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Respiratory Physiology & Neurobiology 173 (2010) 64–70

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology

journa l homepage: www.e lsev ier .com/ locate / resphys io l

unctional topography of respiratory, cardiovascular and pontine-waveesponses to glutamate microstimulation of the pedunculopontineegmentum of the rat

rina Topchiya,b,e,∗, Jonathan Waxmana,d, Miodrag Radulovacki c, David W. Carleya,b

Center for Narcolepsy, Sleep and Health Research, M/C 802, University of Illinois at Chicago, 845 South Damen Ave, Chicago, IL 60612, USADepartment of Medicine, M/C 719, University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612, USADepartment of Pharmacology, M/C 868, University of Illinois at Chicago, 901 S. Wolcott Ave., Chicago, IL 60612, USADepartment of Electrical Engineering, University of Illinois at Chicago, Chicago, IL 60612, USAA.B. Kogan Research Institute for Neurocybernetics, Rostov State University, 194/1 Stachka Ave, Rostov-on-Don 344090, Russia

r t i c l e i n f o

rticle history:ccepted 14 June 2010

eywords:edunculopontine tegmentumpnea

a b s t r a c t

Functionally distinct areas were mapped within the pedunculopontine tegmentum (PPT) of 42ketamine/xylazine anesthetized rats using local stimulation by glutamate microinjection (10 mM,5–12 nl). Functional responses were classified as: (1) apnea; (2) tachypnea; (3) hypertension (HTN);(4) sinus tachycardia; (5) genioglossus electromyogram activation or (6) pontine-waves (p-waves) acti-vation.We found that short latency apneas were predominantly elicited by stimulation in the lateral

unctional anatomyleep

portion of the PPT, in close proximity to cholinergic neurons. Tachypneic responses were elicited fromventral regions of the PPT and HTN predominated in the ventral portion of the antero-medial PPT. Weobserved sinus tachycardia after stimulation of the most ventral part of the medial PPT at the bound-ary with nucleus reticularis pontis oralis, whereas p-waves were registered predominantly followingstimulation in the dorso-caudal portion of the PPT. Genioglossus EMG activation was evoked from the

pporr fun

medial PPT.Our results surespiration, cardiovascula

. Introduction

The pedunculopontine tegmentum (PPT) is known to partici-ate in a wide range of state-regulating and behavioral functions.PT plays an important role in rapid eye movement (REM) sleepnd REM sleep-related phenomena, including EEG activation, hip-ocampal theta rhythm and pontine-waves (p-waves) (Datta andobson, 1995; Garcia-Rill, 1991; Rye, 1997; Shouse and Siegel,992; Steriade and McCarley, 1990; Vertes et al., 1993). Injection

f glutamate into the PPT increases REM sleep and wakefulness forours in unanesthetized rats (Datta et al., 2001a,b) and injection ofarbachol increases REM sleep for days in cats (Calvo et al., 1992;atta et al., 1991) and rats (Carley and Radulovacki, 1999). Lesions

Abbreviations: PPT, pedunculopontine tegmentum; EEG, electroencephalogram;CG, electrocardiogram; EMG, electromyogram; BP, blood pressure; HTN, hyperten-ion; p-wave, spontine waves; REM, rapid eye movement; AP, antero-posterior; ML,edio-lateral; DV, dorso-ventral; NTS, nucleus of the solitary tract; VLM, ventro-

ateral medulla; RVLM, rostro-ventro-lateral medulla; PB, parabrachial complex.∗ Corresponding author at: University of Illinois at Chicago, Department ofedicine, Section of Pulmonary, Critical Care and Sleep Medicine, 840 South Wood

treet, M/C 719, IL 60612-7323, USA. Tel.: +1 312 996 3573; fax: +1 312 996 4665.E-mail address: itopchiy@uic.edu (I. Topchiy).

569-9048/$ – see front matter. Published by Elsevier B.V.oi:10.1016/j.resp.2010.06.006

t the existence of the functionally distinct areas within the PPT affectingction, EEG and genioglossus EMG.

