[1] 16w Cell discharge correlates of posterior hypothalamic theta rhythm. Recipe for success in recording stable field potential

[1] 179w The theta rhythm discovered in the posterior hypothalamus area (PHa) differs from theta observed in the hippocampal formation. In comparison to hippocampal spontaneous theta, the theta recorded in the PHa is rarely registered, has lower amplitude, often disappears, and sometimes returns after a few minutes. These features indicate that spontaneous theta recorded in the PHa is not an appropriate experimental model to search for the correlation between PHa cell discharges and local field potential. In this paper we present standard experimental conditions necessary to record theta-related cells in the PHa in anesthetized rats. Three pharmacological agents were used in the experiments to induce PHa theta rhythm in urethanized rats: carbachol (CCH), carbenoxolone and kainic acid, which are potent enough to induce well-synchronized PHa theta. However, CCH was found to be the best pharmacological tool to induce PHa theta oscillations, due to its longest duration of action and lack of preliminary epileptogenic effects. It seems that CCH-induced theta can be the most suitable pharmacological model for experiments with the use of protocol of long-lasting recordings of PHa theta-related cell discharges.

[1] 18w Electrical recordings from deep brain structures using wire electrodes are one of the relatively old methods in neuroscience.

[2] 101w The combination of local field potential measurements with recordings of neuronal discharges is the best experimental tool available to study the behavior of small populations of neurons. Since the membrane current generated by single neurons passes electronically through extracellular space it can be measured by electrodes placed outside neurons. Field potentials recorded at any given site reflect the linear sum of fields generated by current sources and sinks distributed along multiple cells. If an electrode is placed close to a neuron, recorded extracellular currents will provide information about the discharges of this given cell and neighboring neurons (Nádasdy et al., 1998).

[3] 82w One of the field potentials which has been intensively studied since the early 1950s is theta rhythm (Green and Arduini, 1954;Liberson and Cadilhac, 1954;MacLean et al., 1952). It is one of the most synchronized electroencephalographic (EEG) activities that can be recorded in several brain regions, for example: the cingulate cortex (Landfield and McGaugh, 1972), the entorhinal cortex (Alonso andGarcía-Austt, 1987a, 1987b) or the hippocampal formation (HPC), which is considered to be the main structure involved in the generation of theta (Bland, 1986).

[4] 212w Previous studies reported that the generation of theta field potential recorded in different brain structures is accompanied by a characteristic pattern of cell discharges (Bland and Colom, 1993;Colom and Bland, 1987;Ford et al., 1989;Kowalczyk et al., 2013aKowalczyk et al., , 2013b)). Taking into consideration the spike train dynamics of a given cell in relation to changes in simultaneously occurring theta and large irregular activity (LIA), hippocampal neurons were classified as theta-related and theta non-related. Theta-related cells were classified as "theta-on" and "theta-off" (Colom and Bland, 1987). As the names implied, "theta-on" cells increased their activity during theta field activity, whereas "theta-off" cells decreased their activity during theta. Both "theta-on" and "theta-off" cells were subclassified as phasic and tonic (Colom and Bland, 1987). Some theta-related neurons can also be classified as gating cells (Konopacki et al., 2006;Kowalczyk et al., 2013b). All types of theta-related cells were described in the HPC, but in subsequent experiments they were also observed in the medial septum (Colom and Bland, 1991;Ford et al., 1989), the supramammillary (SuM) nucleus (Bland et al., 1995;Kirk et al., 1996;Kocsis and Vertes, 1994), posterior hypothalamus nucleus (PH; Bland et al., 1995;Kirk et al., 1996), and entorhinal cortex (Dickson et al., 1994(Dickson et al., , 1995) ) during simultaneously recorded hippocampal theta field activity.

[5] 104w Just recently Kowalczyk et al. (2014) demonstrated for the first time that theta activity could be also observed in the posterior Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/brainres hypothalamus area (PHa) in anesthetized rats. Furthermore, they showed that in vivo theta recorded in the PHa was produced independently of simultaneously occurring HPC theta. Experimental evidence indicates that the PHa, primarily the posterior hypothalamic and supramammillary nuclei of the posterior hypothalamic area (Vertes, 1982), comprise a critical part of the ascending brainstem synchronizing pathway (Oddie et al., 1994;Thinschmidt et al., 1995;Vertes and Kocis, 1997;Kirk, 1998;Bocian andKonopacki, 2001, 2004;Pan and McNaughton, 2004;Bocian and Konopacki, 2007).

