Serotonergic modulation of hippocampal theta activity in relation to hippocampal information processing

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1 Exp Brain Res (2013) 230: DOI /s x Review Serotonergic modulation of hippocampal theta activity in relation to hippocampal information processing María Esther Olvera Cortés Blanca Erika Gutiérrez Guzmán Elisa López Loeza J. Jesús Hernández Pérez Miguel Ángel López Vázquez Received: 1 May 2013 / Accepted: 7 August 2013 / Published online: 30 August 2013 Springer-Verlag Berlin Heidelberg 2013 Abstract Hippocampal theta activity is the result of the concerted activity of a group of nuclei located in the brain stem and the caudal diencephalic area, which are together referred to as the synchronizing ascending system. Serotonin is recognized as the only neurotransmitter able to desynchronize the hippocampal electroencephalographic. A theory has been developed in which serotonin, acting on medial septal neurons, modulates cholinergic/gabaergic inputs to the hippocampus and, thus, the cognitive processing mediated by this area. However, few studies have addressed the relationship between serotonin modulation of theta activity and cognition. In this review, we present a summary and analysis of the data relating serotonin and its theta activity modulation with cognition, and we also discuss the few works relating serotonin, theta activity and cognition as well as the theories regarding the serotonin regulation of memory processes organized by the hippocampus. We propose that serotonin depletion induces impairment of the relays coding the frequency of hippocampal theta activity, M. E. Olvera Cortés (*) B. E. Gutiérrez Guzmán J. J. Hernández Pérez Laboratorio de Neurofisiología Experimental, Centro de Investigación Biomédica de Michoacán, Instituto Mexicano del Seguro Social, Morelia, México maesolco@yahoo.com E. López Loeza M. Á. López Vázquez Laboratorio de Biofísica, Instituto de Física y Matemáticas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, México M. Á. López Vázquez Laboratorio de Neuroplasticidad de los Procesos Cognoscitivos, Centro de Investigación Biomédica de Michoacán, Instituto Mexicano del Seguro Social, Morelia, México whereas depletion of the relays in which frequency is not coded induces improvements in spatial learning that are related to increased expression of high-frequency theta activity. Keywords Hippocampus Theta activity Serotonin Learning Memory Introduction Theta activity is a sinusoidal slow rhythmic activity recorded predominantly in hippocampal EEGs (Leranth et al. 1999; Vanderwolf 1969). In rats, theta activity ranges from 3 to 13 Hz (Hayes et al. 2004; Vanderwolf 1969), whereas in humans, hippocampal theta activity ranges between 2 and 8 Hz (Cornwell et al. 2008; Watrous et al. 2011), which are lower frequencies than those observed for cortical theta activity recorded from scalp (ranging from 4 to 7 Hz) (Bastiaansen et al. 2002; Klimesch et al. 1996). Hippocampal theta activity has been related to information processing in both humans (Watrous et al. 2011; Ekstrom et al. 2005) and rats (Ammassari-Teule et al. 1991; Andersen et al. 1979; McNaughton et al. 2006; Mitchell et al. 1982; Olvera- Cortes et al. 2004); furthermore, correlations between theta activity and movement speed (Hinman et al. 2011; Li et al. 2012), movement selection, sensorimotor integration (Bland and Oddie 2001; Oddie and Bland 1998), and the recency of experiences (Hinman et al. 2011) have been shown in rats. Hippocampal theta activity results from the concerted activity of a group of nuclei located in the brain stem and the caudal diencephalic area, which are together referred to as the synchronizing ascending system (SAS)

2 408 Exp Brain Res (2013) 230: (Andersen et al. 1979; Kirk and McNaughton 1991; Pan and McNaughton 1997; Vertes 1986; Vertes and Kocsis 1997). Serotonin is recognized as the only neurotransmitter that is able to desynchronize hippocampal EEGs (Assaf and Miller 1978; Leranth and Vertes 1999; Maru et al. 1979; Vertes and Kocsis 1997; Vinogradova et al. 1999). A theory has been developed in which serotonin acting on medial septal neurons modulates cholinergic/gabaergic inputs to the hippocampus and, thus, the cognitive processing mediated by this area (Jeltsch-David et al. 2008). However, few studies have addressed the relationship between serotonergic modulation of theta activity and cognition. In this review, we present a summary and analysis of the data relating serotonin and theta activity modulation to cognition. The few works relating serotonin, theta activity, and cognition are also presented and considered as are the theories from studies regarding the regulation of memory processes organized by the hippocampus. Theta activity Hippocampal theta activity was most likely first described by Jung and Kornmüller (1938). Later, Green and Arduini (1954) extensively described the theta activity from cats, rats, and monkeys both in acute and in chronic preparations under arousal and sensorial stimulation. Vanderwolf (1969) remarked on the relationship of theta activity with movement and proposed the existence of a sinusoidal slow wave (theta) related to the execution of voluntary movements; however, two classes of findings cast doubt on this relationship: the presence of voluntary movement after hippocampal lesions and the presence of theta activity during immobility (Bennett and French 1977; Montoya et al. 1989; Sainsbury et al. 1987). Later, Leung et al. (1982) quantitatively analyzed theta activity and described it as rhythmical slow activity with a frequency peak at 6 9 Hz. Based on the studies in which a predominant presence of theta activity was related to exploration, rearing, head movements and alert immobile states in the rat, it was proposed that this activity is related to voluntary movements that function in the acquisition of information concurrent with these types of behavior (Vinogradova 1995). Numerous studies have assessed hippocampal theta activity in relation to mnemonic processes. Among these studies, some are relevant for elucidating the attributes of theta activity that are necessary for the occurrence of hippocampal information processing. Although most of the available information is correlational, together, this