between the hippocampus and the cerebellum and between the hippocampus and the mpfc without contamination from the possible effects of learning, the combined paired and unpaired group data from only the first session was analyzed. Binomial test (P.01) showed significant PS only in the theta band ( 6 Hz) oscillation occurring simultaneously in the hippocampus and in the cerebellar cortex (Fig. 4A, C). In response to external stimulation, hippocampo cerebellar theta band PS was further increased (tone: t(26) 5.67, P.001; airpuff: t(26) 5.47, P.001; binomial test P.001) (Fig. 4A). The explicit lack of hippocampo cerebellar PS was evident in all the other frequency bands (delta, alpha, beta, and gamma) and during all three conditions (pre-stimulus, Behavioral results The percentage of CRs (Fig. 2) increased across paired treatment but remained stable across unpaired treatment (block: F [4,100] 17.71, P.001; group: F [1,25] 11.25, P.01; block group: F [4,100] 4.91, P.01; paired group, block: F [4,64] 22.45, P.001; unpaired group, block: F [4,36] 3.27, ns). Theta ( 6 Hz) oscillation in the rabbit cerebellum A clear theta band oscillation visible to the naked eye and verified by FFT was found in the cerebellar cortex (lobule HVI, Fig. 3). FFT showed that when the hippocampal theta ratio was high (Fig. 3A, C), the cerebellar theta oscillation was also strong, and when the hippocampal theta ratio Fig. 2. Conditioned responding increased in the paired but not in the unpaired group.

4 J. Wikgren et al. / Neuroscience 165 (2010) Fig. 3. The power of the theta-band activity was high/low in the cerebellum (HVI) when it was high/low in the hippocampus (HPC). The results of fast fourier transform (FFT, upper panels) run on hippocampal and cerebellar LFPs (lower panels, representative examples) during periods of high relative power of hippocampal theta activity (theta/[delta theta]) are shown in A (C) and during low relative power of hippocampal theta activity in B (D). tone, and airpuff), as shown by binomial test (P.001) (Fig. 4A). No statistically significant PS was found between the hippocampus and the mpfc (Fig. 4B). In fact, the binomial test showed a significant lack of PS in all bands (P.05/.001). Hippocampo cerebellar theta band PS remains statistically significant across training, but decreases slightly Significant main effects of training block (5) and condition (3) were found when the hippocampo cerebellar PS during the pre-stimulus period and in response to the tone and airpuff across training was examined in both the unpaired group (block: F [4,108] 7.85, P.001; condition: F [1,27] 4.36, P.05) and paired group (block: F [4,192] 7.51, P.001; condition: F [1,48] 16.24, P.001) (Fig. 5). In both groups, hippocampo cerebellar PS was higher in response to stimuli compared to the pre-stimulus period, but decreased linearly across the training. The effect of training type (unpaired vs. paired) and learning (fast vs. slow learners) was also tested, but no connections were found. In addition, no significant correlations were found between hippocampo cerebellar PS and learning rate (trials to 5th CR and trials to criterion) in the paired group (n 17), although a clear predictive connection was found between the hippocampal theta ratio recorded during the first session and the number of trials needed to reach the 5th CR (pre-cs theta ratio 5th CR: r.48, P.05 and post-cs theta ratio 5th CR: r.63, P.01). Based on the lack of significant group differences, data from the paired (n 17) and unpaired (n 10) groups were again combined (n 27) for further analyses. Phase synchrony is stronger during episodes of a high hippocampal theta ratio but increases in response to external stimuli irrespective of the on-going theta state When the trials were divided into two classes based on the degree of the hippocampal theta ratio (high vs. low) during the absence of stimulation (pre-stimulus period), the PS between the hippocampus and cerebellar cortex was statistically significant only when the hippocampal theta ratio was high (binomial test P.001) (Fig. 4D, E). However, regardless of whether the hippocampal theta ratio was high or low (i.e. the PS during the pre-stimulus period was significant or not), the presentation of the tone or the airpuff always elicited significant theta band PS between the hip-

