The effect of descending theta rhythmic input from the septohippocampal system on firing in the supramammillary nucleus

The supramammillary nucleus (SUM) is part of an ascending pathway conveying behavior-dependent drive to the septal generator of limbic theta rhythm. The SUM is, however, reciprocally connected to the septohippocampal system and there is strong evidence that both septum and SUM are capable of generating theta rhythmic activity. The present study examined the possible role of a descending rhythmic input to the SUM using simultaneously recorded hippocampal EEG and SUM neuronal activity in anesthetized rats. Fourier based phase analysis was performed on recordings in which fast theta rhythmic activity was elicited by tail pinch and in which a slower theta rhythm persisted after cessation of the sensory stimulus. It was found that the firing of a subpopulation of SUM neurons followed the hippocampal theta waves with a constant time delay, rather than a constant phase, suggesting that during deceleration associated with a shift from sensory-elicited theta to spontaneous theta rhythm they followed a descending rhythmic input, most likely from the medial septum. Neurons of a second group, which fired at the hippocampal theta peaks, did not show such relationship demonstrating heterogeneity in the population of rhythmic SUM neurons and their possible roles in theta generation. Combined with previous studies focusing on the role of the ascending theta drive from the SUM, these results demonstrate dynamic bidirectional coupling between subcortical theta generators. Thus, during certain states, rhythmically firing SUM neurons lead the septal theta oscillator, in others the direction may reverse and SUM follows a theta drive of septal origin.     

Glutamatergic synaptic transmission participates in generating the hippocampal EEG

The participation of ionotropic glutamatergic synapses in the generation of hippocampal electroencephalography (EEG) of behaving rats has not been systematically studied. In this study, field potentials in hippocampal CA1 were recorded following injection of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists, or vehicle control, either into the lateral ventricles or directly into the hippocampus or the medial septum. Intraventricular (i.c.v.) AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 5-10 microg) decreased the commissural evoked potential and the amplitude of the hippocampal EEG, including the theta rhythm. Theta frequency was decreased by 10 microg, but not 5 microg DNQX i.c.v. Unilateral intrahippocampal injection of DNQX (5 microg) only decreased the amplitude, but not the frequency, of the theta rhythm near the site of injection, without affecting theta amplitude or frequency at the opposite hippocampus. Other than theta, the large irregular activity (with a delta frequency peak at 1-2 Hz) and gamma EEG (30-100 Hz) were also decreased by i.c.v. and intrahippocampal injections of DNQX. Intrahippocampal injection of NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV, 2.5 microg) decreased the amplitude of the theta rhythm and, less consistently, the gamma EEG. The frequency of the theta rhythm and the peak of the commissural evoked potential were not significantly affected by intrahippocampal D-APV injection. Medial septal injections of D-APV or D,L-APV (2.24 microg in 0.4 microl), but not DNQX (10 microg in 0.4 microl), decreased the amplitude of the hippocampal theta significantly, but theta frequency was not significantly affected. It is concluded that both NMDA and AMPA receptors in the hippocampus are involved in generating the amplitude of the hippocampal EEG of theta and gamma frequencies, while NMDA receptors in the medial septum are involved in controlling the amplitude of theta and gamma EEG in the hippocampus. Excitatory glutamatergic synaptic currents, activated by afferents from the entorhinal cortex and CA3, are suggested to participate in hippocampal EEG activities.     

Respiratory cycle entrainment of septal neurons mediates the fast coupling of sniffing rate and hippocampal theta rhythm

Memory for odour information may result from temporal coupling between the olfactory and hippocampal systems. Respiration defines the frequency of olfactory perception, but how the respiratory rate affects hippocampal oscillations remains poorly understood. The afferent connectivity of the medial septum/diagonal band of Broca complex (MS/DB) proposes this region as a crossroads between respiratory and limbic pathways. Here we investigate if the firing rates of septal neurons integrate respiratory rate signals. We demonstrate that approximately 50% of MS/DB neurons are temporally correlated with sniffing frequency. Moreover, a group of slow‐spiking septal neurons are phase‐locked to the sniffing cycle. We show that inter‐burst intervals of MS/DB theta cells relate to the sniff rate. Intranasal odour infusion evokes sniff phase preference for the activity of fast‐spiking MS/DB neurons. Concurrently, the infusion augments the correlation between sniffing and limbic theta oscillations. During periods of sniffing–theta correlation, CA1 place cells fired preferentially during the inhalation phase, suggesting the theta cycle as a coherent time frame for central olfactory processing. Furthermore, injection of the GABAergic agonist muscimol into medial septum induces a parallel decrease of sniffing and theta frequencies. Our findings provide experimental evidence that MS/DB does not merely generate theta rhythm, but actively integrates sensorimotor stimuli that reflect sniffing rate. Such integration may provide temporal oscillatory synchronisation of MS/DB‐innervated limbic structures with the sniffing cycle.     