evidence shapes an important body of data that sustains the role of hippocampal theta activity in learning. Hippocampal theta activity is generated by the rhythmic fluctuation of the membrane potentials of hippocampal neurons, and this fluctuation is driven by subcortical inputs (Bland and Whishaw 1976; Buzsaki 2002; Freund and Antal 1988; Hangya et al. 2009; Huh et al. 2010; Vertes and Kocsis 1997). Studies of electroencephalographic (EEG) activity have shown that hippocampal activity exhibits two zones of maximum amplitude between the surface and deep strata [the oriens pyramidal strata in CA1 and the molecular layer of the dentate gyrus (DG), respectively] that are 180 out of phase with one other (Bland and Whishaw 1976; Buzsaki 2002; Monmaur and Thomson 1986). Theta activity ranging from 6 to 9 Hz was referred as high-frequency theta or theta type 1 and was described as occurring when animals display voluntary behaviors, such as running, head movements, rearing, jumping, visual exploration, manipulation of objects with their forelimbs, and swimming (type I behaviors). Theta type 2 was described ranging from 4 to 7 Hz and occurring with short durations when reflective behaviors, such as grooming, eating, chewing, and licking are displayed (type II behaviors). During these behaviors, a large amplitude irregular activity predominates (Vanderwolf 1988). Although a pharmacological dissociation was proposed in which type 2 theta activity would depend on the cholinergic system, while type 1 theta would depend on another neurotransmitter system (proposed serotonergic) (Kramis et al. 1975; Vanderwolf 1988; Vanderwolf and Baker 1986), further advances in the theta activity research showed that the proposal of a non-cholinergic theta activity was unsustainable (Assaf and Miller 1978; Kitchigina et al. 1999; Maru et al. 1979; Vinogradova et al. 1999). Additionally, it was shown that the theta activity during immobility can spread over a broader range of frequencies (4 9.5 Hz) in proportions similar to theta activity while mobile (Olvera-Cortes et al. 2002). In spite of this finding, based on the association of theta activity with voluntary motor behaviors closely related to environmental information, it was proposed that theta activity recorded under these conditions may underlie the processing of environmental information by the hippocampus (Eichenbaum et al. 1992; Gavrilov et al Vinogradova 1995). Theta activity and the synchronizing ascending system (SAS) Hippocampal theta activity is controlled by a network of nuclei extending from the brain stem reticular formation to the septum and the hippocampus. This system principally includes the reticular pontis oralis nuclei (RPOn), the supramammillary nucleus (SUMn), the medial septum/ diagonal band of Broca complex (MS/DBB) (Vertes 1981; Vertes et al. 2004; Vertes and Kocsis 1997; Vertes and Linley

3 Exp Brain Res (2013) 230: ), and the posterior hypothalamic nuclei (PHn) (Bland et al. 1990; Kirk et al. 1996; Leranth and Vertes 1999). The RPOn of the rostral pontine reticular formation is directly involved in the generation of hippocampal theta activity; the stimulation of this structure induces, and inactivation suppresses, hippocampal theta activity in anesthetized rats (Vertes et al. 1993). Furthermore, microinjections of carbachol into the RPOn or the tegmental pedunculopontine nuclei (TPPn) induce hippocampal theta activity (Kinney et al. 1998; Vertes et al. 1993), and the injection of procaine into the TPPn eliminates hippocampal theta activity in anesthetized rats (Nowacka et al. 2002). Both nuclei are considered to be sites involved in theta generation; the RPOn first triggers theta onset, and the PPTn activity acts to maintain hippocampal theta by activating other brain stem nuclei (Takano and Hanada 2009). RPOn is composed mainly for glutamatergic neurons, whereas TPPn contain a large proportion of cholinergic neurons (Satoh et al. 1983; Semba and Fibiger 1989) intermixed with glutamatergic and GABAergic neurons (Clements and Grant 1990; Martinez-Gonzalez et al. 2012; Mena-Segovia et al. 2009; Wang and Morales 2009). The SUMn is a relay of the RPOn that influences hippocampal EEGs; both electrical stimulation and carbachol injections into the RPOn induce theta-related cell firing in SUMn neurons (Bland et al. 1995; Kirk and McNaughton 1991; Kocsis and Vertes 1994; Vertes and Kocsis 1997); moreover, procaine injections in subregions of the SUMn reduce the amplitude and frequency of hippocampal theta activity evoked by RPOn stimulation, and reversible blockade of MS/DBB with procaine infusion does not alter SUMn theta-related activity evoked by RPOn stimulation (Kirk et al. 1996). The SUMn is reciprocally connected with the hippocampus, and the MS/DBB may influence hippocampal theta activity both directly through glutamatergic contacts with granular (DG), pyramidal, and GABAergic neurons (from CA) (Kiss et al. 2000) and indirectly via synapses on cholinergic and GABAergic neurons of the MS/DBB (Leranth and Kiss 1996). It has been shown that the SUMn and PHn play important roles in controlling hippocampal theta activity, principally in anaesthetized animals (Kirk et al. 1996). The PHn projects to the MS (Smythe et al. 1991), and the activation of this nucleus with carbachol injections produces hippocampal theta activity and the activation of theta-on cells of the MS/DBB (Bland et al. 1990, 1994; Oddie et al. 1994; Smythe et al. 1991). PHn inactivation with procaine injection blocks hippocampal theta activity generated by RPOn stimulation (Oddie et al. 1994) and septal theta activity related to phasic cell discharges (Bland et al. 1994). The pacemaker status of the MS was proposed, and well established, by Stumpf s group (Gogolak et al. 1967, 1968; Petsche and Stumpf 1960; Petsche et al. 1962; Sailer 409 and Stumpf 1957; Stumpf et al. 1962). More recent works added insights into the mechanisms of theta generation by the septum and SAS (Vertes and Kocsis 1997) and into the cholinergic participation in theta modulation (Vinogradova 1995). Among other findings, it was shown that electrolytic lesions of the MS eliminate this activity (Andersen et al. 1979; Leung et al. 1994; Winson 1978), and inhibition of the activity of MS cells with procaine or muscimol