The amygdala and the pedunculopontine tegmental nucleus: Interactions controlling active (rapid eye movement) sleep

Experimental Neurology 238 (2012) 44–51

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The amygdala and the pedunculopontine tegmental nucleus: Interactions controlling active (rapid eye movement) sleep Mingchu Xi a, b,⁎, Simon J. Fung a, b, Jianhua Zhang a, b, Sharon Sampogna a, Michael H. Chase a, b, c a b c

Websciences International, Los Angeles, CA 90024, USA VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA UCLA School of Medicine, Los Angeles, CA 90025, USA

a r t i c l e

i n f o

Article history: Received 11 May 2012 Revised 23 July 2012 Accepted 1 August 2012 Available online 17 August 2012 Keywords: REM sleep Amygdala PPT Electrical stimulation Intracellular recording

a b s t r a c t There is a consensus that active sleep (AS; i.e., REM sleep) is produced by cholinergic projections from the pedunculopontine tegmental nuclei (PPT) that activate AS-on neurons in the nucleus pontis oralis (NPO) that are components of the AS-Generator. However, there is a growing body of evidence indicating that other sites, such as the amygdala, also participate in the control of AS by inducing the discharge of AS-Generator neurons. In this regard, we recently reported that there are direct, excitatory (glutamatergic) projections from the central nucleus of the amygdala (CNA) to presumptive AS-Generator neurons in the NPO. We therefore hypothesized that the CNA and the PPT act alone, as well as in concert, to promote AS. To test this hypothesis, the effects of stimulation of the CNA and the PPT on the activity of NPO neurons, recorded intracellularly, were examined in urethane-anesthetized rats. Stimulation of either the CNA or the PPT evoked short-latency excitatory postsynaptic potentials (EPSPs) in the same neurons within the NPO. The amplitude of PPT-evoked EPSPs that were recorded from NPO neurons increased by 20.1 to 58.6% when stimulation of the PPT was preceded by stimulation of the CNA at an interval of 0 to 12 ms: maximal potentiation occurred at an interval of 4 to 6 ms. Concurrent subthreshold stimulation of the CNA and the PPT resulted in the discharge of NPO neurons. NPO neurons that were activated following CNA and/or PPT stimulation were identified morphologically and found to be multipolar with diameters > 20 μm; similar neurons in the same NPO site have been previously identified as AS-Generator neurons. The present data demonstrate the presence of converging excitatory synaptic inputs from the CNA and the PPT that are capable of promoting the discharge of AS-Generator neurons in the NPO. Therefore, we suggest that the occurrence of AS depends upon interactions between cholinergic projections from the PPT and glutamatergic projections from the CNA as well as inputs from other sites that project to AS-Generator neurons. Published by Elsevier Inc.

Introduction It is well established that active sleep (AS), also known as rapid eye movement (REM) sleep, is induced by excitatory cholinergic projections from the pedunculopontine tegmental nucleus (PPT) as well as the laterodorsal tegmental nucleus (LDT). These projections excite executive active sleep-on (AS-on) neurons that are located in and/or in the vicinity of the nucleus pontis oralis (NPO). These AS-on neurons in the NPO are critical components of the AS-Generator that is responsible for the generation and maintenance of AS (see reviews of Chase and Morales, 2005; Jones, 2004; Lydic and Baghdoyan, 2005; McCarley, 2007; Steriade and McCarley, 2005). There is also a growing body of evidence indicating that AS is not solely “triggered” by cholinergic projections from the PPT and the LDT, but that other sites also participate in the control of AS by promoting ⁎ Corresponding author at: WebSciences International, 1251 Westwood Blvd., Los Angeles, CA 90024, USA. Fax: + 1 310 235 2067. E-mail address: [email protected] (M. Xi). 0014-4886/$ – see front matter. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.expneurol.2012.08.001

the discharge AS-Generator neurons. In this regard, physiological and functional studies have shown that the amygdala, specifically its central nucleus (CNA), is capable of influencing the generation of AS, especially the occurrence of phasic phenomena that arise during this state (e.g., ponto-geniculo-occipital (PGO) waves, muscle twitches, etc.). For example, electrical stimulation of the CNA produces not only a significant increase in the duration of AS (Smith and Miskiman, 1975) but also results in an increase in the frequency as well as the amplitude of PGO waves, which are one of the most prominent phasic events that occur during AS (Calvo et al., 1987; Deboer et al., 1998, 1999). There is also a complementary elevation in neuronal and metabolic activity within the amygdala during AS (Maquet et al., 1996; Nofzinger et al., 1997; Smith and Miskiman, 1975) which is accompanied by an increase in the discharge rate of CNA neurons (Jha et al., 2005; Zhang et al., 1986). In addition, pharmacological data demonstrate that functional activation of the CNA promotes AS. Thus, microinjections into the CNA of the cholinergic agonist carbachol (Calvo et al., 1996) and the vasoactive intestinal peptide (VIP) (Simon-Arceo et al., 2003), as well as GABAA antagonists, result