Electrical activity of the cingulate cortex. II. Cholinergic modulation

The role of the cholinergic innervation in the modulation of cingulate electrical activity was studied by means of pharmacological manipulations and brain lesions. In the normal rat, an irregular slow activity (ISA) accompanied with EEG-spikes was recorded in the cingulate cortex during immobility as compared to walking. Atropine sulfate, but not atropine methyl nitrate, increased ISA and the frequency of cingulate EEG-spikes. Pilocarpine suppressed ISA and EEG-spikes during immobility, and induced a slow (4-7 Hz) theta rhythm. Unilateral or bilateral lesions of the substantia innominata and ventral globus pallidus area using kainic acid did not significantly change the cingulate EEG or its relation to behavior. Large electrolytic lesions of the medial septal nuclei and vertical limbs of the diagonal band generally decreased or abolished all theta activity in the cingulate cortex and the hippocampus. However, in 5 rats the cingulate theta rhythm increased while the hippocampal theta disappeared after a medial septal lesion. The large, postlesion cingulate theta, accompanied by sharp EEG-spikes during its negative phase, is an unequivocal demonstration of the existence of a theta rhythm in the cingulate cortex, independent of the hippocampal rhythm. Cholinergic afferents from the medial septum and diagonal band nuclei are inferred to be responsible for the behavioral suppression of cingulate EEG-spikes and ISA, and partially for the generation of a local cingulate theta rhythm. However, an atropine-resistant pathway and a theta-suppressing pathway, possibly coming from the medial septum or the hippocampus, may also be important in cingulate theta generation.     

Hippocampal theta rhythm in behaving rats following ibotenic acid lesion of the septum

The effects of ibotenic acid lesion of the septum were studied in rats implanted with chronically indwelling electrodes and septal cannula. Each rat served as its own control and the properties of the hippocampal theta rhythm were studied before and after ibotenic acid and control saline infusion into the medial septal area. Ibotenic acid preferentially killed neurons in the lateral septum, and significantly attenuated the hippocampal theta rhythm about 50% bilaterally, at both surface and deep electrodes. The coherence and the phase of the theta rhythm at the CA1 apical dendrites, with respect to a superficial electrode, also declined significantly after ibotenic acid lesion. Pilocarpine (25 mg/kg i. p.) induced a theta rhythm of 7–9 Hz during immobility in the lesioned rats that was significantly higher in frequency than that induced in intact rats (4–6 Hz). In lesioned rats, the theta rhythm during tail pinch under urethane anesthesia was largely abolished, and the theta during walking was attenuated by atropine sulfate (50 mg/kg i. p.). Phencyclidine (10 mg/kg i. p.) or parachlorophenylaline (PCPA) alone, which was inferred to abolish an atropine-resistant theta input, did not affect the power of the walking theta rhythm in either the lesioned or the normal rat. It was concluded that the theta in the behaving rats after ibotenic acid lesion in the septum has a strong astropine-sensitive component, and that it is not predominantly atropine-resistant, as suggested previously. The lack of PCPA effect on the theta phase in intact and lesioned rats also suggested a different view of the atropine-resistant theta in hippocampal region CA1. One possible mechanism of the atropine-resistant theta at the distal dendrites of pyramidal cells may result from rhythmic inhibition by stratum lacunosum-moleculare interneurons which may be activated by either serotonergic or cholinergic inputs. © 1994 Wiley-Liss, Inc.     