injections leads to the suppression of theta oscillations in the hippocampus (Bland et al. 1994, 1996; Lawson and Bland 1993). Whereas the infusion of carbachol into the MS induces hippocampal theta activity in urethane anesthetized rats (Oddie et al. 1994), cholinergic septal lesions fail to eliminate hippocampal theta activity in freely moving rats (Lawson and Bland 1993; Lee et al. 1994). The MS/DBB contains cholinergic, GABAergic, and glutamatergic neurons (Brashear et al. 1986; Huh et al. 2010; Sotty et al. 2003; Vertes 1991), which are all most likely involved in the pace of hippocampal theta activity (Freund and Antal 1988; Huh et al. 2010; Lee et al. 1994; Li et al. 2007; Yoder and Pang 2005). Both glutamatergic and GABAergic neurons display pacemaker activity in vitro (Hangya et al. 2009; Huh et al. 2010). The MS/DBB complex projects to the DG, CA1, CA2, and CA3 and entorhinal cortex (Crutcher et al. 1981; Gaykema et al. 1990). The septal cholinergic neurons project to hippocampal pyramidal neurons and interneurons (Frotscher and Leranth 1985), whereas the septal GABAergic neurons (containing parvalbumin) synapse on GABAergic interneurons of the hippocampus; thus, their activation leads to disinhibition of the pyramidal hippocampal cells that drive hippocampal theta (Freund and Antal 1988; Hangya et al. 2009; Toth et al. 1997). A recently described population of glutamatergic neurons from MS/DBB projects to DG, CA1, and CA3, but the neuronal targets of their efferents are unknown (Colom et al. 2005). However, the excitation of pyramidal cells of CA3 by stimulation of glutamatergic MS/DBB neurons has been described in mice (Huh et al. 2010). The hippocampus sends descending afferents to both the MS/DBB and the lateral septum. Glutamatergic hippocampal axons contact GABAergic septal lateral neurons, whereas a population of GABAergic hippocampal neurons projects to GABAergic and cholinergic neurons of the MS/DBB (Alreja et al. 2000; Jakab and Leranth 1995). Notably, many of the results regarding the role of SAS in hippocampal theta drive were obtained in anesthetized animals; thus, different contributions of each relay of the SAS to the modulation of hippocampal theta activity may occur in behaving rats. However, the characteristics of the SAS components in behaving rats have not been thoroughly explored.

4 410 Exp Brain Res (2013) 230: Theta activity and hippocampal information processing Porter et al. (1964) assessed the hippocampal theta activity underlying learning in cats. EEG was recorded in several sites (CA1 to CA3) in the hippocampus during the training of a bright discrimination task using a T-maze. The cats were trained to find food in the bright zone of the maze (randomly assigned), and when their performance reached 100 % correct responses for several days, they were trained for reversal learning (the food was placed in the dark zone) until the new task was learned. The cats received 40 daily trials, and EEG records were taken during each trial. The results showed theta oscillations related to exploration of the environment with 4 5 Hz frequencies. For each training day, the averaged traces of the 40 daily trials were obtained, and they showed predominant theta activity with a frequency of 6 Hz. When the animals performed correctly less than 50 % of the time, the EEGs showed very little synchrony (through the trials, phase-locked by the initiation of a task-related stimulus), and the synchrony increased over the training days and was most pronounced when the performance of the animals was rapidly improving (days 4 8). When the performance of the cats reached 90 % correct responses, the synchronicity diminished from trial to trial. In the reversal learning, the cats EEGs showed prominent theta activity with a marked increase in synchrony on the first day of reversal training, but theta activity decreased by the time the cats reached 50 % of correct responses, despite the predominance of theta activity. When the cats performance began to increase above 50 %, the theta synchronicity increased again, similar to that which occurred in the initial task. After small lesions of the hippocampus, the cats showed intact ability to re-acquire the task (upon retraining in the first task); however, one cat that was trained only in the first task and then lesioned was deficient in the reversal acquisition (required 400 trials in comparison with 280 for the first task) (Porter et al. 1964). Although deficiencies in the behavior were not observed after lesions of hippocampus, this could be due to the small extent of the lesions or because the associations were learned pre-lesion. This work was among the first showing evidence of theta synchronicity related to the acquisition of a discrimination task in EEG activity recorded simultaneously to the training. In more recent works, an important body of evidence has shown the crucial role of hippocampal theta activity in tasks involving associative learning, such as eye blink conditioning. In these types of associative learning, hippocampal and prefrontal functions are required, in addition to the cerebellar participation, when a delay between the conditioned and unconditioned stimuli is present (Kalmbach et al. 2009). In these tasks, training contingent on the spontaneous occurrence of hippocampal theta activity produces increased learning as evidenced by greater numbers of conditioned responses in rabbits (Berry and Thompson 1978; Nokia et al. 2008). When conditioning trials are given to the animal during hippocampal theta activity, learning improves, whereas when training trials occur in the absence of theta activity, learning is impaired (Asaka et al. 2005; Griffin et al. 2004; Hoffmann and Berry 2009). It has been suggested that increases in performance are due to greater coherence in the theta band between the cerebellum, interpositus nucleus, and the hippocampus (Hoffmann and Berry 2009). Hippocampal theta activity has also been related to other types of information processing, such as spatial learning and navigation (Buzsaki 2005), performance of short-term memory tasks (Vertes 2005), and novelty detection (Aggleton and Brown 1999; Vinogradova 2001). The appearance of theta activity upon the perception of new and significant information and during the