[6] 181w Typically, in rodents hippocampal theta rhythm occurs spontaneously in short epochs divided by large irregular activity. Kowalczyk et al. (2014), demonstrated that theta rhythm observed in PHa differs significantly from theta recorded in the HPC. Firstly, the PHa theta field potential is much lower in amplitude (approximately 7-8 times) than the HPC signal. Secondly, when the appropriate level of anesthesia is applied a spontaneous transition from theta into irregular activity and vice versa can be observed. This effect is well pronounced in the HPC, whereas in PHa theta oscillations are unstable, i.e. theta activity can occur in the PHa for several seconds and then may disappear for a few minutes. These findings indicates that spontaneous theta activity recorded from the posterior hypothalamic area is not an appropriate experimental model to search for the correlation between PHa cell discharges and local theta field potential. Indeed, the in vivo extracellular recording requires a specific experimental conditions including carefully controlled level of anesthesia in which rats typically cycle spontaneously between theta and LIA field activity, each lasting for periods of 5-10 s or longer.

[7] 28w In this paper we present the standard experimental conditions necessary for recordings of theta-related local cell discharges localized in the posterior hypothalamic and supramammillary nuclei in anesthetized rats.

[1] 66w Histological analysis revealed that in all rats the electrodes for recordings of posterior hypothalamic field activity were located either in PH or SuM and microinjection cannula tracks were localized in PHa in the frontal plane between the range of 3.6-3.8 mm (Paxinos and Watson, 2014). Moreover, histological procedures verified that all recorded neurons were localized in the PH or SuM nuclei of the posterior hypothalamic area.

[2] 242w Four doses of CCH were tested: 0.5 μg/0.5 μl, 1.0 μg/0.5 μl, 1.5 μg/0.5 μl and 2.0 μg/0.5 μl, respectively. Intrahypothalamic injection of the two highest doses (1.5 μg/0.5 μl and 2.0 μg/0.5 μl) induced only epileptiform discharges (data not shown). CCH applied in a concentration of 1.0 μg/0.5 μl was recognized as effective in producing repeatable epochs of PHa theta rhythm (Fig. 1). In the first stage of postinjection recordings (15 min) continuous and well-synchronized epochs of theta rhythm were observed. In the second stage (30 min), typically a few seconds epochs of theta were separated by large irregular activity (Fig. 1, left panel). Amplitude of theta rhythm measured 30 min postinjection was higher than amplitude observed in control conditions (251.978.1 μV vs. 169.37 18.7 μV; Fig. 1, left panel). CCH-induced theta rhythm was still very well-developed 90 min postinjection. The effect of CCH injection was reversible after 120 min, i.e. theta rhythm observed in this postinjection time resembled theta activity recorded in the control conditions (Fig. 1, left panel). It is worth noting that intrahypothalamic injection of CCH not only facilitated theta rhythm in anesthetized rats that produced spontaneous theta, but also induced theta rhythm when spontaneous activity in theta band was not observed in control conditions (Fig. 1, right panel). The lowest concentration of CCH (0.5 μg/0.5 μl) was found to be a subthreshold dosage: it did not induce any apparent changes in PHa field potential (data not shown).

[3] 192w In separate experiments two doses of CBX were tested: 25 μg/ 0.5 μl and 50 μg/0.5 μl, respectively. Intrahypothalamic administration of CBX in a concentration of 25 μg/0.5 μl never produced PHa theta rhythm (data not shown). A twice higher concentration of CBX (50 μg/0.5 μl) was found to be effective and in the first postinjection period (15 min) induced long-lasting and well-synchronized epochs of theta rhythm in the PHa (Fig. 2, left panel). In the second postinjection period (30 min) typically, a few seconds of theta epochs were separated by large irregular activity (Fig. 2, left panel). Amplitude of CBX-induced theta, measured 30 min postinjection, was higher than the amplitude observed in control conditions (234.5 79.4 mV vs. 148.5 711.2 mV). The effect of CBX injection was reversed after 60 min, i.e. theta rhythm observed in this postinjection time resembled theta activity recorded in control conditions before drug injection (Fig. 2, left panel). It is worth noting that intrahypothalamic injection of CBX not only facilitated theta rhythm when spontaneous theta was observed in control conditions, but also evoked theta rhythm which was not observed in control conditions (Fig. 2, right panel).