5 1542 J. Wikgren et al. / Neuroscience 165 (2010) Fig. 4. Statistically significant phase synchrony (PS) was found only between the hippocampus and the cerebellar cortex and only in the theta-band ( 6 Hz). Binomial statistics showed significant PS only in the theta oscillation occurring simultaneously in the hippocampus and in the cerebellar cortex (A). In response to external stimulation, this hippocampo cerebellar theta-band PS was increased. No statistically significant PS was found between the hippocampus and the mpfc (B). The hippocampo cerebellar theta-band PS increased in response to both stimuli (tone and airpuff, A and C), irrespective of the relative power of the hippocampal theta oscillation (low vs. high theta) immediately preceding stimulus onset (D and E). The PS calculated from the real data are shown in the uppermost panel of each subplot, the PS calculated from the surrogate data obtained by shuffling real data are shown in the middle panel, and the standardized PS is shown in the lowest panel. The vertical lines indicate the onset of the stimulus. The asterisk reflects statistical significance of paired samples t-tests: *** P.001. The horizontal dotted line shows the threshold value of 1 (one) corresponding to statistical significance at the level of P.05. pocampus and the cerebellar cortex (binomial test P.001) (Fig. 4D, E). DISCUSSION The two main findings of this study were (1) a prominent theta band oscillation in the cerebellar cortex of the rabbit and (2) an increased and high degree of PS between the theta band oscillation in the cerebellum and that in the hippocampus during the presentation of external stimuli. Hippocampo cerebellar PS was especially strong when the hippocampal theta ratio was high, but presenting the animal with external stimuli increased the theta band PS regardless of the ongoing theta ratio immediately preceding the stimulus presentation. However, we did not observe any changes in the hippocampo cerebellar theta band PS specific to paired training or learning, nor did we detect any correlation between the level of spontaneous hippocampo cerebellar theta-band PS and learning rate. To our knowledge, this is the first report on theta oscillation ( 6 Hz) in the rabbit cerebellum. In rats, D Angelo et al. (2001) have shown that granule cells in the cerebellar cortex exhibit bursting and resonance at the theta frequency range (3 12 Hz). Such rhythmicity might play an important role in cerebellum-mediated learning. Indeed, they have also shown that long-term potentiation/depression (LTP/LTD) is present in the mossy fiber-granule cell synapses in the cerebellum when high-frequency stimulation of the former is paired with membrane depolarization in the latter (D Angelo et al., 1999; Armano et al., 2000; Mapelli and D Angelo, 2007). Whereas the relative magni-

6 J. Wikgren et al. / Neuroscience 165 (2010) Fig. 5. The overall level of hippocampo cerebellar PS decreased across training, but remained higher in response to external stimuli compared to the pre-stimulus period at all times in both the unpaired (A) and paired (B) group. tude of the hippocampal theta band oscillation affects the behavioral learning rate and learning-related unit activity during eyeblink conditioning (see, for example, Nokia et al., 2008), the phase of hippocampal theta oscillation affects synaptic plasticity when LTP procedures are used (Huerta and Lisman, 1995; Hölscher et al., 1997). Stimulating the hippocampus via the Schaffer collaterals during the positive phase of hippocampal CA1 theta oscillation produced LTP, whereas stimulation given during the negative phase reversed the previously established LTP (Huerta and Lisman, 1995; Hölscher et al., 1997). In sum, the responsiveness and plasticity of hippocampal output, as well as that of the cerebellar input neurons, is synchronized by theta oscillation, now also shown to exist in the cerebellar LFPs. Not only did our present study show a theta oscillation in the cerebellar LFPs but it also demonstrated that the theta oscillation in the cerebellar cortex is in concert with that in the hippocampus, as indicated by the significant PS. Synchronous activity is thought to reflect the formation of transient functional assemblies by anatomically distant brain structures (Fries, 2005). Related to the role of the hippocampal theta oscillation in learning and memory, in healthy humans undergoing tasks requiring working memory, coherent