M. Xi et al. / Experimental Neurology 238 (2012) 44–51

in an increase in AS. On the other hand, inactivation of the CNA following microinjections of the GABAA agonist muscimol and tetrodotoxin selectively decreases the occurrence of AS (Sanford et al., 2002, 2006; Tang et al., 2005). We have recently provided data demonstrating that there are direct, excitatory (glutamatergic) projections from the CNA to the NPO and that these amygdalar projections exert a powerful excitatory postsynaptic drive that is capable of activating NPO neurons which comprise the AS-Generator. These data arose from intracellular studies in which electrical stimulation of the CNA produced short-latency, fast-rising, and large amplitude EPSPs in neurons within the NPO; in addition, an increase in the intensity of CNA stimulation resulted in the discharge of these neurons (Xi et al., 2011b). These studies are complemented by our anatomical explorations wherein we have shown that glutamatergic neurons within the CNA project directly to the NPO (Fung et al., 2011). The preceding findings indicate that projections from the CNA are capable of directly activating the neurons of the AS-Generator in the NPO. The activation of these projections results in the generation of AS in a manner that is separate from cholinergic AS-inducing inputs from either the PPT or the LDT. Accordingly, we believe that although the CNA can act independently in determining the activity of the AS-Generator in the NPO, it likely interacts with inputs from the PPT (and the LTD) and possibly other sites as well in promoting the occurrence of AS. Therefore, we hypothesize that converging excitatory synaptic inputs from the CNA and the PPT act in concert, as well as independently, to facilitate the discharge of AS-Generator neurons in the NPO.1 To test this hypothesis, we examined the response of NPO neurons, which was recorded intracellularly, following stimulation of the CNA alone and in conjunction with the concurrent excitation of PPT neurons. The results of these experiments indicate that projections from the CNA and the PPT converge on AS-Generator neurons in the NPO and that alone, and in concert, they play an important role in the generation of AS. Preliminary data have been presented in abstract form (Xi et al., 2011a). Materials and methods Animals and surgical procedures Adult male Sprague–Dawley rats (body weight: 280–430 g; n = 7), which were used in the present experiments, were in good health as determined by veterinarians of the VA Greater Los Angeles Healthcare System (VAGLAHS). All experimental procedures were conducted in accord with the guidelines established by the Guide for the Care and Use of Laboratory Animals (National Academies Press, Eighth Edition, Washington DC, 2011), and were approved by the Institutional Animal Care and Use Committee of VAGLAHS. Appropriate measures were taken to minimize pain, discomfort or stress of the animals; only the minimum number of animals was utilized that were necessary to produce reliable scientific data. Surgical procedures, which were carried out under urethane anesthesia, have been described, in detail, in a previous paper (Xi et al., 2011b). Briefly, anesthesia was initially induced with isoflurane (2.5%) for approximately 5 min to provide for the stress-free, intraperitoneal injection of urethane (1.4 g/kg). The animals were then placed in a stereotaxic apparatus and holes (2–3 mm in diameter) were drilled in the skull to provide access to the NPO, the PPT and the CNA. Supplementary doses of urethane (0.7 g/kg, i.p.) were administered throughout the duration of the experiment as needed to maintain an appropriate level of anesthesia, which was determined by the presence of a stable heart and respiratory rate in response to a paw pinch. Rectal temperature was maintained at 35–36 °C throughout 1 In this paper we examined interactions between the CNA and the PPT with respect to the control of the activity of NPO neurons, although we recognize that similar pathways and functions are ascribed to the LDT, or more commonly, to the combined LDT/PPT.