[4] 217w In separate experiments the effect of two concentrations of KA on the posterior hypothalamic EEG activity was evaluated: 0.1 μg/ 0.5 μl and 0.25 μg/0.5 μl. The concentration of 0.1 μg/0.5 μl was found to be a subthreshold one. It has never produced PHa theta rhythm (data not shown). Following intrahypothalamic injection of KA in concentration of 0.25 μg/0.5 μl in locally recorded EEG, two different patterns of field potentials were observed: epileptiform discharges and theta activity (Fig. 3). Epileptiform discharges were recorded immediately after compound administration and maintained at least for 15 min (Fig. 3, left panel). After this time well-synchronized epochs of theta rhythms were observed. Recorded 30 min postinjection, theta was characterized by increased amplitude in comparison to the control conditions (213.8 77.2 μV vs. 157.8 714.7 μV; Fig. 3, left panel). KA-induced theta rhythm was still very well-developed 60 min postinjection. The effect of KA injection in concentration 0.25 μg/0.5 μl was reversible after about 90 min, i.e. theta rhythm observed in this postinjection time resembled theta activity recorded in control condition (Fig. 3, left panel). Intrahypothalamic injection of KA not only facilitated theta rhythm in anesthetized rats that produced spontaneous theta but also evoked theta rhythm when spontaneous activity in theta band was not observed in control conditions (Fig. 3, right panel).

[5] 122w Interestingly, the analysis of the rat age revealed that the age per se could be also the essential factor that determines success in recording hypothalamic spontaneous theta activity. We noted that the probability of recording posterior hypothalamic spontaneous theta rhythm gradually increased with age. Specifically, among the animals aging 40-45 days only 3 out of 20 rats (15.0%) generated spontaneous PHa theta rhythm (Fig. 4). When the rat's age reached 50-65 days the probability amounted to 73.9% (17 out of 23 rats generated spontaneous PHa theta rhythm, Fig. 4). Surprisingly, in the third group of rats aging 70-75 days, the probability of recording spontaneous theta again decreased to 30.8% (only 4 out of 13 animals generated spontaneously PHa theta rhythm; Fig. 4).

[1] 213w Theta rhythm is one of the most synchronized EEG activities that can be recorded mainly from the hippocampal formation (Bland, 1986), entorhinal cortex (Alonso andGarcía-Austt, 1987a, 1987b) and cingular cortex (Landfield and McGaugh, 1972). Recently, Kowalczyk et al. (2014) indicated that this EEG pattern could also be observed in the posterior hypothalamic area, both in vivo and in vitro conditions. Mapping studies showed that the PH and SUM nuclei were the most effective PHa sites in generating theta field activity in anesthetized rats. Theta recorded in the PHa was produced independently of simultaneously occurring hippocampal theta. Detailed analysis of the recorded PHa theta revealed that the tested EEG pattern differs significantly from theta recorded from HPC. Posterior hypothalamic theta activity, in contrast to HPC theta, has a much lower amplitude (usually 7-8 times than HPC signal). In addition, PHa theta oscillations are rather unstable, that is, they often disappeared and sometimes returned after a few minutes. Furthermore, it cannot be induced by sensory stimulus, for example a tail pinch. All these features make spontaneous PHa theta to be rather hard to register. This resulted in a relatively low percentage of successful experiments (about 50%), i.e. experiments in which epochs of spontaneous PHa theta were observed throughout the entire experiment (Kowalczyk et al., 2014).

[2] 144w In a light of the above-mentioned data the question arises whether PHa spontaneous theta rhythm recorded in anesthetized rats can be recognized as a useful experimental model for studying local cell discharges. Simultaneously recording theta rhythm and theta-related cell behavior requires specific experimental conditions, among which well-developed theta activity and a pronounced transition from theta mode into irregular activity mode are absolutely necessary. Taking into consideration these requirements, PHa spontaneous theta per se does not seem to be an appropriate model to search for the correlation between cell discharges and locally recorded theta activity. Hence we attempted in the present study to induce repeatable epochs of theta rhythm with the use of some specific agents earlier recognized to be potent enough to induce theta (Diamond et al., 1992;Garner et al., 2005;Kowalczyk et al., 2013aKowalczyk et al., , 2013b;;Margineanu and Klitgaard, 2001;Sinfield and Collins, 2006).