Published by Elsevier B.V.

or pharmacological blockade of the PPT diminishes wakefulnessand eliminates or alters expression of the tonic and phasic com-ponents REM sleep, including p-waves and rapid eye movements(Shouse and Siegel, 1992). The PPT also participates in regulation ofmotor control (Garcia-Rill, 1991; Winn, 2006), modulation of sen-sation (Reese et al., 1995), orientation and attention (Rostron et al.,2008), reaction time, and learning and memory (Garcia-Rill, 1991;Datta, 1997; Datta and Hobson, 1995).

The PPT also may play an important role in autonomic reg-ulation. Continuous electrical stimulation within the PPT evokedrespiratory depression in anesthetized cats (Lydic and Baghdoyan,1993), whereas we demonstrated that carbachol injection into thePPT increased respiratory dysrhythmia during sleep in consciousrats (Carley and Radulovacki, 1999; Radulovacki et al., 2004). Wefurther showed that microinjection of glutamate into the PPT ofanesthetized rats initiated respiratory disturbances characterizedby irregular alternations between tachypnea and bradypnea/apnea(Saponjic et al., 2003, 2005, 2006). Stimulation of the PPT can also

evoke cardiovascular reactions, characterized by increased bloodpressure (BP) (Padley et al., 2007).

A variety of evidence shows that PPT neurons are, to someextent, anatomically organized into subregions according to theirfunction (Rye, 1997). For example, anatomically a specific antero-

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entro-medial region of the PPT reciprocally innervates the basalanglia (Rye et al., 1987), whereas a dorso-caudal portion of thePT has specific connectivity to the limbic ventral tegmental areaOakman et al., 1995; Rye, 1997; Rye et al., 1987). Lesions of thenterior and posterior PPT produced opposite effects on nicotine-nduced locomotion and self-stimulation behavior in rats (Aldersont al., 2008), and electrical stimulation specific to the anteriorPT prior to training improved subsequent learning (Andero et al.,007).

Our laboratory has demonstrated that local injection oflutamate in anesthetized rats can evoke respiratory dys-hythmia, electroencephalogram (EEG) activation, hippocampalheta-rhythm, or increased phasic events such as p-waves (Saponjict al., 2003, 2005, 2006). Further, these studies suggested that suchhenomena are at least partially differentiable, according to stimu-

ation site, suggesting a functional topography. However, a detailedunctional mapping of these activities within the pedunculopontineegmentum has not been performed. The aim of the present studyas to use glutamate microinjections to determine the functionalap of respiratory, cardiovascular and electroencephalographic

henomena within the PPT.

. Methods

Experiments were performed on 42 spontaneously breathingdult male Sprague–Dawley rats, weighing 200–300 g, maintainedn a 12-h light–dark cycle, and housed at 25 ◦C with free access toood and water. Principles for the care and use of laboratory animalsn research were strictly followed, as outlined by the Guide for theare and Use of Laboratory Animals (National Academy of Sciencesress, Washington, DC, 1996).

.1. Surgical preparation

The rats were anesthetized with a combination of 80 mg/kgetamine (Abbott Laboratories, North Chicago, IL) and 5 mg/kgylazine (Phoenix Scientific Inc., St. Joseph, MO) by intraperitonealnjection. After a surgical plane of anesthesia was achieved, rats

ere placed in the stereotaxic apparatus (David Kopf Inst., model62 A Tujunga, CA). The level of anesthesia was monitored by BP,eart rate and reflexes (corneal and toe-pinch). Supplemental dosef 40 mg/kg ketamine/2.5 mg/kg xylazine were used as necessaryo maintain a stable plane of anesthesia. No injections were madeor at least 15 min after any supplemental dose of anesthesia.