The rhythmicity of cells of the medial septum/diagonal band of Broca in the awake freely moving rat: relationships with behaviour and hippocampal theta

Chronic extracellular recordings were obtained from cells of the medial septum and diagonal band of Broca in rats performing a simple behavioural task. The cells were found to display a variety of bursting patterns phase-locked to hippocampal theta rhythm to a greater or lesser degree. Among phase-locked cells, no systematic distribution in preferential phase could be found, and these cells were shown to maintain their preferential phase for extended periods. Cells were classified into those which showed signs of a broadening of the repolarization phase of their action potential (‘inflected’: putative cholinergic) and those without (‘non-inflected’: putative GABAergic). Non-inflected cells tended to fire rhythmic bursts while inflected cells mostly fired in an irregular fashion, although still significantly phase-locked to hippocampal theta. In neither population did the phase-locked cells show any coherent distribution of their preferential phase. Sixty-five per cent of the rhythmically bursting cells showed a significant correlation between the interburst frequency and the animal’s running speed. Five cells displayed rhythmic activity only when the rat ran in a specific direction. These results have implications for models of septohippocampal function and the effects of variable septal rhythmicity on the production of hippocampal theta rhythm.     

Control of the neuronal rhythmic bursts in the septal pacemaker of theta-rhythm: Effects of anaesthetic and anticholinergic drugs

The drugs, described as blocking the high-frequency (pentobarbital) or low-frequency (scopolamine, atropine) theta rhythm of the hippocampal electroencephalogram, were tested upon the rhythmically bursting septal cells. Three groups of chronic, unanaesthetized rabbits were used for the experiments: with intact septum; with septohippocampal disconnection; with complete basal undercutting of the septum, depriving it of ascending brainstem influences (MFB lesion). While the frequency and other parameters of theta bursts did not differ in the first two groups (5.2-5.5 Hz), in MFB-lesioned septum their frequency was significantly lower (3.5 Hz). Intravenous injection of pentobarbital suppressed theta bursts in some cells with unstable, periodic rhythmic activity and lowered the frequency of the bursts in continuously bursting cells. The parameters of bursts in intact and hippocampectomized septum under pentobarbital did not differ from those of undercut septum in undrugged state. Acetylcholine-blocking drugs suppressed theta modulation in some intermittently bursting cells, but only slightly decreased regularity of the bursts in some cells with continuous theta bursting even in sublethal doses; physostigmine has the opposite effect. Neither scopolamine and atropine, nor physostigmine influenced frequency of theta bursts in any way. Sensory or reticular stimulation could temporarily restore both the theta rhythm of hippocampal EEG and the rhythmic bursting of some septal cells under pentobarbital or anticholinergic drugs. On the basis of the experiments a unitary concept of theta rhythm origin is proposed. Pentobarbital influences ascending excitatory input to the septum, which results in a decrease of the burst frequency in the limited group of septal cells, regarded as endogenous bursting pacemakers, and in restriction of the population of high-threshold secondary rhythmic cells, synaptically involved in the rhythmic process. Anticholinergic drugs do not influence the pacemaker cells, but block intraseptal and septohippocampal cholinergic transmission. Both cholinergic and non-cholinergic neurons projecting to the hippocampus exist among septal cells synaptically involved in the rhythmic activity.     

Medial septal modulation of hippocampal theta cell discharges

The effect of small electrolytic lesions in various areas of the septum on the behavioral correlates and firing repertoires of hippocampal theta cells, was investigated in the freely moving rabbit. Lesions localized to the medial septum were found to abolish both slow wave theta and the rhythmic firing of CA1 and dentate layer theta cells, in both the type 1 theta (movement) and type 2 theta (sensory processing) behavior conditions. Small lesions of the diagonal band, lateral septum and fimbria/fornix regions only affected rhythmicity to the extent that they also involved the medial septal region. The same medial septal lesions that abolished rhythmicity were also shown to reduce the mean discharge rate of theta cells occurring during the type 1 movement condition by approximately 50%, while the discharge rate occurring during the type 2 sensory processing condition did not change significantly. Behavioral changes were also only observed for lesions involving the medial septum. The importance of afferent input from the medial septum in the generation of hippocampal theta cell rhythmicity was discussed.     

The role of the septal and entorhinal inputs in generating hippocampal electrical activity in the wakefulness-sleep cycle of the cat

The effect of septal lesion and section of the entorhinal cortex on hippocampal electrical activity was studied in the sleep-wakefulness cycle of chronic cats. It was demonstrated that the medial part of the septum participates in the generation of the hippocampal electrical activity. The principal effect of the septal lesion is the complete abolishment of the hippocampal theta rhythm during active wakefulness and paradoxical sleep. During the slow wave sleep the septal lesion effect is evidenced by a slight reduction of the power of the dominating frequency (1 Hz). Section of the entorhinal cortex results in a sharp increase of the hippocampal theta rhythm during wakefulness and paradoxical sleep. The slow wave sleep causes a sharp reduction of delta and subdelta rhythms. Under normal conditions the entorhinal input of the hippocampus is supposed to have a modulating effect on the genesis of the hippocampal theta rhythm.     