execution of orienting responses has been observed in rats (Jacobson et al. 2013; Klimesch 1999; Segal 1976). Moreover, theta activity has been related to the coding of new information (Buzsaki et al. 1994; Segal 1976). And, reductions in theta frequency (from to Hz) have been reported when rats are exposed to new environments, and there is a slow recuperation of frequency with the familiarization process (Jeewajee et al. 2008; Sambeth et al. 2009). The involvement of the hippocampus in place-learning ability in both rodents and humans has been established by experimental and clinical studies (Jarrard 1993; Milner 1972; Stubley-Weatherly et al. 1996). Lesions of the hippocampus and related brain structures lead to deficits in cognitive function during experimental place-learning tests performed in the Olton eight-arm radial maze or in the Morris water maze (Floresco et al. 1997; Jarrard 1993; Stubley-Weatherly et al. 1996). Suppression or alteration in hippocampal theta activity adversely affects animal performance in tests that require the establishment of spatial maps (Ammassari-Teule et al. 1991; Winson 1978). For example, lesions of the fornix in rats that have been submitted to the radial arm maze spatial task produce alterations in search patterns and increases in theta frequency after the lesion. Clonidine (α-noradrenergic agonist) administration causes a reduction in theta activity frequency to normal values and restores the behavioral performance of fornixlesioned rats (Ammassari-Teule et al. 1991; Maho et al. 1988). Furthermore, deficient spatial memory processing is associated with reductions in hippocampal theta activity (McNaughton et al. 2006; Pan and McNaughton 1997; Winson 1978). Both place-learning and theta activity rhythmicity (7.7 Hz) were restored using the SUMn oscillation to rhythmically stimulate the fornix and restore the rhythmicity in the hippocampus (McNaughton et al. 2006). In addition, Pan and McNaughton (1997) observed that the CDP infusion into SUMn induced a modest reduction in

5 Exp Brain Res (2013) 230: the peak theta frequency in hippocampus, which was associated with a mild impairment in a one-day Morris water maze test; thus, a relationship between changes in the theta frequency band and place-learning impairment has been observed. In a later work, Ruan et al. (2011) studied the relationships between the field potentials of hippocampus and SUMn during a spatial learning test using the Morris maze, based on directed transfer function analysis. The task was performed on 1 day with 16 consecutive trials in which the time that the rats remained on the platform was considered as the inter-trial interval. The authors reported an initial weak influence from hippocampus on SUMn, and that the direction of the influence between areas changed with the progression of the training trials, toward a weak control of the hippocampus by SUMn, which was concurrent with a learning-related increase in coherence that was observed in the last four trials. This result implies that SUMn participates when the consolidation of place-learning takes place in the last training trials in the task, as was previously suggested (Pan and McNaughton 1997; Shahidi et al. 2004). However, the authors grouped the results by quadrants when comparing the EEG data and it is possible that an analysis considering the training trial as a whole would be valuable. Such an analysis would allow information about the learning process to be observed across all training trials, assuming that the processing of information by the rats occurs similarly throughout the maze area, at least until a representation of the place and the position of the sunken platform is acquired and stored. There is evidence that damage to the cholinergic septohippocampal system disrupts hippocampal theta rhythms, and cholinergic neurotransmission disturbances are sufficient to impair orientating abilities (McNamara and Skeleton 1993; Nilsson and Bjorklund 1992; Nilsson et al. 1992; Vinogradova et al. 1995). Furthermore, in rat models of temporal lobe epilepsy, the initial insult caused by pilocarpine (muscarinic receptors agonist) induces immediate reductions in hippocampal theta power and deficiencies in place-learning ability independent of interictal activity (Chauviere et al. 2009). To determine whether hippocampal theta activity changes concurrently with the acquisition of spatial information in intact rats, we recorded CA1 EEGs during training in the Morris water maze over six training days. During place-learning training in the Morris water maze, concurrent recordings of rat EEGs showed increases in highfrequency theta activity ( Hz) during searching trials over training days; these increases were significant when place-learning occurred, as determined by significant reductions in distances traveled. This high-frequency learning-related theta increase is most likely related to the consolidation of spatial memory because it has been observed when rats are trained in the standard Morris task, 411 but not when cue-learning or egocentric learning strategies are displayed. Moreover, a significant correlation between high-frequency theta activity and escape latency exists in place-learning-trained rats; greater high-frequency theta activity power is associated with shorter platform-finding latency (Olvera-Cortes et al. 2002, 2004, 2012). These changes are absent in aged rats with decreased placelearning efficiencies, the swimming velocity increased with the training days only the last day in the old group, but not changes were observed in the young group (Olvera-Cortes et al. 2012). Furthermore, the absence of hippocampal theta activity associated with disruptions in place-learning acquisition in the Morris maze has been observed in rats after blockade of neuronal septal activity (McNaughton et al. 2006). Theta reset is a phenomenon that occurs when electrical stimulation of hippocampal afferents or sensorial stimulation induces the reset of the ongoing theta and unitary activity in the hippocampus (Buno et al. 1978; Vinogradova 1995; Williams and Givens 2003). The electrical stimulation of the fornix, medial and lateral septum, posterior hypothalamus, reticular mesencephalic formation, and entorhinal area evoked the phase-lock of theta activity