[3] 118w Since Kowalczyk et al. (2014) reported that PHa theta activity shares a similar neurochemical (cholinergic-muscarinic) profile with hippocampal theta, that is, it was antagonized by muscarinic blocker (atropine sulphate) we injected into posterior hypothalamus a carbachol, cholinergic agonist. It was found that the intrahypothalamic injection of this agent in a concentration of 1.0 μg/0.5 μl induced well-synchronized theta rhythm. CCH-induced theta field potentials appeared immediately after compound administration and cycles of theta rhythm-LIA were recorded regularly for almost two hours. Of the four tested CCH concentrations, the dosage of 1.0 μg/0.5 μl was demonstrated to be the most suitable for an experiment in which long-lasting recordings of PHa cell discharges and local theta epochs are necessary (Fig. 5A).

[4] 356w In separate experiments the gap junction blocker and agonist of mineralocorticoid receptor (MR)carbenoxolone was injected into or PH or SuM nuclei. Many studies conducted on different experimental models revealed that the interaction of CBX with gap junction evoked an inhibitory or abolishing effect on hippocampal theta (Bocian et al., 2009;Gołębiewski et al., 2006;Konopacki et al., 2004). Additionally, it was shown with use of lateral geniculate slices that CBX reversibly depressed the peak power of local theta band activity produced by activation of the metabotropic glutamate receptor (Hughes et al., 2004). Garner et al. (2005) also demonstrated a reduction in power of kainate-induced theta band oscillations in medial septum-diagonal band slices after the application of CBX. These findings are in complete contrast to the effect of CBX observed in the present studies. Posterior hypothalamic injection of CBX induced long-lasting, well-synchronized epochs of theta rhythm in the PHa. CBX-induced theta appeared usually in the first 5 min of compound administration, and was reversible after 60 min. The excitatory effect of CBX on PHa theta in the present studies could also be analyzed in the light of CBX activation of mineralocorticoid receptors. Immunohistochemical and cytogenetic studies have shown that the brain mineralocorticoid receptors are present in the hippocampus, cortex, brain stem, spinal cord, pituitary and also in the hypothalamus (Gomez-Sanchez, 2014; Moisan et al., 1990). Interestingly, MR are coexpressed with 11β-hydroxysteroid dehydrogenase (11β-HSD), which metabolizes corticosterone to inactive 11-dehydrocorticosterone and thus protects the MR from exposure to corticosterone in vivo (Gomez-Sanchez et al., 2008). This means that 11β-HSD may regulate the access of corticosterone to cerebral mineralocorticoid receptors and modulate the effect of corticosteroid on brain functions. It was reported that CBX has the ability to block 11β-HSD, thereby permitting corticosterone to activate MR and induce an excitatory effect (Zhang et al., 2006). The abovementioned data allows us to suggest that the excitatory effect of CBX on the PHa theta field potential is mediated by mineralocorticoid receptors. Of the two tested CBX concentrations, the dosage of 50 μg/0.5 μl was found to be the most suitable for experiments with recordings of PHa theta epochs and cell discharges (Fig. 5B).

[5] 264w In the last series of experiments we studied the effect of local injection of kainic acid on the posterior hypothalamic EEG activity. Many cytogenetic studies have demonstrated the widespread expression of all types of glutamate (AMPA, kainate and NMDA) receptors in the hypothalamus (Biziere et al., 1980;Eyigor et al., 2001Eyigor et al., , 2012;;Van den Pol et al., 1994;Wisden and Seeburg, 1993). The presence of many different types of ionotropic glutamate receptors throughout the hypothalamus suggests that multiple modes of ion channel regulation by glutamate probably operate in this structure what provides support for the importance of excitatory transmitter glutamate in hypothalamic regulation. In the present study the intrahypothalamic injection of KA (0.25 μg/ 0.5 μl) evoked two different patterns of field potentials in locally recorded EEG: epileptiform discharges and theta activity. The first pattern was recorded immediately after compound administration and maintained in EEG less than half an hour. After this time, the second EEG pattern, i.e. theta rhythm, was recorded. KA-induced theta was well-synchronized and theta rhythm-LIA cycles were clearly visible in the recording of posterior hypothalamic EEG activity up to 90 min. Of the two tested KA concentrations, the dosage of 50 μg/0.5 μl was found to be the most suitable for experiments with long-lasting recordings of PHa theta epochs and cell discharges (Fig. 5C). It is worth noting that the intrahypothalamic injection of KA, as well as CBX and CCH, not only facilitated theta rhythm in anesthetized rats that produced spontaneous theta, but also evoked theta rhythm when spontaneous activity in the theta band was not observed in control conditions.