Distribution of polysialylated neural cell adhesion molecule in rat septal nuclei and septohippocampal pathway: transient increase of polysialylated interneurons in the subtriangular septal zone during memory consolidation

During memory consolidation neuroplastic events in the mediotemporal corticohippocampal pathway are accompanied by transient increases in the frequency of neurons expressing polysialylated neural cell adhesion molecule (NCAM PSA), a posttranslational modification associated with morphofunctional change. As a bidirectional pathway between the hippocampus and the septal nuclei also influences memory processing, we have determined the distribution of NCAM PSA within this system before and after learning in the adult Wistar rat. The most intense NCAM PSA immunoreactivity was observed in the medial and triangular septal nuclei, regions that regulate hippocampal theta rhythm during memory consolidation. Within the fimbria, NCAM PSA was expressed only in a subpopulation of fibres, most likely cholinergic projections from the medial septum to the hippocampus. Grey level analysis or direct cell counting revealed no learning-specific change in NCAM PSA expression in these septal subregions after avoidance conditioning or spatial training. A population of discrete polysialylated neurons in the subtriangular septal zone, however, exhibited a transient twofold frequency increase at 12 hr after training in either task. Immunohistochemical analysis revealed these cells to be gamma-aminobutyric acid (GABAergic) interneurons co-expressing vasoactive intestinal peptide. The unique location of these interneurons is proposed to provide a natural plexus by which bidirectional communication between the septum and hippocampus may be modified during memory consolidation.     

Septal projections to nucleus incertus in the rat: Bidirectional pathways for modulation of hippocampal function

Projections from the nucleus incertus (NI) to the septum have been implicated in the modulation of hippocampal theta rhythm. In this study we describe a previously uncharacterized projection from the septum to the NI, which may provide feedback modulation of the ascending circuitry. Fluorogold injections into the NI resulted in retrograde labeling in the septum that was concentrated in the horizontal diagonal band and areas of the posterior septum including the septofimbrial and triangular septal nuclei. Double‐immunofluorescent staining indicated that the majority of NI‐projecting septal neurons were calretinin‐positive and some were parvalbumin‐, calbindin‐, or glutamic acid decarboxylase (GAD)?67‐positive. Choline acetyltransferase‐positive neurons were Fluorogold‐negative. Injection of anterograde tracers into medial septum, or triangular septal and septofimbrial nuclei, revealed fibers descending to the supramammillary nucleus, median raphe, and the NI. These anterogradely labeled varicosities displayed synaptophysin immunoreactivity, indicating septal inputs form synapses on NI neurons. Anterograde tracer also colocalized with GAD‐67‐positive puncta in labeled fibers, which in some cases made close synaptic contact with GAD‐67‐labeled NI neurons. These data provide evidence for the existence of an inhibitory descending projection from medial and posterior septum to the NI that provides a "feedback loop" to modulate the comparatively more dense ascending NI projections to medial septum and hippocampus. Neural processes and associated behaviors activated or modulated by changes in hippocampal theta rhythm may depend on reciprocal connections between ascending and descending pathways rather than on unidirectional regulation via the medial septum. J. Comp. Neurol. 523:565–588, 2015. © 2014 Wiley Periodicals, Inc.     

GABAB receptor blockade enhances theta and gamma rhythms in the hippocampus of behaving rats