and neuronal bursts of theta-firing units only when the ongoing activity presented theta frequency. Lesions of the fornix and the septal medial area prevent the theta reset induced by electric stimulation of the mentioned sites. The frequency of the post-stimulus theta was dependent on the stimulated structure; after fornix and septal stimulation, the following theta was low frequency (3 4 Hz), but after the stimulation of entorhinal cortex or the mesencephalic reticular formation, high-frequency theta activity was observed (6 7 Hz) (Buno et al. 1978). In later works, Givens (1996) investigated the theta reset in relation to working memory and reference memory tasks in rats. The working memory task consisted of a conditional continuous discrimination task using a box with two levers, in which the rats were trained to press one lever if a stimulus (tone or light) was similar to the previous, whereas the other lever must be pressed when the previous and the actual stimulus did not match. The lever press was associated with a reward of water. The reference memory task consisted of the association of one lever with the light presentation and the other with the tone presentation. The rats received daily trials. In both tasks, stimuli were separated by variable intervals. Dentate gyrus records were taken during the training on three sessions separated by at least 3 days, from which 1 min of pre-stimulus and 1 min of post-stimulus periods were analyzed and averaged to obtain a single spectral density plot per session. Additionally, the raw EEG data were averaged after being time-locked to the stimulus onset. The spectral density of the theta activity in the 6 9 Hz band was high in both tasks for the pre-stimulus period, but only the working-memory-trained rats showed a peak of power in the

6 412 Exp Brain Res (2013) 230: post-stimulus period in this band. In the analysis of the averaged raw data aligned by stimulus onset, the theta reset was only observed in the working-memory-trained rats. From these results, it is important to note that both tasks used similar sensory stimuli, but only the stimulus linked to working memory processes was associated with the theta reset of the dentate gyrus EEG. It was suggested that this mechanism allows the temporal coupling of septal and entorhinal inputs to dentate gyrus under behaviorally relevant conditions. In this sense, it was initially proposed that the theta reset could reflect a descending output from the hippocampus that is related to the establishment of temporal references used in motor sequences. Because both ascending and descending influences can reset theta activity, it was proposed that rhythmical time-locked feedback is required to establish temporal relationships between structures in complex tasks linking sensory inputs with motor responses (Velluti and Buño 1973). Later, it was proposed that theta reset could be related to the optimal conditions for long-term potentiation in hippocampus, because stimulation applied at the peak of the theta reset evoked by one working-memory-associated stimulus induced LTP, whereas stimulation at the trough of the theta reset failed to induce LTP (McCartney et al. 2004). In addition, theta reset has been proposed as a mechanism for selective attention to relevant stimuli, in which both the theta reset and the phase-locking of theta activity to relevant stimuli avoidance responses to other, non-relevant stimuli permit the storage in memory of relevant stimuli (Kitchigina 2010). Taken together, the previous results support the idea that theta activity attributes, such as power, frequency, coherence, and phase (between hippocampus and in relation to other relays of the SAS), could underlie hippocampaldependent learning and memory. Small changes in frequency or decreases in theta power are thus related to deficiencies in place-learning ability. Differential proportions of high- and low-frequency theta activities may be related to different functional conditions of the central nervous system; thus, dominant high-frequency theta activity (type 1, 6 12 Hz) or predominantly low-frequency theta activity (type 2, Hz) can be observed during walking and voluntary movement or during anesthesia and stimulation of the RPOn, respectively (Gavrilov et al. 1995; Vanderwolf 1988). Thus, it is possible that serotonin, which is involved in desynchronization of theta activity, may modulate learning processes through the underlying learningrelated changes in hippocampal EEGs. Serotonin as desynchronizer of hippocampal theta activity Medial raphe nucleus (MRn) serotonergic neurons are dispersed throughout the nucleus in small populations (10 12 μm) of oval neurons with short and coarse dendrites located dorsally. Ventrally are medium-sized (15 22 μm) and oval- or spindle-shaped long-dendrite neurons ( μm) (Vertes and Linley 2007). The hippocampus receives monosynaptic projections from the MRn (Arita et al. 1993; Conrad et al. 1974) through the medial forebrain bundle. These fibers pass through the diagonal band of Broca, the septal nuclei or cingulum bundle (CB), and the fimbria fornix (FF) to reach the subiculum and the hippocampus (Azmitia 1978; Conrad et al. 1974). GABA B receptors are selectively present on serotonergic neurons of the MRn (Varga et al. 2002). In addition to serotonergic neurons, the MRn contains glutamatergic neurons, cells that are immuno-positive for both serotonin and GAD (glutamate decarboxylase) (Forchetti and Meek 1981; Fremeau et al. 2002), and a significant number of GABAergic neurons (Forchetti and Meek 1981) that inhibit serotonergic neurons (Nishikawa and Scatton 1985). MRn fibers predominantly innervate the midline structures of the brain stem; i.e., the hypothalamus, thalamus, basal forebrain, and the hippocampus (Vertes et al. 1999). Certain components of the SAS, such as the RPOn, SUMn, PHn, and MS/DBB, and the hippocampus receive serotonergic fibers from the MRn (Assaf and Miller 1978; Azmitia 1978; Leranth and Vertes 1999; Vertes et al. 1999; Vertes and Linley 2007). Thus, the MRn can act on the SAS through many points or directly on the hippocampus to regulate theta activity (Fig. 1). A proportion of MRn neurons (8 12 %) send projections to both the hippocampus and the MS (McKenna and Vertes 2001). Although