[6] 106w Considering the three pharmacological agents used in the present study, we can conclude that all of them, when are applied in proper concentrations, are potent enough to induced wellsynchronized PHa theta. However, there are substantial differences between the agents' time effect and ability to induce epileptic activity. CCH, in concentration of 1 μg/0.5 μl, has the longest time duration and does not cause of epileptogenic effects and is the best pharmacological compound for the induction of PHa theta oscillations. Hence, it seems that CCH-induced theta can be the most suitable pharmacological model for experiments with use of protocol of long-lasting recordings of PHa theta-related cell discharges.

[7] 431w One more issue should to be discussed at the end. Kowalczyk et al. (2014) suggested that the most effective sites in generating theta field activity in anesthetized rats were the PH and SUM nuclei. However, even if the localization of the recording electrode is proper (i.e. it is precisely positioned in the PH or SUM nuclei) the probability of theta recordings remains still only about 50%. In the present study we accidentally observed that the animal age could be an additional factor that determines the success in recording spontaneous hypothalamic theta. The probability of recording PHa theta gradually increased with age. Specifically, when the rats aged 60-65 days, the probability amounted to over 70%. We suppose that this phenomena is associated with the postnatal ontogenesis of the neuronal network in the hypothalamus. Indeed, numerous data has revealed that postnatal ontogenetic processes are held intensively in different areas of the hypothalamus in the first two-three weeks of life (Borisova et al., 1991;Huang et al., 2010;Sumová et al., 2008;Ugrumov et al., 1989). For example, Sumová et al. (2008) reported that synchronized molecular oscillations in suprachiasmatic nuclei develop gradually in the early postnatal period. It is possible that the same process occurs in the case of the neuronal network connected with theta generation in PHa. Interestingly, in the hippocampal formation, considered as the main structure involved in theta production, activity in the theta band appears progressively in the EEGs of young rats (Creery and Bland, 1980;Leblanc and Bland, 1979). Cholinergic theta field potential was observed in rats between 10 and 14 days. The ability to register hippocampal theta at this time could be associated with the postnatal development of the cholinergic system. Immunohistochemical studies have clearly demonstrated that the concentration of muscarinic-like binding sites in HPC increase progressively with age (Falkeborn et al., 1983). Unfortunately, the above-proposed ontogenetic hypothesis does not explain the decrease in the probability of spontaneous PHa theta recoding in animals aging 70-75 days. Rats in this the age can be considered to be adult, but not old. Hence, the decrease in probability of recording PHa theta in 70-75 days of age rats is definitively not related with processes of ageing. On the other hand it should be emphasized that rats aging 70-75 days and weighing over 250-300 g usually generate more technical problems related with surgery than younger animals. Specifically, the bleeding is more intense, particularly during jugular cannulation and drilling trepanation holes. The loss of blood may have an indirect effect on anesthetized rats and thus the quality of the recorded EEG signal and the probability of theta registration.

[8] 41w In conclusion, we have described the effect of three different pharmacological agents on the posterior hypothalamus field potential. CCH-induced theta rhythm is the most suitable pharmacological model for experiments which use the protocol of longlasting recordings of PHa theta-related cell discharges.

[1] 26w All experiments described below were monitored by a Local Ethic Commission (permission no. 74/ŁB 688/2013 in accordance with European Communities Council Directive of 24 November 1986).