activity at the theta-band between different cortical sites has been observed by recording electroencephalogram (EEG; for a review, see Sauseng and Klimesch, 2008). In addition, in an early experiment by Livanov (1977), visual stimulation caused limb movements at a probability increasing along with the degree of coherence between EEG signals measured above the visual and the motor cortex in rabbits. Similarly, Schoffelen et al. (2005) showed that subjects readiness to respond in a simple reaction-time task closely correlated with the degree of gamma-band coherence between neurons in the motor cortex and spinal cord. Given that neural plasticity in both the output area of the hippocampus (Huerta and Lisman, 1995; Hölscher et al., 1997) and in the input area of the cerebellar cortex (D Angelo et al., 1999; Armano et al., 2000; Mapelli and D Angelo, 2007) are related to the phase of the theta cycle, and that theta oscillations in both areas occur in a fixed temporal relation to each other, it is likely that hippocampo cerebellar theta band PS is relevant to neural communication between these structures. It is also to be noted that presenting a stimulus to the animal actually decreased the amount of theta band synchrony for a brief time period. This observation conforms to the theory of large-scale integration of neural activity (Rodriguez et al., 1999) which states that transiently formed cognitive states are separated by brief moments of desynchronization, i.e. active uncoupling of neural activity. The hippocampo cerebellar theta band PS observed in our study suggests the formation of transient neural assemblies between the structures, but, in the absence of accompanying single-unit analyses, unfortunately is not able to demonstrate it. However, evidence from other lines of research indicates that hippocampal theta oscillation might be the pacemaker for neuronal activity in learningrelevant regions. For instance, neuronal activity in prefrontal regions becomes synchronized with hippocampal theta in the early phase of appetitively motivated trace conditioning (Paz et al., 2008). Similarly, Berke et al. (2004) found coherent theta oscillations in the hippocampal CA1 and the ventral/medial striatum, and showed that the striatal neurons tended to fire more frequently during the descending phase of hippocampal theta oscillation, thus manifesting a link between hippocampal theta and motor areas relevant in consummatory behavior. Furthermore, Sirota et al. (2008; see also Sirota and Buzsáki (2005) recently suggested that oscillatory synchrony might be the mechanism through which the information flow between the hippocampus and the neocortex is controlled. There is also evidence in anesthetized animals that cells in the neocortex are synchronized with cells in the cerebellum, albeit not at the theta band (Ros et al., 2009). Thus, further studies are needed to investigate the possible connections between the hippocampo cerebellar theta oscillations and unit-activity in the cerebellar cortex/deep nuclei. Much to our surprise, we found no connection between hippocampo-cerebellar theta band PS and learning. This is at odds with previous studies, which have shown a clear predictive connection between the relative magnitude of hippocampal theta oscillation and learning rate, as well as changes across training/learning during trace eyeblink

7 1544 J. Wikgren et al. / Neuroscience 165 (2010) conditioning (Griffin et al., 2004; Nokia et al., 2009). This difference in results could be explained by the difference in the nature of the analyses used: in the present study the phase of theta oscillation was examined whereas in previous studies analyses were targeted at the relative amplitude of theta oscillation. It is possible that hippocampo cerebellar theta band PS is a fundamental and rather stable mediator of communication between the two structures, and thus does not show changes across training/ learning. In other words, the pattern of activation leading to synchronized activity and mediating efficient communication remains stable, whereas learning-related changes occur, for instance in the number of cerebellar neurons firing in relation to the theta band oscillation. It could well be that cerebellar unit activity either increases during periods of strong hippocampal theta oscillation, or becomes more sharply synchronized with it. Further investigation of