the MRn projections appear to be mainly serotonergic, a glutamatergic projection originating in the MRn cells has been reported (Sotty et al. 2003) that also reaches hippocampal and septal medial targets (Aznar et al. 2004; Colom et al. 2005; Sotty et al. 2003). Serotonergic axons form perisomatic and peridendritic asymmetric contacts on parvalbumin-positive GABAergic neurons (Aznar et al. 2004). Non-serotonergic neurons target septal and hippocampal calbindin-positive and parvalbumin-positive interneurons (Aznar et al. 2004). Dorsal raphe nucleus (DRn) fibers reach the SUMn, lateral septum, and entorhinal cortex and form moderate contacts with the hippocampal formation (Vertes 1991) and TPPn (Steininger et al. 1997). In the septum, DRn and MRn axons target segregated regions (Vertes et al. 1999). In the medial septum, MRn neurons principally target GABAergic cells (Aznar et al. 2004) and excite them, inhibiting pacemaker GABAergic and cholinergic septohippocampal neurons (Alreja 1996). Although both the DRn and MRn innervate the hippocampus, the projections of the MRn are considerably heavier than those from the DRn. MRn fibers target all regions of the dorsal and ventral hippocampus and are

7 Exp Brain Res (2013) 230: Fig. 1 Diagrammatic representation of some of the constituents of the SAS on which 5-HT may act to modulate hippocampal theta activity, with special focus on the evidence relating serotonin, hippocampal theta activity, and learning. Abbreviations are the same as those used in the text. Serotonin depletion in relays that code the frequency of hippocampal theta activity induces impairments in spatial learning, whereas depletion in relays in which frequency is not coded induces improvements. The traces into the boxes represent the power of the high-frequency theta band recorded in the hippocampus while searching for the platform or reward, and the segmented line into the boxes indicates the power level on the first day of training for a control rat more dense in the outer molecular layer (stratum lacunosomolecular) of CA1, CA3, and the inner molecular and granular cell layers of the DG (Vertes et al. 1999). Both electrical stimulation of the MRn and application of serotonin produce strong inhibition of the firing of hippocampal neurons, in rats, and this effect disappears when rats receive p-chlorophenylalanine (PCPA), a serotonin synthesis blocker (Segal 1975). This effect implies a mainly serotonergic mechanism. Furthermore, highfrequency electric stimulation of the MRn desynchronizes hippocampal EEGs (i.e., it induces low-voltage fast activity) in anaesthetized cats and rats (Assaf and Miller 1978; Macadar et al. 1974), and the effect was mediated by serotonin (Assaf and Miller 1978), whereas the electrolytic lesion of the MRn enhances the amplitude of hippocampal theta activity and elicits persistent and continuous theta (also during immobility, when its appearance is few frequent in normal rats) (Maru et al. 1979). Reductions in the frequency after MRn lesions have been reported for movement-related theta activity (from 7.4 to 6.8 Hz) and paradoxical sleep ( Hz). The immobility-related theta activity produced by MRn lesion is eliminated by atropine sulfate administration, whereas the theta activity produced during movement in lesioned MRn rats is resistant to atropine. As a result of a series of studies and based on findings showing that the double-lesion of serotonergic (through intra-raphe infusion of 5,7-DHT or ip administration of PCPA) and cholinergic (through ip administration of scopolamine or atropine) systems was able to eliminate all hippocampal theta activity in awake rats (both during immobility and walking), the neurochemical dissociation of a cholinergic-dependent and a serotonergic-dependent theta activity was proposed (Vanderwolf 1988; Vanderwolf and Baker 1986). The cholinergic-dependent theta activity would be present both during type 1 behavior (voluntary behavior) and type 2 behavior (reflexive behavior), whereas the serotonergic-dependent theta activity would occur only during type 1 behavior (Vanderwolf 1988). Further studies supported a desynchronizing role of serotonergic median raphe neurons on hippocampal EEG (see below). However, those results must be considered in light of more recent studies in which it has been suggested that the glutamatergic raphe neurons participate in theta generation through their influences on septal nocholinergic neurons (Crooks et al. 2012). These results revisit some early studies showing that the stimulation of MR in freely moving rats induces freezing behavior and hippocampal theta activity (with frequencies below 7 Hz) (Graeff et al. 1980; Peck and Vanderwolf 1991), which was facilitated by the administration of methysergide (serotonin antagonist) or p-chlorophenylalanine (Graeff et al. 1980). In line with this finding, it was shown that in rats under anesthesia, high-frequency MRn stimulation

8 414 Exp Brain Res (2013) 230: (100 Hz), which desynchronizes the hippocampal EEG, induces the reset of hippocampal theta activity, and concomitantly inhibits the activity of both theta-on and thetaoff phasic neurons, whereas theta burst stimulation (bursts of 100 Hz with duration of 80 ms and frequencies ranging from 30 to 170 ms) induced theta activity phase-locked to the stimulation. Because theta burst stimulation is a more similar pattern of neuronal activity, the authors proposed that at least some MRn neurons could participate in synchronizing the hippocampal EEG activity (Jackson et al. 2008). Although it is unknown whether these effects were mediated by serotonin, it is more probably that nonserotonergic neurons would participate as promoters of hippocampal theta, in view of the previous findings and considering the accumulation of evidence sustaining the desynchronizing effect of serotonin on the hippocampal EEG. The desynchronizing effect of serotonin on the hippocampal EEG was observed after injections of pharmacological compounds that suppress serotonergic neuron activity in the MRn (agonists 5-HT1 A and GABA) or reduce the excitatory functions of these neurons (i.e., antagonists of excitatory amino acids) that induce hippocampal theta activity with short latency and long duration. This finding has been observed, for example, with