Animals were instrumented to record bilateral electroen-ephalograms (EEGs) using stainless steel screw electrodes locatedn the frontal (AP, +2.5 mm to bregma, ML, 2 mm to midline; DV,mm ventral to the surface of the brain) and parietal cortex (AP,2.5 mm to bregma; ML, 2 mm to midline; DV, 1 mm ventral to

he surface of the brain), referenced to a screw electrode in theasion. A bipolar twisted electrode made of teflon-coated stain-

ess steel wires with uninsulated tips of 1 mm and with a 1 mmeparation between the uninsulated tips was stereotaxically tar-eted into the contralateral PPT to record the pontine EEG (AP,7.8 mm to bregma; ML 1.8 mm to midline; DV, 7 mm ventral to

he surface of the brain) (Paxinos and Watson, 2004). The genioglos-us electromyogram (EMG) was obtained using teflon-coated wirelectrodes with uninsulated tips of 2 mm inserted bilaterally intohe base of the tongue 1–2 mm lateral to the frenulum. The electro-ardiogram (ECG) was registered by needle electrodes placed in the

eft axilla and the right flank. The femoral artery was catheterizedo monitor blood pressure using Transpac IV transducer (Hospira,ake Forest, IL).

A unilateral burr-hole osteotomy provided access to the ros-ral lateral pons, contralaterally to the twisted electrode and the

& Neurobiology 173 (2010) 64–70 65

dura was carefully removed. Three-barrel micropipettes were builtusing standard glass with filament (1 mm × 0.25 mm, A-M Systems,Carlsborg, WA) and a vertical puller (Model No 50-239, HarvardApparatus Ltd., Kent, England) to achieve an overall tip diameter of10–20 �m. A milli-pulse pressure injector (MPPI-2, ASI, Eugene, OR)was used to inject glutamate (10 mM l-glutamic acid monosodiumsalt in 0.2 M PBS; ICN Biomedicals, Aurora, OH), 0.2 M PBS, or oilred-O dye (Sigma, St. Louis, MO; solution of 7 mg in 1 ml ethanol)to aid histological verification of injection sites.

The MPPI-2 was outfitted with a backpressure unit to preventinjected fluids or extracellular fluid from entering the multibar-rel assembly between injections. Typical injection pressure was∼70 psi, while back pressure was ∼0.1 psi.

2.2. Recording procedure

Throughout each experimental protocol, we performed a 10-channel recording comprising: right and left monopolar frontalcortical EEG; right and left monopolar parietal cortical EEG; pon-tine bipolar EEG; genioglossus EMG; respiration recorded by athoracic piezoelectric sensor (VelcroR Tab-Infant-Ped; SleepmateR

Technologies); ECG; arterial BP and an injection marker (logic levelpulse) provided by the pressure injector.

After conventional amplification and filtering (1–20 Hz band-pass for EEG and BP, 1–100 Hz for EMG and ECG, and 1–10 Hz forrespiration; CyberAmp 380, Axon instruments/Molecular Devices,Sunnyvale, CA) the analog data were digitized (sampling fre-quency 200/s) and recorded using SciWorks for Windows software(Datawave Systems, Longmont, CO). Each recording began with a10 min registration of the baseline activity prior to any injections,which were separated by at least 5 min.

2.3. Experimental protocol

The pipette was stereotaxically positioned at the expected dor-sal margin of the PPT region (Paxinos and Watson, 2004) and wasadvanced ventrally in 100 �m increments. At each site 5–12 nl of l-glutamate was injected. Injection volumes were directly measuredusing a dissecting microscope (Wild Heerbrugg, model M5) with acalibrated reticule to observe the movement of the fluid meniscuswithin the pipette barrel. The dose of the glutamate was chosenaccording to our previous work (Saponjic et al., 2003, 2005, 2006),which showed the effectiveness of this volume and concentrationto evoke a prominent respiratory reaction from the PPT. Further,work by Nicholson (1985) suggests that within the first 35 s theeffective diffusion radius is limited to approximately 150 �m.