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the experiment. Vital signs (heart rate, hemoglobin saturation [SpO2] and end-expiratory CO2) were continuously monitored with a SurgiVet V90041 pulse oximeter and were within a normal physiological range. Electrical stimulation procedures Electrical stimulation of the CNA and the PPT was carried out with stainless steel electrodes (0.5 MΩ AC impedance) which were placed, stereotaxically, in the CNA and PPT. The stereotaxic coordinates for the CNA and the PPT were AP: −2.6 (Bregma at zero), L: 4.5, H: −7.0 and AP: −8.0, L: 2.0, H: −6.0 (Paxinos and Watson, 1998), respectively. Electrical stimulation consisted of a single-pulse cathodal stimulus or a train of 2 to 3 stimuli at intensities ranging from 20 to 300 μA with a duration of 0.2 ms that were delivered at a rate of 1 Hz with a Grass S88 stimulator (Grass Instruments, Quincy, MA) and a Grass photoelectric constant current isolation unit. Intracellular recording procedures Intracellular recordings from NPO neurons were performed using glass micropipettes filled with 3 M KCl (30 to 50 MΩ). An Ag–AgCl wire, which was embedded subcutaneously in the neck musculature, was used as the indifferent electrode. A micromanipulator was employed to position the tip of the recording micropipette within the NPO (AP: − 7.5 to − 9.0, L: 1.0 to 2.0, H: − 7 to − 8; Bregma zero). Highgain (× 100) DC and low-gain (× 10) DC intracellular activities were displayed on an oscilloscope following amplification with a capacity-neutralized preamplifier (Axoclamp 2A). Data were stored on a video cassette recorder by means of a PCM recording adapter (Vetter Co. Model 4000). In selected experiments, NPO neurons that were studied electrophysiologically were also marked with neurobiotin in order to subsequently determine their location in the NPO and analyze their morphological profiles (Xi et al., 2011b). Accordingly, an intracellular recording micropipette was filled with a solution of neurobiotin (2% in 1 M KCl). After electrophysiological data had been obtained, the recorded neurons were filled with neurobiotin by injecting depolarizing current pulses (1.0–5.0 nA, 200 ms, 50% duty cycle) for a period of 2 to 8 min. Data analysis Measurements were made of the following waveform parameters of the averaged postsynaptic potentials (PSPs): (1) peak amplitude, which was determined as the voltage difference between the potential preceding the onset of the stimulus artifact and the peak of the PSP; (2) slope, which was defined as the first derivate (i.e., mV/ms) of the PSP; and (3) latency, which was calculated from the onset of the stimulus artifact to the onset of the PSP. Data were digitized off-line at 20 kHz and analyzed with an Apple microcomputer (Power Macintosh G4) utilizing AxoGraph X (AxoGraph Scientific, Sydney, Australia). Experimental data values were expressed as means±SEM. The statistical level of significance of the difference between sample means was evaluated using the two-tailed, unpaired Student's t-test (pb 0.05). Histological procedures At the completion of each experiment, the sites of stimulation in the CNA and the PPT were marked by an electrolytic lesion by passing 50 μA of anodal DC current through the stimulating electrode for 30 s. The animals were then deeply anesthetized with sodium pentobarbital and perfused transcardially with heparinized saline followed by 500 ml of a fixative (4% paraformaldehyde, 15% picric acid, 0.25% glutaraldehyde in 0.1 M PB at pH 7.4). After overnight post-fixation in fresh fixative at 4 °C, the forebrain and brainstem were cryoprotected in a solution of sucrose (25%) in 0.1 M PB at pH 7.4. Serial coronal sections were cut at a

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M. Xi et al. / Experimental Neurology 238 (2012) 44–51

thickness of 40 μm on a Leica CM3050 S cryostat. Free-floating serial brainstem sections were rinsed in phosphate buffered saline (PBS) and then placed in an endogenous blocking solution of 3% methanol and 0.9% hydrogen peroxide in PBS for 30 min. The sections were rinsed several times with PBS and placed in 0.3% Triton X 100 in PBS and kept overnight at 4 °C. Subsequently, tissue sections were incubated in reagents of the ABC Elite Kit (1:200; Vector Laboratories, Burlingame, CA) for 2 h. After rinsing, in order to visualize the black reaction product that indicates the presence of neurobiotin, the sections were processed with a mixture of diaminobenzidine (DAB; 0.2%), 0.05% hydrogen peroxide, and 0.6% nickel ammonium sulfate in 50 mM Tris buffer (pH 7.6). Serial sections of the forebrain and brainstem were examined under light microscopy to verify the sites of stimulation. Histological studies revealed that all stimulation sites were located within the CNA and PPT (Fig. 1).