this matter is needed. Another explanation for the lack of learning-related changes in PS between the cerebellar cortex and hippocampus in the present study could be that the cerebellar cortex actually does not critically contribute to this form of learning. For instance, it has been shown that impaired cerebellar cortical function has a detrimental effect specifically on delay but not trace eyeblink conditioning in mice (Kishimoto et al., 2001; Woodruff-Pak et al., 2006). As hippocampal function modulates learning also during delay conditioning, replicating the present study but using the delay conditioning paradigm instead might, therefore, result in changes in PS as a function of training or learning. In relation to more technical issues, it might be argued that the hippocampo cerebellar theta band PS observed in this study resulted from volume conduction. However, PS was seen only in the theta band, and only between the hippocampus and the cerebellum, and not between the hippocampus and the mpfc (which resides spatially closer to the hippocampus), making it unlikely that volume conduction could explain the results. It is more probable, since the rhythm of hippocampal theta oscillation is in part generated in the medial septum-diagonal band of Broca (see Buzsáki, 2002), that it also plays a part in pacing cerebellar theta oscillation. In the knowledge that both the hippocampus and the cerebellum are connected with the neocortex, it might be suggested that it provides a link between them. Whether this is the case and, if so, through which pathways, remains, however, yet to be determined. CONCLUSION In conclusion, the theta band PS found here between the hippocampus and the cerebellum might reflect a mechanism by which these two structures can interact efficiently. Via theta band PS the hippocampus might transiently form neural assemblies with its target area in the cerebellum, possibly enhancing synaptic modification and thus learning. In future studies it would be of importance to investigate whether firing in the cerebellar neurons is also synchronized with the phase of hippocampal theta oscillation. Showing synchrony between cerebellar plasticity and hippocampal theta oscillation would provide strong evidence for the functional significance of cerebellar theta oscillation. Acknowledgments Supported by grants from the Academy of Finland to JW ( and ) and from the Emil Aaltonen foundation to MSN (Young researcher s grant). The authors wish to thank Matias Palva for advice on phase synchrony calculation and Michael Dutton and Michael Freeman for language checking. REFERENCES Armano S, Rossi P, Taglietti V, D Angelo E (2000) Long-term potentiation of intrinsic excitability at the mossy fiber granule cell synapse of rat cerebellum. J Neurosci 20: Berke JD, Okatan M, Skurski J, Eichenbaum HB (2004) Oscillatory entrainment of striatal neurons in freely moving rats. Neuron 43: Berry SD, Seager MA (2001) Hippocampal theta oscillations and classical conditioning. Neurobiol Learn Mem 76: Berry SD, Thompson RF (1978) Prediction of learning rate from the hippocampal electroencephalogram. Science 200: Beylin AV, Gandhi CC, Wood GE, Talk AC, Matzel LD, Shors TJ (2001) The role of the hippocampus in trace conditioning: temporal discontinuity or task difficulty? Neurobiol Learn Mem 76: Bland BH (1986) The physiology and pharmacology of hippocampal formation theta rhythms. Prog Neurobiol 26:1 54. Bures J, Petran M, Zachar J (1967) Electrophysiological methods in biological research. New York: Academic Press. Buzsáki G (2002) Theta oscillations in the hippocampus. Neuron 33: Buzsáki G (2005) Theta rhythm of navigation: link between path integration and landmark navigation, episodic and semantic memory. Hippocampus 15: D Angelo E, Rossi P, Armano S, Taglietti V (1999) Evidence for NMDA and mglu receptor-dependent long-term potentiation of mossy fiber granule cell transmission in rat cerebellum. J Neurophysiol 81: D Angelo E, Nieus T, Maffei A, Armano S, Rossi P, Taglietti V, Fontana A, Naldi G (2001) Theta-frequency bursting and resonance in cerebellar granule cells: experimental evidence and modeling of a slow K -dependent mechanism. J Neurosci 21: De Zeeuw CI, Yeo CH (2005) Time and tide in cerebellar memory trace formation. Curr Opin Neurobiol 15: Fries P (2005) A mechanism for cognitive dynamics: neuronal communication through neuronal coherence. Trends