[2] 159w Fifty-six male Wistar rats weighting from 120 g to 300 g (animals aging 40-75 days) were used in the experiments. Rats were initially anesthetized (Dragger vapor 19.3, USA) with halothane (Narcotan, Sigma Chemical Co., St. Louis, USA) while a jugular cannula was inserted. Halothane was then discontinued, and urethane (0.6 g/ml Sigma Chemical Co., St. Louis, USA) was administered via the jugular cannula in order to maintain anesthesia throughout the experiment. Next, the rats were placed in a stereotaxic frame, with the plane between the bregma and lambda levelled to horizontal. The anesthesia level was maintained such that theta field potentials and the transition from theta to large irregular activity could occur spontaneously (Bocian et al., 2009(Bocian et al., , 2011)). Body temperature was maintained at 36.5 °C (70.5 °C) by a heating pad, and heart rate was monitored constantly throughout the experiment. To avoid dehydration, at the beginning of experiments all animals received physiological saline (10 ml/kg sc).

[3] 89w Fifty-six experiments were performed to develop the proper pharmacological model allowing for the correlation between theta-related cells activity and posterior hypothalamic EEG. Three different pharmacological agents were used in separate experiments to induce PHa theta rhythm. In the first step of experiments four doses (0.5, 1.0, 1.5 and 2.0 μg/0.5 μl) of cholinergic agonistcarbachol (CCH, Sigma Chemical Co., St. Louis, USA) were tested in 4 groups of animals (7 rats each). In the next step, two doses (25 μg and 50 μg/0.5 μl) of carbenoxolone (Sigma Chemical Co., St.

[4] 64w Louis, USA), recognized as a gap junction blocker and mineralocorticoid agonist receptor were tested in 2 groups of animals (7 rats each). Finally, kainic receptor agonist -kainic acid (KA, Sigma Chemical Co., St. Louis, USA) was administered in concentrations of 0.1 μg/0.5 μl and 0.25 μg/0.5 μl in 2 groups of animals (7 rats each). All agents were dissolved in pharmacological saline (Polpharma, Poland).

[5] 30w To characterize the effect of CCH, CBX and KA on the posterior hypothalamic EEG activity, 5 min continuous, control recordings of local spontaneous activity were made prior to each microinjection.

[6] 77w Tested agents were injected into the PH or SuM nuclei of posterior hypothalamic area even when posterior hypothalamic spontaneous theta rhythm was not observed. After administration of CCH, CBX and KA 5 min recordings were continued in successive postinjection time periods (after CCH injection in 15,30,45,60,90 and 120 min;after CBX injection in 15,30,45,and 60 min;after KA injection in 15,30,45,60 and 90 min). Ten 2-s EEG samples containing theta rhythms were selected for analysis from successive time periods.

[7] 60w Data obtained from present experiments was analyzed off-line using the Spike 2.7 software computing system (Cambridge Electronic Design, Cambridge, GB). Two-second samples of PHa EEG activity (from PH or SuM), recorded after the injection of different pharmacological agents, were subjected to power/frequency (FFT) analysis. The mean peak-to-peak amplitude of the posterior hypothalamic theta rhythm was determined directly from EEG recordings.

[8] 75w The pattern of discharging neurons was classified in relation to ongoing PHa field potentials. Cell firing patterns recorded in the PHa were analyzed off-line as described previously (Colom and Bland, 1987;Ford et al., 1989). Each neuron was classified as a theta-related or theta non-related cell, according to the discharge pattern manifested during spontaneous theta epochs and LIA states. Neurons that discharged irregularly during both theta epochs and LIA states were classified as theta non-related cells.

[9] 118w Theta-related cells were divided into two types: theta-on and theta-off neurons. Cells were categorized as theta-on if they met the following criterion: the mean discharge rate occurring during theta epochs was greater than that occurring during LIA. Within this broad definition of a theta-on cell there were two subtypes. The first was termed phasic. These cells discharged in a rhythmic manner, phase-locked with ongoing theta rhythm. The second subtype was termed tonic. Tonic cells discharged in an arrhythmic pattern with no relation to the phase of simultaneously recorded theta field activity. Cells were classified as theta-off if they met the following criterion: the mean discharge rate occurring during the theta epochs was lower than that occurring during LIA.

[10] 93w According to our previous studies (Konopacki et al., 2006;Kowalczyk et al., 2013aKowalczyk et al., , 2013b)), some neurons were also classified as gating cells if they met one of the following criteria: (i) the cell discharges occurred precisely in the beginning and at the end of each theta epoch (gating cell A); (ii) the cell began to discharge just before the transition from non-theta interval/LIA into the theta epoch (gating cell B); (iii) the cell began to discharge just after the transition from the theta epoch into non-theta interval/ LIA (gating cell C).