EEG (cortical and pontine), EMG, BP and respiration were reg-istered prior to and after each glutamate injection. An “effectivesite” was determined by visually evident perturbations in any ofthese parameters after the glutamate injection. In this case a mini-mum 5-min interval was provided prior to advancing the pipette. Inall animals at least two repeated glutamate injections were madeat any effective site to better document the duration and repro-ducibility of the response. A sham control also was obtained ateach effective site by injection 5–12 nl of PBS. The PBS injectionwas made either prior to or after the second glutamate injectionat the effective site. In every experiment, subsequent glutamateinjections reproduced the initial response, whereas all PBS injec-tions failed to do so. Although application of the back pressure tothe barrel containing O-red dye raises potential concern of alcohol-leakage to the injection site between injections, the reproducibility

of the responses after the repeated glutamate injections argues thatthis effect was not of significant concern to the interpretation of thepresent observations.

The effective sites were injected with oil O-red dye for histolog-ical verification. In some cases the point of the reaction was marked

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y biotinylated dextran amine (BDA, Invitrogen, 5% in 0.1 M PBS) athe end of the experiment. BDA was delivered iontophoretically.

The initial target sites within the PPT were varied among exper-ments in order to provide a balanced sampling of responses acrosshe anatomical extent of the PPT region.

.4. Data processing

Respiratory pattern analysis was conducted using softwareeveloped in our laboratory. An automated adaptive thresholdlgorithm was used to detect and quantify the inspiratory and expi-atory phases of the respiratory cycle, providing the inspiratory,xpiratory and total duration of each breath. Apnea was defineds any breath with a duration exceeding the mean of the previ-us 10 breaths by at least 50%. Tachypnea persisted for at leastbreaths, was determined by visual scoring. Hypertension (HTN)as assessed visually as a transient BP increase with a return to the

aseline. Sinus tachycardia was assessed visually and had a mini-um duration of 3 beats. Genioglossus EMG activation was visually

ssessed with respect to the preceding 10 s baseline. p-waves wereisually assessed from the pontine EEG tracings according to theollowing criteria: (1) a major deflection with the same polarityhroughout a recording; (2) a wave duration of 50–150 ms; and (3)large amplitude that was readily discriminable from the back-

round.

.5. Histology

At the end of each experiment rats were deeply anesthetized (andditional dose of 40/2.5 mg/kg of ketamine/xylazine was admin-stered if necessary) and perfused intracardially. The perfusiontarted with a vascular rinse with 0.9% saline until the liver hadleared (at a perfusion speed of 40 ml/min, about 1 min). The per-usion continued with 4% paraformaldehyde solution (300 ml at0 ml/min, then 30 ml/min), and finally with 10% sucrose solu-ion in phosphate buffer (30 ml/min). The brain was extractedn-bloc, cleared of meninges and blood vessels, and immersed in 4%araformaldehyde overnight. Brains were immersed in 30% sucroseolution for several days. Then they were cut in a transverse planen 40 �m thick sections, using a cryostat microtome. To identify theocation of microinjection sites in relation to the cholinergic neu-ons of the PPT, sections were processed for NADPH-diaphorasey incubating for 15 min in a mixture of 2 mg/ml �-NADPH andmg/ml nitroblue tetrazolium (Sigma, St. Louis, MO). Air-dried

lides were cleared in xylene and coverslipped with Permaslip.

. Results

Functional responses were observed following 102 glutamatenjections in a total of 42 rats. Based upon an initial exploratory andeuristic examination of the data, the responses were classified as:1) apnea (49 injections in 22 rats); (2) tachypnea (8 injections inrats); (3) HTN (10 injection in 5 rats); (4) ECG sinus tachycardia

5 injections in 2 rats); (5) genioglossus EMG activation (14 injec-ions in 6 rats); (6) p-waves (6 injections in 5 rats) (Fig. 1A and). This classification scheme captured over 90% of all responsesbserved. However, complex responses, including combined apneand EMG activation (2 cases), apnea and HTN (4 cases), tachypneand HTN (2 cases), HTN and EMG activation (1 case), tachypnea,TN and p-waves (1 case) were observed in 10 rats. Advancing theipette along a dorso-ventral track could elicit responses of differ-

nt modalities or of different latencies within the same modality.esponses included for analysis as illustrated in Fig. 1B, had vari-ble latencies, but occurred within 5–35 s of glutamate injection. Toompile a functional map of the “response regions” we employedhe pipette location associated with the shortest response latency