Results Intracellular recordings were obtained from 22 NPO neurons that exhibited resting membrane potentials equal to or more negative than –50 mV and action potentials greater than 55 mV. All neurons were recorded for a length of time (i.e., more than 5 min) that was sufficient to obtain stable data and conduct a thorough examination of the effects of the individual and combined stimulation of the CNA and the PPT. Electrical stimulation of the CNA evoked a characteristic EPSP (e.g., short latency-to-onset, short latency-to-peak) in NPO neurons following single-pulse stimulation at 300 μA (peak amplitude: 2.6±0.3 mV; latency-to-onset: 3.5±0.2 ms; latency-to-peak: 1.3.±0.2 ms; n=22; Figs. 2A, B). Electrical stimulation of the PPT also elicited a short latencyto-onset EPSP in NPO neurons following single-pulse stimulation at 50 μA (peak amplitude: 2.0±0.3 mV; latency-to-onset: 2.3±0.1 ms; latency-

A) Stimulation Sites PPT

CNA

Bregma -2.6 mm

Bregma -8.0 mm

IC PAG PPT CPu

NPO

ic CNA

DE

B) Recording Site Bregma -8.3 mm

IC

PAG DR

PPT NPO

Fig. 1. Schematic representation of the anatomical location of the sites of stimulation in the CNA and the PPT and of recording in the NPO. A: Coronal sections of stimulation sites (filled circles) in the CNA (from Bregma − 2.6 stereotaxic plane) and PPT (from Bregma − 8.0 stereotaxic plane) are shown, which reveal that the stimulating electrodes were stereotaxically placed in the CNA and the PPT, respectively. The location of the tips of the stimulating electrodes was verified, histologically, by the presence of the Prussian Blue reaction. B: A micropipette for intracellular recording was directed to the NPO (from Bregma − 7.5 to − 9.0 stereotaxic planes). The location of the tip of the recording electrode was verified by immunohistochemistry (see text). CNA stimulation parameters: 0.2 ms (pulse duration); 100–300 μA (intensity). PPT stimulation parameters: 0.2 ms (pulse duration); 10–60 μA (intensity). Abbreviations: CNA, central nucleus of the amygdala; CPu, caudate putamen; DEn, dorsal endopiriform nucleus; DR, dorsal raphe nucleus; IC, inferior colliculus; ic, internal capsule; NPO, nucleus pontis oralis; PPT, pedunculopontine tegmental nucleus.

M. Xi et al. / Experimental Neurology 238 (2012) 44–51

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C) NPO Response to

A) NPO Response to

PPT Stimulation

CNA Stimulation 200 µA

40 µA

300 µA

60 µA

2 mV

2 mV

5 ms

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B) Latency of NPO EPSPs

D) Latency of NPO EPSPs

to CNA Stimulation

to PPT Stimulation 4

Number of neurons

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4 3 2 1 0

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2

3

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6

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Fig. 2. Excitatory responses elicited by stimulation of the CNA and the PPT. A: An excitatory postsynaptic potential (EPSP) with a latency of 3.1 ms was evoked in a representative NPO neuron by single-pulse stimulation of the CNA (200 μA). The amplitude of the EPSP greatly increased following stimulation at a higher intensity (300 μA). B: Histogram of the distribution of the latencies of EPSPs evoked in NPO neurons by single-pulse stimulation of the CNA at an intensity of 300 μA. The latency-to-onset of the EPSPs was measured from the beginning of stimulation to the initial rising deflection of each EPSP. The mean latency of amygdala-induced responses was 3.5 ± 0.2 ms, as indicated by the arrow. C: An EPSP is shown with a latency of 2.0 ms that was evoked by single-pulse stimulation of the PPT. The amplitude of the EPSP increased following stimulation at a higher intensity (60 μA). D: Histogram of the distribution of latencies of EPSPs evoked in NPO neurons by single-pulse stimulation of the PPT at an intensity of 50 μA. The arrow in D indicates the mean value of the latency of PPT-induced responses (2.3 ± 0.1 ms). Note that the mean latency of the CNA-induced EPSPs is approximately 1 ms longer than that of the PPT-induced responses. Each trace in A and C is an average of 10 consecutive trials. CNA and PPT stimulation: 0.2 ms.

to-peak: 1.4.±0.1 ms; n=22; Figs. 2C, D). The synaptic potentials recorded in NPO neurons following both CNA and PPT stimulation exhibited a pattern of response that was typical of EPSPs, i.e., their amplitude decreased when their membrane potential was depolarized and their amplitude increased when the cell was hyperpolarized. Interactions between projections from the CNA and the PPT were then examined with respect to the control of the activity of NPO neurons. Fig. 3 presents representative records from a typical NPO neuron which demonstrate that stimulation of the CNA facilitated EPSPs that were evoked by stimulation of the PPT. Stimulation of the CNA with 2 pulses of 100 μA (conditioning stimulus) evoked almost no PSPs (Fig. 3A), whereas stimulation of the PPT with 50 μA (test stimulus) evoked a small EPSP (Fig. 3B). When stimulation of the CNA and the PPT was combined at an interval so that conditioning stimulation of the CNA preceded test stimulation of the PPT by 3 ms, there was a great increase in the amplitude and the slope of the EPSP (Fig. 3C). The magnitude of the facilitation of NPO EPSPs that occurred when CNA stimulation preceded PPT stimulation varied according to the interval between the conditioning (CNA) and test (PPT) stimuli. The amplitude and slope of PPT-evoked EPSPs in NPO neurons increased by 20.3 to 61.1% and 24.7 to 65.8% when CNA stimulation preceded stimulation of the PPT at intervals of 0 and 12 ms, respectively (Figs. 3C and D). Maximal facilitation occurred when the CNA and