muscimol (GABA A, agonist) injections into the MRn, and it has been proposed that GABA inhibits serotonergic neurons (Kinney et al. 1995; Varga et al. 2002; Vertes et al. 1994). Furthermore, the administration of the 5-HT1 A /7 agonist 8-OH-DPAT (8-hydroxy-2-(di-n-propyl-amino)- tetralin) or buspirone into the MRn, but not the DRn, produces persistent hippocampal theta activity with short latency and long duration in anesthetized rats; this finding is in contrast to administration of 8-OH-DPAT into other nearby areas where no effect was observed (Vertes et al. 1994). The systemic blockade of serotonin receptors via the administration of methysergide (a non-selective 5-HT antagonist) in freely moving rats changes the threshold frequency with which septal stimulation current induces hippocampal theta activity from 7.7 to 6.9 Hz (Gyermek 1961). The same effect has been observed after selective serotonin depletion in the hippocampus by 5,7-dihydroxytryptamine (5,7-DHT) injection in the BC and FF. This effect is dependent on the reduction in serotonin, and extensive depletions cause a reduction in the threshold frequency for the induction of theta by septal stimulation (from 7.7 to 6.9 Hz) (McNaughton et al. 1980a, b). Furthermore, bilateral intra-cerebroventricular administration of fluoxetine (an inhibitor of serotonin reuptake) in awake rabbits induces a reduction in hippocampal theta power of at least to 50 % without changing the frequency peak (Kudina et al. 2004), an effect that supports the inhibitory role of serotonin on hippocampal synchronization. However, it has been reported that an inhibitor of serotonin reuptake, fluvoxamine, administered intravenously to anesthetized rats does not produce alterations in the single unit firing of MS/DBB neurons or changes in the frequency or power of hippocampal theta activity (Hajos et al. 2003a, b). The EEG and unitary activity of hippocampal neurons have been recorded after microinjections of 8-OH-DPAT into the MRn of rats foraging in different environments. This agonist produces behavioral arousal and increases the amplitude of hippocampal EEGs. This treatment causes GABAergic interneurons to show variable changes in unitary activity (mainly increases the firing rate), whereas the spatially related firing of the pyramidal cells remains unaffected (Nitz and McNaughton 1999). Crooks et al. (2012) studied the MRn modulation of hippocampal theta activity in anesthetized rats with simultaneous records of hippocampal theta activity and the unitary activity of septal neurons. These recordings were made both during spontaneous theta episodes and persistent theta that was induced by 8-OH-DPAT infusions into the MRn. In this condition, results similar to those of inactivation of the MS/DBB by lidocaine infusion or cholinergic blockade were realized. The persistent theta generated by 5-HT1 A stimulation of the MRn can be blocked by septal inactivation with lidocaine, indicating that the MS/DBB is crucial for persistent theta generation after serotonin reduction under anesthesia. Theta persistence is not blocked by concurrent infusion of scopolamine in the MS/DBB, implying that glutamatergic MRn neurons are relevant for theta generation. Because it has been shown that the agonist 8-OH-DPAT does not inhibit the firing rates of non-serotonergic neurons in the MRn (Calizo et al. 2011), it has been proposed that a putative glutamatergic input to non-cholinergic neurons of the MS/DBB may mediate the induction of cholinergicindependent hippocampal theta activity, and, together with serotonergic inhibition, act on both cholinergic and nocholinergic neurons in the MS/DBB to regulate hippocampal theta activity (Crooks et al. 2012). Thus, the MS/DBB is crucial for the serotonergic modulation of hippocampal synchrony. Another relay of the SAS in which an important serotonin influence has recently been observed is the TPPn. The injection of the 5-HT1 A receptor agonists 8-OH-DPAT or 5-carboxamidotryptamine (5-CT) into the TPPn of anesthetized rats induces persistent theta activity in the hippocampus with short onset latencies. It has been suggested that the inhibition of cholinergic neurons acts as the trigger for this persistent theta activity (Matulewicz et al. 2010). We do not know of any other works that assess the serotonergic modulation of theta activity via different relays of the SAS. However, the previously described results suggest that the

9 Exp Brain Res (2013) 230: effects of serotonin on hippocampal theta synchronization may be accomplished through different relays in the SAS (Table 1). With respect to the modulation of theta activity by subtypes of serotonin receptors, some research has evaluated the effects of systemic manipulation of agonists and antagonists. 5-HT2 C receptors are widely distributed in regions related to theta activity generation and expression, such as CA1, CA2, and CA3 of the hippocampus, and the MS/DBB (Clemett et al. 2000). The activation of 5-HT2 C receptors inhibits, whereas antagonists of these receptors facilitate, theta oscillations in MS/DBB neurons and hippocampal theta activity, in anesthetized rats; these findings imply that the suppressive action of serotonin on hippocampal theta may be mediated by 5-HT2 C receptors located in the MS/DBB (Hajos et al. 2003a, b). Furthermore, the effects of systemic administration of mcpp (m-chlorophenylpiperazine, 5-HT2 C agonist) in freely moving and anesthetized rats have been evaluated. mcpp in increasing doses or in combination with SB (6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridine-3-yl carbamoyl]indoline, 5-HT2 C antagonist) has been administered to rats, and EEGs have been obtained under both anesthesia and awake sleep conditions. mcpp reduces spontaneous theta activity and theta evoked by RPOn stimulation in dose-dependent manners; this effect is blocked by SB Theta activity associated with motor activity was reduced (20 %), and more robust effects have been observed during REM sleep (reduction of 60 %) after mcpp administration (Sorman et al. 2011). However, the antagonist SB does not affect theta activity during REM sleep (Kantor et al. 2005). These results highlight the relevance of the behavioral conditions in which theta expression and modulation are evaluated (i.e., anesthetized