The participation of GABA(B) receptors in hippocampal EEG generation was studied by intracerebroventricular (icv) and intracerebral infusions of GABA(B) receptor antagonist p-(3-aminopropyl)-p-diethoxymethyl-phosphinic acid (CGP35348) in freely behaving rats. During awake-immobility, icv CGP35348 induced a theta rhythm and increased gamma waves (30-100 Hz) in the hippocampus. The immobility theta peaked at 6-7 Hz and had a theta phase in CA1 stratum radiatum of approximately 160 degrees with reference to the theta at the alveus, when compared with approximately 130 degrees during walking. Immobility theta power peaks at 6-7 Hz was also found in normal rats, and it was detected in 27% of the EEG segments during immobility. Incidence of immobility theta increased to 87.5% after 480 nmol of CGP35348 icv. Muscarinic antagonist scopolamine (5 mg/kg, ip) suppressed the induction of immobility theta and the gamma power increase after icv CGP35348. CGP35348 icv did not significantly change the hippocampal theta power at 7-8 Hz during walking (theta fundamental), but it increased power at 12-15 Hz, at the second harmonic of theta. CGP35348 icv also increased 30-50 Hz gamma power during walking. Medial septal infusion of CGP35348 (12 nmol in 0.4 microl) increased the power and the frequency of the hippocampal theta second harmonic during walking, but did not increase gamma activity. Infusion of CGP35348 (8 nmol in 0.4 microl) in the hippocampus increased the local gamma activity at 30-100 Hz, but did not induce immobility theta or affect the walking theta rhythm. In conclusion, icv GABA(B) receptor blockade increased an atropine-sensitive input that generated an immobility theta rhythm, while GABA(B) receptor blockade of the medial septum increased atropine-resistant theta harmonics possibly generated by apical dendritic spikes. GABA(B) receptor blockade may enhance cognitive task performance by activating hippocampal theta and gamma rhythms in behaving rats.     

Dynamic changes in the direction of the theta rhythmic drive between supramammillary nucleus and the septohippocampal system

Neurons in the supramammillary nucleus (SUM) of urethane-anesthetized rats fire rhythmically in synchrony with hippocampal theta rhythm. As these neurons project to the septum and hippocampus, it is generally assumed that their role is to mediate ascending activation, leading to the hippocampal theta rhythm. However, the connections between SUM and the septohippocampal system are reciprocal; there is strong evidence that theta remains in the hippocampus after SUM lesions and in the SUM after lesioning the medial septum. The present study examines the dynamics of coupling between rhythmic discharge in the SUM and hippocampal field potential oscillations, using the directionality information carried by the two signals. Using directed transfer function analysis, we demonstrate that during sensory-elicited theta rhythm and also during short episodes of theta acceleration of spontaneous oscillations, the spike train of a subpopulation of SUM neurons contains information predicting future variations in rhythmic field potentials in the hippocampus. In contrast, during slow spontaneous theta rhythm, it is the SUM spike signal that can be predicted from the preceding segment of the electrical signal recorded in the hippocampus. These findings indicate that, in the anesthetized rat, SUM neurons effectively drive theta oscillations in the hippocampus during epochs of sensory-elicited theta rhythm and short episodes of theta acceleration, whereas spontaneous slow theta in the SUM is controlled by descending input from the septohippocampal system. Thus, in certain states, rhythmically firing SUM neurons function to accelerate the septal theta oscillator, and in others, they are entrained by a superordinate oscillatory network.     

Hippocampal theta rhythm is reduced by suppression of the H-current in septohippocampal GABAergic neurons

Hippocampal learning and memory tasks are tightly coupled to the hippocampal theta rhythm, which is critically dependent on the medial septum/diagonal band of Broca (MSDB) although the underlying mechanisms remain unclear. The MSDB sends both cholinergic and GABAergic projections to the hippocampus. Here we show that: (i) septo-hippocampal GABAergic but not cholinergic neurons have a pacemaking current, the H-current, and that its selective blockade by ZD7288 reduces their spontaneous firing in rat brain slices; and (ii), local infusions of ZD7288 into the MSDB reduce exploration and sensory evoked hippocampal theta bursts in behaving rats. Thus, the H-current in septohippocampal GABAergic neurons modulates the hippocampal theta rhythm.     

The effect of atropine administered in the medial septum or hippocampus on high- and low-frequency theta rhythms in the hippocampus of urethane anesthetized rats

Cholinergic mechanisms are critical for the generation of hippocampal theta rhythm. Cholinergic innervation of the hippocampus originates from the medial septum (MS) and cholinergic receptors are expressed in both the MS and hippocampus. In this study, we compared the effects of the muscarinic receptor antagonist atropine in the MS and the hippocampus on theta generation. Hippocampal theta rhythm was elicited by electrical stimulation of the pontine reticular formation using series of stimuli with varying intensities. Atropine was administered either systemically (50 mg/kg i.p.) or locally in the MS (microdialysis; 25 and 75 mM for 30 or 90 min) or in the hippocampus on one side (microinjection; 20 or 40 ug). The relative power at the peak theta frequency was calculated and averaged over episodes of low-intensity and high-intensity stimulations. We found that atropine drastically reduced theta rhythmic synchronization when injected in either location. After MS administration of atropine, however, high-frequency theta elicited by high-intensity stimuli was more resistant (58% and 67% decrease after 25 mM and 75 mM atropine, respectively) than slow theta elicited by low-intensity stimuli (86% and 91% decrease). There was no significant difference between the powers of the two oscillations after hippocampal injections (70-75% decrease). We conclude that the theta suppressing effect of atropine involves both hippocampal and septal mechanisms and that low-frequency theta as compared with fast theta rhythm is more sensitive to muscarinic acetylcholine receptor antagonism in the MS but not in the hippocampus.     