the PPT were stimulated at an interval of 4 to 6 ms (peak amplitude: 1. 8 ± 0.3 mV vs. 2. 9 ± 0.3 mV; slope: 205.7 ± 15.9 mV/ms vs. 341.1 ± 21.6 mV/ms, before and following concurrent stimulation of the CNA and the PPT, n = 22; p b 0.01). EPSP facilitation was not observed when the stimulus interval was longer than 15 ms. Combined stimulation of the CNA and the PPT facilitated the discharge of NPO neurons. Fig. 4 illustrates the response of a representative NPO neuron following stimulation of the CNA (Fig. 4A) and the PPT (Fig. 4B). When delivered separately, subthreshold stimulation of each site was ineffective with respect to the production of action potentials in NPO neurons. However, when subthreshold stimulation of the CNA preceded subthreshold stimulation of the PPT at intervals ranging from 3 to 18 ms, NPO neurons responded with at least one or more action potentials with a discharge index of 100% (Fig. 4C). Almost all NPO neurons (87.5%) exhibited action potentials with a discharge index of 100%; only 2 neurons had a discharge index of 50 to 70%. Seven NPO cells that were recorded were filled with neurobiotin; histological analysis revealed that they all contained neurobiotinfilled somata and processes and were situated within the NPO. The majority of these neurons, which were located in the dorsolateral part of the NPO (Fig. 5A), were multipolar, medium to large in size with diameters that were equal to or larger than 20 μm (Fig. 5B). The morphology and electrophysiological profile of NPO neurons that were activated following stimulation of the CNA and/or PPT were

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M. Xi et al. / Experimental Neurology 238 (2012) 44–51

A) CNA

B) PPT

C) CNA + PPT

D) CNA + PPT

2 mV 5 ms Fig. 3. Amygdalar facilitation of EPSPs in NPO neurons evoked by stimulation of the PPT. A and B: Sample records of responses in a representative NPO neuron following stimulation of the CNA (marked with filled triangles) and the PPT (marked with an open triangle), respectively. Note that stimulation of the CNA with two pulses of 100 μA evoked no distinct EPSPs. C: The amplitude of the PPT-evoked EPSPs greatly increased when both the CNA and the PPT were stimulated, i.e., there was spatial facilitation of PPT-evoked EPSPs in conjunction with stimulation of the CNA. D: CNA facilitation of PPT‐evoked responses at different time intervals. The amplitude of PPT-evoked EPSPs increased by 15.6 to 61.6% when stimulation of the PPT was preceded by stimulation of the CNA at intervals of 1 to 15 ms. Arrows in B to D indicate EPSPs evoked in this NPO neuron following stimulation of the PPT.

similar to previously identified glutamatergic, AS-on neurons that were located in the NPO (Brown et al., 2004, 2006). Discussion In the present study, we demonstrate that stimulation of either the CNA or the PPT produces short-latency, fast-rising EPSPs in neurons within the NPO. Importantly, we found that conditioning stimulation of the CNA potentiates the effects of stimulation of the PPT. Maximal facilitation of PPT-evoked EPSPs in NPO neurons occurs when stimulation of the PPT is preceded by stimulation of the CNA at an interval of 4 to 6 ms. In addition, simultaneous subthreshold stimulation of the CNA and the PPT results in the discharge of NPO neurons. These data demonstrate that converging excitatory inputs from the CNA and the PPT are capable of facilitating the activity and discharge frequency of neurons in the NPO that appear, on the bases of their morphology and electrophysiological characteristics as well as their location, to be AS-on cells which are critical components of the AS-Generator that is responsible for the generation of AS. Therefore, we propose that the CNA is capable of promoting AS by activating AS-Generator neurons in the NPO not only via direct projections to the NPO, but also by its interactions with projections from the PPT. There is no doubt that cholinergic projections to the NPO that originate in the PPT as well as the LDT play a crucial role in the generation of AS (see reviews of Chase and Morales, 2005; Jones, 2004; Lydic and Baghdoyan, 2005; McCarley, 2007; Siegel, 2005; Steriade and McCarley, 2005). Anatomical studies utilizing retrograde tracers have shown that cholinergic cells of the LDT/PPT send direct projections to the cholinoceptive site of the AS-Generator in the NPO (Mitani et al., 1988; Shiromani et al., 1988). In addition, stimulation of the PPT increases acetylcholine release in the region of the AS-Generator