[11] 42w The rats were sacrificed by an overdose of urethane for histological examination. The brains were removed and stored in 10% formalin. Frozen brain sections (30 mm) were taken serially and mounted on glass slides for the reconstruction of PHa theta recording sites.

[1] 58w In separate experiments PHa cell discharges and local theta epochs were recorded in the presence of local injection of CCH (n ¼7), CBX (n ¼7) and KA (n ¼7). Fig. 5 demonstrates that three pharmacological agents used in these testing experiments can be used in experimental protocol for successful recording PHa theta field potentials and local cell discharges.

[2] 160w A tungsten electrode (0.1-0.9 MΩ) for recording posterior hypothalamic area field activity was implanted either in the right posterior hypothalamic nucleus or the supramammillary nucleus. These nuclei have been earlier recognized as the most effective sites in generating theta field activity (Kowalczyk et al., 2014). The stereotaxic coordinates for the PH were the following: from 4.3 mm posterior to bregma, 0.5-0.7 mm lateral to midline, and 7.2-7.7 mm ventral to the dural surface (Paxinos and Watson, 2014). The stereotaxic coordinates for the SuM were the following: from 4.3 mm posterior to bregma, 0.5-0.7 mm lateral to midline, and 8.0-8.5 mm ventral to the dural surface (Paxinos and Watson, 2014). The recording electrode was implanted into SuM only when spontaneous theta activity was not observed after placement of the electrode in PH. Additionally, an uninsulated tungsten wire placed in the cortex, 2 mm anterior from the bregma, served as an indifferent electrode, and the stereotaxic frame was connected to a ground.

[3] 84w During all experiments AC amplifiers (P-511, Grass-Astromed, West Warwick, USA) were used for recording PHa field potentials, with the low filter set at 1 Hz and the high filter set at 0.3 kHz. The field activity was displayed using a digital storage oscilloscope (TDS 3014B; Tektronix, Beaverton, USA). EEG signals were digitalized by Micro 1401 interface (Cambridge Electronic Design, Cambridge, GB) and recorded onto a computer hard disk for subsequent off-line analysis using the Spike 2.7 software computing system (Cambridge Electronic Design, Cambridge, GB).

[4] 209w Glass microelectrodes (6-9 MΩ) made from Kwik-Fill capillaries (Word Precision Instruments, Sarasota, FL, USA) to record single units were filled with 0.5 M sodium acetate mixed with 2% Pontine Sky Blue (Sigma Chemical Co., St. Louis, USA). Tip locations (blue dots) of the microelectrodes for each cell recorded were marked by passing 15 mA current for 14 min (7 min cathodal, 7 min anodal; S48 stimulator, Grass-Astromed). The glass recording electrode was positioned with the use of a micropositioner (model 660; Kopf, USA) in the left the PH nucleus according to the following coordinates: 4.3 mm posterior to bregma, 0.5-0.7 mm lateral to midline, 7.2 mm ventral to the dural surface or in left part of the SuM nucleus according to the following coordinates: 4.3 mm posterior to bregma, 0.5-0.7 mm lateral to midline, 8.5 mm ventral to the dural surface (Paxinos and Watson, 2014). Cell activity signals were passed to the Grass-Astromed model P511 wide-band AC preamplifier with the low filter set at 300 Hz and the high filter set at 3 kHz. The field and cell activities were displayed simultaneously using a Tektronix TDS 3014B digital storage oscilloscope. All signals were stored on hard disk for subsequent off-line computer analysis (Spike 2.7, Cambridge Electronic Design, Cambridge, GB).

[5] 127w We did not find any evidence for lateralization of the effects of injection of drugs into the posterior hypothalamic area on locally recorded theta activity. Therefore, all drugs were always injected into the right PH or SuM nuclei of the posterior hypothalamic area (26 gauge, 5 μl Hamilton 701N microsyringe). When the recording electrode was placed in PH the coordinates of Hamilton cannula were as follows: 3.7 mm posterior from bregma, 0.5 mm lateral from the midline, and 7.5 mm ventral to the dural surface (Paxinos and Watson, 2014). If the electrode was implanted in SuM the coordinates of Hamilton cannula were as follows: 3.7 mm posterior from bregma, 0.5 mm lateral from the midline, and 8.3 mm ventral to the dural surface (Paxinos and Watson, 2014).