or during behavior). Furthermore, the results indicate that 5-HT2 C receptors suppress behavior-associated theta activity, likely via the MS/DBB, although results obtained following the systemic administration of drugs imply that these effects could be due to the additive effects in each relay of the SAS. Additionally, there is evidence relating 5-HT3 receptors and theta activity modulation. The systemic administration of the 5-HT3 receptor antagonist ondansetron increases hippocampal theta frequency dose dependently (Staubli and Xu 1995). Serotonin acting on 5-HT3 receptors has been shown to excite GABAergic hippocampal and septal interneurons (Alreja 1996; McMahon and Kauer 1997). Accordingly, it has been shown that ondansetron causes reductions in the firing rates of a subgroup of hippocampal interneurons (Reznic and Staubli 1997). These effects on both the MS and the hippocampus must influence changes in frequency of theta activity. 415 Lastly, the role of 5-HT6 receptors in the modulation of theta activity was recently addressed. The hippocampus has high levels of 5-HT6 receptor expression principally on GABAergic interneurons (Woolley et al. 2004). The effects of administration of the 5-HT6 agonist (EMD386088, 5-chloro-2-methyl-3-(1,2,3,6-tetrahydro-4-pyridinyl)- 1H-indole hydrochloride) and antagonist (SAM-531) on theta activity in anesthetized (evoked by reticular stimulation) and freely moving rats (trough the sleep awake states) has been observed. 5-HT6 activation decreases the theta peak frequency in freely moving and anesthetized rats, and this effect is blocked by the co-administration of an antagonist. However, the administration of the antagonist alone does not cause changes in theta activity in freely moving or anesthetized rats; thus, serotonin does not exert an inhibitory effect on hippocampal synchrony through the activation of this receptor (Ly et al. 2013). As evidenced by the previous section, despite the several hypotheses involving the serotonergic desynchronization of hippocampal EEG as a mechanism of selectivity in information processing (Jeltsch-David et al. 2008; Vertes and Kocsis 1997; Vinogradova et al. 1999), there is little available data regarding theta modulation by serotonin in behaving animals, particularly during learning processes and at the specific sites of action in the SAS (Table 1). The functional impact of serotonin s influence on each relay of the SAS needs to be investigated both in anesthetized and in behaving animals; the latter is a field that is relatively unexplored. The available evidence regarding serotonin s modulation of learning and memory creates a complex picture. Serotonin and hippocampal processing of information Serotonin has been implicated in several behavioral functions and pathologies. The involvement of serotonin in mood regulation, aggression, impulsivity, and anxiety is well documented (Andrews et al. 1994; Deakin and Graeff 1991), and disturbances of this neurotransmitter system have been implicated in pathologies of depression, bipolar disorder, obsessive compulsive disorder, and schizophrenia (Coffey and Shechter 2006; Dursun et al. 2000; Meltzer 1989; Tibrewal et al. 2010). However, evidence relating the serotonergic system to cognition is lacking. In healthy human volunteers, reductions in cerebral serotonin by acute tryptophan depletion (ATD) cause deficiencies in long-term memory performance that are evident during delayed recall (30 min) of a word list (Riedel et al. 2002), visually presented words, pictures, and abstract figures (Harrison et al. 2004; Schmitt et al. 2000). It has been proposed that serotonin is involved in consolidation of information in long-term memory in

10 416 Exp Brain Res (2013) 230: Table 1 Summary of some representative evidences about the desynchronizing effect of serotonin and the NRM on hippocampal theta activity, considering the behavioral Experimental strategy Cerebral area Behavior Septal effect Hippocampal effect References Electrical stimulation MRn Anaesthetized rats Deszynchronization Segal (1975), Anchel and Lindsley (1972), Maru et al. (1979) Electrolytic lesion MRn Anaesthetized rats Synchronization, theta persistent Maru et al. (1979) Behaving rat Presence of theta activity during immobility Reduction in frequency of movement-related theta 5-HT1 A agonist (8-OH-DPAT) MRn Anaesthetized rats Synchronization, theta persistent Vertes et al. (1994) 5-HT1 A agonist (8-OH-DPAT) MRn Foraging rats GABAergic neurons changed their properties Nitz and McNaughton (1999) of firing Principal neurons did not change 5-HT1 A agonist (8-OH-DPAT) MRn Anaesthetized rats Intraseptal lidocaine Blockade of 8-OH-DPAT theta induced Crooks et al. (2012) Intraseptal atropine No changes in 8-OH-DPAT theta induced Muscimol (GABA A agonist) MRn MRn Theta persistent Kinney et al. (1995) Facilitation of theta induction Gyermek (1961) Blockade of 5-HT receptors (methysergide) Inhibitor of 5-HT reuptake (fluvoxamine) Inhibitor of 5-HT reuptake (fluoxetine) 5-HT2 C agonists (m-cpp, Ro ) Systemic Anaesthetized rats Reduction in frequency threshold to induce theta Systemic Anaesthetized rats No changes in unitary No changes Hajos et al. (2003a, b) firing ICV Immobile awake rabbit Reduction in theta power Kudina et al. (2004) Intravenous Anaesthetized rats Abolition of theta oscillations 5-HT2 C antagonist (SB ) Enhanced theta oscillations 5-HT2 C agonist (m-cpp) Systemic Anaesthetized rats/awake sleep Abolition of theta oscillations of neurons and theta desynchronization Enhancement of theta oscillation of neurons and enhanced theta activity Reduction in spontaneous and RPO-stimulation-evoked theta in dose-dependent manner 5-HT2 C antagonist (SB ) Increase in theta activity in freely moving but not during REM sleep 5-HT6 agonist (EMD386088) Systemic Anaesthetized rats/awake sleep Decreased theta peak frequency both in anaesthetized and in awake rats Hajos et al. (2003b) Sorman et al. (2011) Ly et al. (2013) 5-HT6 antagonist (SAM-531) No effect 5-HT1 A agonist (8-OH-DPAT, TPPn Anaesthetized rats Theta persistent Matulewicz et al. (2010) 5-CT) Facilitation of theta induction McNaugthon et al. (1980a, b) Serotonin depletion (5,7-DHT in CB and FF) Hippocampal Anaesthetized rats Reduction in frequency threshold to induce theta