(Lydic and Baghdoyan, 1992). Intracellular recordings have demonstrated that stimulation of the LDT produces cholinergic, monosynaptic EPSPs in the neurons of the AS-Generator (Imon et al., 1996; Xi et al., 2003). Unit recording experiments have also shown that LDT/PPT neurons discharge prior to and during AS (El Mansari et al., 1989; Steriade et al., 1990). Consistent with the preceding data, the microinjection of cholinergic, muscarinic agonists or anticholinesterases into the region of the ASGenerator elicits, with a short latency, both individual elements of AS (e.g., atonia, PGO waves, rapid eye movements, etc.) as well as the complete state of AS, with all of its attendant physiological patterns of activity (Baghdoyan et al., 1989; George et al., 1964; Shiromani et al., 1992; Vanni-Mercier et al., 1989; Yamamoto et al., 1990a, 1990b; Yamuy et al., 1993). In further support of the concept that AS is initiated by the activation of cholinergic mechanisms, studies that employ microdialysis techniques have demonstrated that acetylcholine release is enhanced during pharmacologically-induced as well as naturally-occurring AS (Kodama et al., 1990; Leonard and Lydic, 1997; Lydic et al., 1991). Finally, there is an increase in the discharge of a majority of NPO neurons during AS (McCarley and Hobson, 1971; McCarley et al., 1995; Sakai, 1988). Taken together, these data provide compelling evidence that cells in and/ or in the vicinity of the NPO function as executive neurons that constitute the AS-Generator that is responsible for the generation and maintenance of AS, and that its constituent neurons are activated by projections from cholinergic neurons of the LDT/PPT. On the other hand, lesion studies have also demonstrated that the loss of cholinergic cells in the LDT/PPT results in a significant decrease in AS but, importantly, not the complete elimination of this state (Webster and Jones, 1988). Thus, although cholinergic processes are capable of activating cells of the AS-Generator and initiating AS (ibid.), it is clear that AS can also occur in the absence of LDT/PPT

M. Xi et al. / Experimental Neurology 238 (2012) 44–51

A) CNA

49

C) CNA + PPT

B) PPT

20 mV 10 ms Fig. 4. Facilitation of the discharge of intracellularly-recorded NPO neurons following stimulation of the CNA. A: No distinct synaptic responses were evoked in this representative NPO neuron following stimulation of the CNA (left panel, paired pulses shown by filled triangles). B: A small synaptic response of the same NPO neuron was induced following single-pulse stimulation of the PPT (middle panel, single pulse which was marked with an open triangle). When delivered separately, each of these stimuli was ineffective (i.e., subthreshold) in producing an action potential. C: Combined concurrent subthreshold stimulation of the CNA and the PPT induced action potentials (indicated by arrows) with a discharge index of 100%. Each panel consists of three consecutive traces of recordings from this NPO neuron.

inputs to the AS-Generator. In this regard, data from a number of anatomical studies provide a neuronal substrate for the present electrophysiological results and support our hypothesis that in addition to the PPT and the LDT, other sites, such as the amygdala, are important in the generation of AS. For example, tract-tracing studies, including those from our laboratory using both anterograde and retrograde tracers, have shown that CNA projection neurons innervate, directly, with ipsilateral predominance, neurons in the NPO in the rat (Boissard et al., 2003; Krettek and Price, 1978; Takeuchi et al., 1988; Yasui et al., 2004), guinea pig (Fung et al.,

A

2011), cat (Hopkins and Holstege, 1978; Krettek and Price, 1978) and monkey (Price and Amaral, 1981). In particular, our recent anatomical study provides morphological evidence that a glutamatergic, excitatory pathway exists that projects from the CNA directly to the NPO (Fung et al., 2011). Using spatial facilitation, and by varying the interval of stimulation between the CNA and the PPT, the time course of the interactions between these two inputs vis-à-vis activation of NPO neurons was determined. To the best of our knowledge, this is the first presentation of in vivo intracellular electrophysiological data which