PHA-L analysis of projections from the supramammillary nucleus in the rat.

The projections of the supramammillary nucleus (SUM) were examined in the rat by the anterograde anatomical tracer Phaseolus vulgaris leucoagglutinin (PHA-L). The majority of labeled fibers from SUM ascended through the forebrain within the medial forebrain bundle. SUM fibers were found to terminate heavily in the hippocampal formation, specifically within the granule cell layer and immediately adjoining molecular layer of the dentate gyrus. In addition, SUM fibers were shown to distribute densely to several structures with strong connections with the hippocampus, namely, the nucleus reunions of the thalamus, the medial and lateral septum, the entorhinal cortex, and the endopiriform nucleus. SUM fibers were also shown to project significantly to several additional subcortical and cortical sites. The subcortical sites were the dorsal raphe nucleus, the midbrain central gray, the fields of Forel/zona incerta, the dorsomedial hypothalamic area, midline/intralaminar nuclei of the thalamus (posterior paraventricular, rhomboid, central medial, intermediodorsal, and mediodorsal), the medial and lateral preoptic areas, the bed nucleus of the stria terminalis, the substantia innominata, the vertical limb of the diagonal band nucleus, and the claustrum. The cortical sites were the occipital, temporal, parietal, and frontal cortices. Some notable differences were observed in projections from the lateral as compared to the medial SUM. For example, fibers originating from the lateral SUM distributed heavily to the hippocampal formation and parts of the cortex, whereas those from the medial SUM projected sparsely to these two regions. The SUM projections to the hippocampal formation and associated structures may serve as the substrate for a SUM involvement in the generation of the theta rhythm of the hippocampus and the gating of information flow through the hippocampal formation.     

Reticular stimulation evokes suppression of CA1 synaptic responses and generation of theta through separate mechanisms

The induction of hippocampal theta by reticular stimulation involves a relay to the hippocampus via the posterior hypothalamic-supramammillary region and then the medial septum. Interestingly, sensory- or behaviour-induced theta is accompanied by suppression of hippocampal field CA1 synaptic responses. In the present study, performed on anaesthetized rats, we observed that reticular stimulation also induced a suppression of the CA1 pyramidal cell population spike and the corresponding dendritic field excitatory postsynaptic potential evoked by field CA3 stimulation. This suppression was observed at stimulation intensity below the threshold for generation of CA1 theta and was maximal at stimulation intensities at the threshold for theta. The frequency and amplitude of theta waves, by contrast, increased further with increasing reticular stimulation voltage. Neural inactivation by microinjection of the local anaesthetic procaine (20% w/v, 0.1-0.2 microL) or the inhibitory ligand gamma aminobutyric acid (0.8 m, 0.5 micro L) in the posterior hypothalamic regions, especially the ipsilateral medial supramammillary region, or the medial septum attenuated both the suppression of CA1 pyramidal cell synaptic excitability and theta generation. However, the effects of microinjection on suppression and theta were not always in parallel. Furthermore, the effect of microinjection of gamma aminobutyric acid on reticularly elicited suppression was observed from relatively fewer sites in the posterior hypothalamus as compared with that on theta activation. These results suggest that reticular stimulation evokes an inhibition of CA1 pyramidal cell excitability that (i) is mediated, at least in part, via medial supramammillary and septal regions, but (ii) involves a separate neural pathway from theta generation.     