Neurobiology 173 (2010) 64–70

for that modality within a track of a given experiment. Polymodalreactions to the glutamate tended to occur in the “transitionalzones” among the unimodal response areas. Fig. 2 maps the shortlatency responses of different modalities from all 42 experimentsonto a schematic representation of transverse brain sections spacedat 400 �m intervals over the anteroposterior extent of the PPT. Theresponses are presented in relation to the locations of the NADPH-diaphorase (+) cholinergic cells (filled circles in the background).The responses of different modalities are indicated by a color sym-bols and mapped to the 400 �m plane nearest to the anatomical40 �m tissue section corresponding to the tip of the pipette.

The semi-transparent circles surrounding the injection sitesdepict the estimated diffusion distance of the glutamate (about300 �m in diameter), predicted theoretically by Nicholson (1985)(see Section 4 for details).

3.1. Apnea

As depicted in Fig. 2, apneic responses to glutamate microin-jection were observed throughout the antero-posterior extent ofthe PPT region (−6.8 to −8.4 mm from bregma). In the most ante-rior portion of the PPT (−6.8 to −7.2 mm from bregma) apnea waselicited from dorsolateral areas associated with cholinergic neu-rons. In the most posterior regions (−8.0 to −8.4 mm from bregma)apneagenic sites were located predominantly in the ventro-lateralpart of the PPT. Between these anterior and posterior poles, apneicsites were distributed through the mediolateral and dorso-ventralextent of the PPT. However, apnea producing sites were found pre-dominantly in the ventro-lateral area. All apneic sites were closelyapproximated to areas of cholinergic cells within the PPT.

Sites for delayed apnea (latency > 35) surrounded the sites asso-ciated with short latency apnea: up to 300 �m dorsally in theanterior PPT; up to 300 �m ventrally in the posterior PPT; andup to 100 �m medially or laterally throughout the PPT. In fiveexperiments, spontaneous apneas were observed during baselinerecordings prior to any injections. However, even in these experi-ments (as shown in Table 1), spontaneous apneas never occurredmore than once during a 5-min interval at baseline and had ashorter duration than glutamate-evoked apneas. Thus, sponta-neous apnea cannot account for the observed PPT evoked apneas.

3.2. Tachypnea

Tachypnea (Fig. 1B) was evoked from several sites at the ventro-lateral margin of the PPT, located −7.6 to −8 mm from bregma andin the ventral part of the PPT −8 to −8.4 from bregma (Fig. 2).Tachypneic responses started from 0 to 15 s after the glutamateinjection and typically persisted for no more than 35 s. In 2 casesthese responses were associated with transient HTN, and eitherthe tachypnea or the HTN could occur first following an individualinjection.

3.3. Cardiovascular disturbance

HTN, lasting approximately 30 s and with a short latency fol-lowing glutamate injection, was elicited from the ventral half ofthe PPT in all but the most anterior regions. HTN was observedin 10 cases, and was most often not associated with respiratorypattern changes. Transient sinus tachycardia was elicited from 2sites residing at the border of the PPT and the nucleus pontis oralis(Fig. 2).

3.4. Genioglossus EMG

Activation of genioglossus EMG was elicited from the medio-posterior part of the PPT (−7.6 to −8 mm from bregma) surrounding

I. Topchiy et al. / Respiratory Physiology & Neurobiology 173 (2010) 64–70 67

Fig. 1. Responses evoked by glutamate microstimulation of the PPT: (A) photomicrographic illustration of various PPT injection sites paired with (B) physiological responsest ainedP ea; (3p espond lectro

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o glutamate microinjection at the depicted site The 40 �m brain sections (A) were stanel (B) illustrated six categories of physiologic response: (1) apnea; (2) tachypn-waves. An injection marker is shown as a vertical rule (at 10 s) on each trace. Rownward deflection from baseline. Resp—respiration; BP—blood pressure; ECG—e

he superior cerebellar peduncules (Fig. 2). EMG responses couldccompany respiratory reactions (2 cases), but also could be evokedeparately from them (4 cases) (Fig. 1B).