B IC

PAG DR

PPT

NPO 50 µm

Fig. 5. Location and morphology of NPO neurons that were recorded and subsequently labeled with neurobiotin. Following stimulation of the CNA and/or the PPT, these neurons exhibited EPSP activity and responses as described in the text. After intracellular data were obtained, these neurons were injected with neurobiotin. A: Schematic distribution of neurobiotin-labeled neurons (filled circles) in the NPO. The schematic coronal plane of the rat brainstem is shown at a level of 8.3 mm posterior to Bregma according to Paxinos and Watson (1998). Note that the majority of these neurons were located in the lateral part of the NPO. B: Photomicrographic example of a representative NPO neuron that was labeled with neurobiotin (indicated by an arrow in A). Note details of the morphology of the neurobiotin-filled neuron. The cell body of this neuron was multipolar with a diameter greater than 30 μm. Abbreviations: DR, dorsal raphe nucleus; IC, inferior colliculus; NPO, nucleus pontis oralis; PAG, periaqueductal gray; PPT, pedunculopontine tegmental nucleus.

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addresses the precise fashion wherein amygdalar projections to the NPO interact with projections from the PPT in a manner that results in enhanced activation of putative AS-Generator neurons in the NPO. Based upon the data presented in this study, as well as previous data from our laboratory and that of others (ibid.), we propose that the CNA is able to act independently vis-à-vis the generation of AS, and that CNA projections also interact with LDT/PPT projections to determine the activity of AS-Generator neurons in the NPO. We suggest that projections from the CNA to the NPO participate primarily in determining the occurrence of the phasic phenomena of this state as a reflection of the emotional (e.g., fear, anxiety, motivational (e.g., feeding, reproduction) and autonomic (i.e., sympathetic and parasympathetic) functions which are within the purview of the amygdala (Pessoa, 2010; Sah et al., 2003). Consequently, we hypothesize that projections from the LDT/PPT to the NPO are responsible for generating and maintaining the tonic aspects (or a “baseline” level) of AS. The concept of multiple “controllers” that activate an “effector site/system” (e.g., the AS-Generator) accords with well-established principles of the functioning of a variety of physiological processes, such as those regulating blood pressure and hormonal secretions (Dampney et al., 2002), wherein there is one set of mechanisms that are responsible for maintaining basic “homeostatic” levels of activity and/or functions (such as the tonic periods of AS by the LDT/PPT), and other sets of control systems that respond to varying changes in the external and/or internal environments (such as the amygdala whose directives are reflected principally by changes in the phasic phenomena of AS, such as PGO waves and muscle twitches) (Calvo et al., 1987; Datta et al., 2008; Deboer et al., 1999). Similarly, wakefulness is generally accepted to occur as a consequence of the activation of neurons that comprise the Reticular Activating System that is controlled by a multitude of interacting projections from a number of sites that employ different neurotransmitter systems (Jones, 2005). There are also certain methodological considerations that involve the present experimental designs that are worthy of being addressed. For example, electrical stimulation was utilized to activate neurons in the CNA and/or in the PPT in the present study. Based on the anatomical dimensions of the CNA and the PPT, relatively low stimulus currents (≤300 μA for the CNA and ≤60 μA for the PPT) were employed in order to confine the effects of stimulation to these sites. In addition, the amplitude of responses was measured when the stimulating electrode was positioned at various distances from the target area in both nuclei. Thus, when the stimulating electrodes were moved 200 to 300 μm or 400 to 500 μm away from the designated target area within the PPT or the CNA, respectively, the responses evoked in NPO neurons were significantly reduced and disappeared when the electrodes were moved beyond the boundary of these nuclei. We therefore believe that the effects which were obtained were due to the discharge of neurons in the PPT and the CNA (see following text). Because of the nature of electrical stimulation, it was not possible in the present study to differentiate between the effects produced by the excitation of cell bodies and/or fibers of passage. Future studies, such as those using optogenetic techniques to selectively activate glutamatergic neurons in the CNA and cholinergic neurons in the PPT, should be able to resolve this issue (Stuber et al., 2011; Zhang et al., 2010). While the chemical nature of the PPT-evoked responses in the NPO in the present study was not determined, we believe that the EPSPs that were obtained were cholinergically mediated via PPT projections on the basis of their waveform parameters (e.g., latency-to-onset, latency-to-peak) and the fact that these responses were comparable to cholinergic EPSPs that have been pharmacologically identified (Imon et al., 1996). Regarding the chemical nature of the CNA-evoked responses, our recent immunohistochemical study demonstrated that a substantial population of CNA neurons that projects directly to the NPO are glutamatergic (Fung et al., 2011). In addition, a small number of CNA neurons that contain neuropeptides (e.g., somatostain, neurotension) also project to the NPO region (Veening et al., 1984). The characteristic