Collateral projections from the supramammillary nucleus to the medial septum and hippocampus

Previous reports have shown that the supramammillary nucleus projects to the medial septum and to the hippocampus, and specifically to the dentate gyrus and the CA2/CA3a region of the hippocampus. The aim of the present study was to examine collateral projections from the supramammillary nucleus to the septum and hippocampus. The fluorescent retrograde tracers, Fluororuby and Fluorogold, were injected into regions of the septum and hippocampus, respectively, and the supramammillary nucleus was examined for the presence of single- and double-labeled neurons. The main findings were: 1) pronounced numbers of single-labeled cells (about 40-60/section) were present in the supramammillary nucleus following retrograde tracer injections in either the septum or hippocampus; 2) single and double retrogradely labeled neurons were intermingled within the supramammillary nucleus and mainly localized to the lateral two-thirds of the supramammillary nucleus; 3) approximately 5-10% of supramammillary cells were double-labeled, ipsilaterally, and 2-4%, contralaterally, with injections in medial or lateral parts of the medial septum and the dentate gyrus of the hippocampus; and 4) approximately 3-5% of supramammillary cells were double-labeled, ipsilaterally, and 1-2%, contralaterally, with injections in the medial septum and CA2/CA3a of the dorsal hippocampus. Cells of the supramammillary nucleus have been shown to fire rhythmically in bursts synchronous with the hippocampal theta rhythm and have been implicated in the generation of the theta rhythm. The supramammillary cells that we identified with collateral projections to the septum and hippocampus may be directly involved in generation of the theta rhythm.     

Distribution and targets of the relaxin-3 innervation of the septal area in the rat

Neural tracing studies have revealed that the rat medial and lateral septum are targeted by ascending projections from the nucleus incertus, a population of tegmental GABA neurons. These neurons express the relaxin-family peptide, relaxin-3, and pharmacological modulation of relaxin-3 receptors in medial septum alters hippocampal theta rhythm and spatial memory. In an effort to better understand the basis of these interactions, we have characterized the distribution of relaxin-3 fibers/terminals in relation to different septal neuron populations identified using established protein markers. Dense relaxin-3 fiber plexuses were observed in regions of medial septum containing hippocampal-projecting choline acetyltransferase (ChAT)-, neuronal nitric oxide synthase (nNOS)-, and parvalbumin (PV)-positive neurons. In lateral septum (LS), relaxin-3 fibers were concentrated in the ventrolateral nucleus of rostral LS and the ventral nucleus of caudal LS, with sparse labeling in the dorsolateral and medial nuclei of rostral LS, dorsal nucleus of caudal LS, and ventral portion nuclei. Relaxin-3 fibers were also observed in the septofimbrial and triangular septal nuclei. In the medial septum, we observed relaxin-3-immunoreactive contacts with ChAT-, PV-, and glutamate decarboxylase-67-positive neurons that projected to hippocampus, and contacts between relaxin-3 terminals and calbindin- and calretinin-positive neurons. Relaxin-3 colocalized with synaptophysin in nerve terminals in all septal areas, and ultrastructural analysis revealed these terminals were symmetrical and contacted spines, somata, dendritic shafts, and occasionally other axonal terminals. These data predict that this GABA/peptidergic projection modulates septohippocampal activity and hippocampal theta rhythm related to exploratory navigation, defensive and ingestive behaviors, and responses to neurogenic stressors.     

Ventral tegmental nucleus of Gudden: A pontine hippocampal theta generator?

It is well-established that rhythmically bursting (RB) activity in the medial septum is crucial for the generation of the hippocampal theta rhythm, but the contribution of other diencephalic-pontine structures is less documented. The ventral tegmental nucleus (VTn) of Gudden is related to the Papez's circuit via its interconnections with the medial mammillary nucleus, and therefore it may play a role in the generation of hippocampal theta. In the present study, extracellular activity from VTn neurons were recorded in unanesthetized restrained rats (n = 9). Hippocampal activity (EEG) and electromyograms were recorded simultaneously to identify sleep-waking states. RB activity was observed in VTn during wakefulness, with periods of hippocampal theta and during rapid eye movement (REM) sleep. Rhythmicity in VTn preceded theta activity in hippocampus. The frequency of RB neurons in VTn was 5.6 Hz during wakefulness and 6.8 Hz during REM sleep. It was similar to that of hippocampal theta. The rhythmicity was particularly stable and the firing rates were strikingly high during REM sleep. RB activity in VTn was also recorded from urethane-anesthetized rates (n = 3). Rhythmic firing (4.0 Hz) was slower than in unanesthetized rats and matched the urethane-related theta frequency. Our results show that neurons in VTn exhibit a marked RB activity during states of vigilance accompanied by hippocampal theta rhythm. They suggest that VTn may be a pontine hippocampal theta generator.