Cogn Sci 9: Gormezano I, Schneiderman N, Deaux E, Fuentes I (1962) Nictitating membrane: classical conditioning and extinction in the albino rabbit. Science 138: Griffin AL, Asaka Y, Darling RD, Berry SD (2004) Theta-contingent trial presentation accelerates learning rate and enhances hippocampal plasticity during trace eyeblink conditioning. Behav Neurosci 118: Hasselmo ME (2005) What is the function of hippocampal theta rhythm? linking behavioral data to phasic properties of field potential and unit recording data. Hippocampus 15: Hölscher C, Anwyl R, Rowan MJ (1997) Stimulation on the positive phase of the hippocampal theta rhythm induces long-term potentiation that can be depotentiated by stimulation on the negative phase in area CA1 in vivo. J Neurosci 17: Huerta PT, Lisman JE (1995) Bidirectional synaptic plasticity induced by a single burst during cholinergic theta oscillation in CA1 in vitro. Neuron 15: Kahana MJ (2006) The cognitive correlates of human brain oscillations. J Neurosci 26: Kishimoto Y, Hirono M, Sugiyama T, Kawahara S, Nakao K, Kishio M, Katsuki M, Yoshioka T, Kirino Y (2001) Impaired delay but normal

8 J. Wikgren et al. / Neuroscience 165 (2010) trace eyeblink conditioning in PLCbeta4 mutant mice. Neuroreport 12: Lachaux JP, Rodriguez E, Martinerie J, Varela FJ (1998) Measuring phase synchrony in brain signals. Hum Brain Mapp 8: Lavond DG, Steinmetz JE (2003) Handbook of classical conditioning. London: Springer. Livanov MN (1977) Spatial organization of cerebral processes. New York: John Wiley & Sons. Mapelli J, D Angelo E (2007) The spatial organization of long-term synaptic plasticity at the input stage of the cerebellum. J Neurosci 27: Moyer JR Jr, Deyo RA, Disterhoft JF (1990) Hippocampectomy disrupts trace eye-blink conditioning in rabbits. Behav Neurosci 104: Nokia MS, Penttonen M, Korhonen T, Wikgren J (2008) Hippocampal theta (3 8 Hz) activity during classical eyeblink conditioning in rabbits. Neurobiol Learn Mem 90: Nokia MS, Penttonen M, Korhonen T, Wikgren J (2009) Hippocampal theta band activity and trace eyeblink conditioning in rabbits. Behav Neurosci 123: Nokia MS, Wikgren J (in press) Hippocampal theta activity is selectively associated with contingency detection but not discrimination in rabbit discrimination-reversal eyeblink conditioning. Hippocampus. Palva JM, Palva S, Kaila K (2005) Phase synchrony among neuronal oscillations in the human cortex. J Neurosci 25: Paz R, Bauer EP, Paré D (2008) Theta synchronizes the activity of medial prefrontal neurons during learning. Learn Mem 15: Rodriguez E, George N, Lachaux JP, Martinerie J, Renault B, Varela FJ (1999) Perception s shadow: long-distance synchronization of human brain activity. Nature 397: Ros H, Sachdev RN, Yu Y, Sestan N, MacCormick DA (2009) Neocortical networks entrain neuronal circuits in cerebellar cortex. J Neurosci 29: Salvatierra AT, Berry SD (1989) Scopolamine disruption of septohippocampal activity and classical conditioning. Behav Neurosci 103: Sauseng P, Klimesch W (2008) What does phase information of oscillatory brain activity tell us about cognitive processes? Neurosci Biobehav Rev 32: Schoffelen JM, Oostenveld R, Fries P (2005) Neuronal coherence as a mechanism of effective corticospinal interaction. Science 308: Seager MA, Johnson LD, Chabot ES, Asaka Y, Berry SD (2002) Oscillatory brain states and learning: impact of hippocampal thetacontingent training. Proc Natl Acad Sci U S A 99: Singer W (1999) Neuronal synchrony: a versatile code for the definition of relations? Neuron 24: Sirota A, Buzsáki G (2005) Interaction between neocortical and hippocampal networks via slow oscillations. Thalamus Relat Syst 3: Sirota A, Montgomery S, Fujisawa S, Isomura Y, Zugaro M, Buzsáki G (2008) Entrainment of neocortical neurons and gamma oscillations by the hippocampal theta rhythm. Neuron 60: Thompson RF, Steinmetz JE (2009) The role of the cerebellum in classical conditioning of discrete behavioral responses. Neuroscience 162: Woodruff-Pak DS, Disterhoft JF (2008) Where is the trace in trace conditioning? Trends Neurosci 31: Woodruff-Pak DS, Green JT, Levin ST, Meisler MH (2006) Inactivation of sodium channel Scn8A (Na-sub(v)1.6) in Purkinje neurons impairs learning in Morris water maze and delay but not trace eyeblink classical conditioning. Behav Neurosci 120: (Accepted 18 November 2009) (Available online 27 November 2009)