waveform parameters (e.g., short latency-to-onset, short latency-topeak) of EPSPs evoked in NPO neurons following stimulation of the CNA indicate that these responses were due to actions that were produced by glutamatergic neurons in the CNA. Another methodological consideration involves the urethaneanesthetized preparation that was used to examine the response of NPO neurons following stimulation of the CNA and/or the PPT. While there are no electrophysiological or pharmacological data regarding the effects of urethane on synaptic pathways to the NPO from either the CNA and/or the PPT, a recent study found no significant effects of urethane on either excitatory or inhibitory synaptic transmission (e.g., there was no difference in the amplitude or decay time course of glutamatergic EPSPs or GABAergic IPSPs or in the frequency, amplitude, or time course of spontaneous synaptic potentials) (Sceniak and Maciver, 2006). In addition, interactions between CNA and PPT projections and the resulting facilitation of EPSPs in NPO neurons by these converging inputs were studied under the same experimental conditions before and after individual stimulation of the CNA or the PPT. Therefore, we believe that the use of urethane did not significantly affect the conclusions that were drawn from the data that were obtained. In summary, the present results demonstrate the importance of excitatory (glutamatergic) projections from the CNA that directly innervate AS-Generator neurons in the NPO, and that interactions between projections from the CNA and cholinergic projections from the PPT determine the occurrence of AS. These findings therefore establish a foundation for future studies to explore the concept that multiple controllers participate in the generation of AS and provide a basis for exploring their control of the tonic and the phasic aspects of this state. Acknowledgments This research was supported by NIH grant NS 60917. We are grateful to Mr. Vincent Lim, Ms. Nichole Stevens and Mr. Daniel Bronson for their excellent technical assistance. References Baghdoyan, H.A., Lydic, R., Callaway, C.W., Hobson, J.A., 1989. The carbachol-induced enhancement of desynchronized sleep signs is dose dependent and antagonized by centrally administered atropine. Neuropsychopharmacology 2, 67–79. Boissard, R., Fort, P., Gervasoni, D., Barbagli, B., Luppi, P.H., 2003. Localization of the GABAergic and non-GABAergic neurons projecting to the sublaterodorsal nucleus and potentially gating paradoxical sleep onset. Eur. J. Neurosci. 18, 1627–1639. Brown, R.E., Basheer, R., Thakkar, M.M., Winston, S., McCarley, R.W., 2004. In vitro electrophysiology of rat subcoerulean neuron. Sleep 27 (Suppl.), A59. Brown, R.E., Winston, S., Basheer, R., Thakkar, M.M., McCarley, R.W., 2006. Electrophysiological characterization of neurons in the dorsolateral pontine rapid-eye-movement sleep induction zone of the rat: intrinsic membrane properties and responses to carbachol and orexins. Neuroscience 143, 739–755. Calvo, J.M., Badillo, S., Morales-Ramirez, M., Palacios-Salas, P., 1987. The role of the temporal lobe amygdala in ponto-geniculo-occipital activity and sleep organization in cats. Brain Res. 403, 22–30. Calvo, J.M., Simon-Arceo, K., Fernandez-Mas, R., 1996. Prolonged enhancement of REM sleep produced by carbachol microinjection into the amygdala. Neuroreport 7, 577–580. Chase, M.H., Morales, F.R., 2005. Control of motoneurons during sleep. In: Kryger, M.H., Roth, T., Dement, W.C. (Eds.), Principles and Practice of Sleep Medicine, vol. WB Saunders, Philadelphia, pp. 154–168. Dampney, R.A., Coleman, M.J., Fontes, M.A., Hirooka, Y., Horiuchi, J., Li, Y.W., Polson, J.W., Potts, P.D., Tagawa, T., 2002. Central mechanisms underlying short- and longterm regulation of the cardiovascular system. Clin. Exp. Pharmacol. Physiol. 29, 261–268. Datta, S., Li, G., Auerbach, S., 2008. Activation of phasic pontine-wave generator in the rat: a mechanism for expression of plasticity-related genes and proteins in the dorsal hippocampus and amygdala. Eur. J. Neurosci. 27, 1876–1892. Deboer, T., Sanford, L.D., Ross, R.J., Morrison, A.R., 1998. Effects of electrical stimulation in the amygdala on ponto-geniculo-occipital waves in rats. Brain Res. 793, 305–310. Deboer, T., Ross, R.J., Morrison, A.R., Sanford, L.D., 1999. Electrical stimulation of the amygdala increases the amplitude of elicited ponto-geniculo-occipital waves. Physiol. Behav. 66, 119–124. El Mansari, M., Sakai, K., Jouvet, M., 1989. Unitary characteristics of presumptive cholinergic tegmental neurons during the sleep-waking cycle in freely moving cats. Exp. Brain Res. 76, 519–529.

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