.5. p-waves

Clusters of p-waves were recorded from the contralateral PPTollowing glutamate injections in the dorso-caudal portion of thePT (Fig. 2). This response comprised bursts of repetitive monopha-

able 1omparison of the apneas at the baseline and post-injection in the group of 8 rats with sp

Apneas

Mean apnea duration (ms) P v

Baseline 1087 ± 590

Post-injectionShort latency apneas 4972 ± 2643 8.8Long latency apneas 4741 ± 2645 9.9Total post-injection 4787 ± 2607 2.7

values were estimated relative to the baseline values and considered significant if ≤0.0

for NADPH-diaphorase to localize cholinergic neurons with a dark reaction product.) hypertension (HTN); (4) sinus tachycardia; (5) genioglossus EMG activation; (6)ses are marked by thin horizontal lines. On the respiratory traces inspiration is acardiogram; EEG—pontine EEG; EMG—genioglossus EMG.

sic waves of the same polarity, with a typical burst duration of1–3 s and with an interwave frequency ranging from 5 to 6 Hz(Fig. 1B). In accordance with our previous findings (Saponjic etal., 2003), the sites where glutamate elicited exclusively p-waves

were located dorsal to sites from which respiratory responsescould be evoked. In 2 cases (in the posterior part of the PPT) bothrespiratory and p-wave responses were evoked from the samesite, but the respiratory response always appeared prior to thep-waves.

ontaneous apneas.

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alue Mean apnea duration (ms) P value

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68 I. Topchiy et al. / Respiratory Physiology & Neurobiology 173 (2010) 64–70

Fig. 2. Schematic map of responses elicited from different sites of the PPT by microinjection of glutamate. Anatomical injection sites were localized with antero-posteriorresolution of ±40 �m (the section thickness). For presentation clarity the diagram pools responses collected across multiple experiments by projecting histologically verifiedpipette locations onto the matching 400 �m coordinate range: 6.8–7.2; 7.2–7.6; 7.6–8 and 8–8.4 mm from bregma. Injection sites are overlaid on a background demonstratingthe location of NADPH-diaphorase positive cholinergic neurons within the PPT. Response types are coded into one of six categories by color. The diameter of each circler �m, s( ns). Smfi FV—cu

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epresents the estimated short-term glutamate diffusion volume (diameter ∼300number within each circle indicate the number of overlapping ineffective injectioeld; PnO—nucleus reticularis pontis oralis; scp—superior cerebellar peduncles; Cn

. Discussion

The present study establishes a systematic functional topog-aphy for respiratory, cardiovascular, electromyographic andlectroencephalographic responses to PPT stimulation by gluta-ate microinjection. Moreover, our findings demonstrate that, toreasonable extent, these functional responses to local PPT stim-lation are anatomically mapped to partially distinct subregions.inally, most responses characterized in this study were elicitedrom the PPT regions associated with numerous cholinergic neu-ons. Because the PPT region participates in a host of integrativeunctions, it is not surprising that local glutamatergic stimulationithin this area elicited a variety of autonomic and electrographic

esponses. However, the various “response regions” revealed a dis-inct spatial organization.

Across all experiments we consistently observed that advancinghe pipette by 100 �m could lead to a change from no response toisually evident response or to a decrease or increase of responseatency. Building the functional map of the “response regions” wasased on the location of the responses with shortest latency (<35 s)ithin a track. These observations also support the likelihood ofistinct “response regions”.

Nicholson used a mathematical model to determine that withinhe first 30 s after the injection, the concentration of the drug (glu-amate) present 300 �m from the injection site would be no morehan 20% of the concentration at the pipette tip (Nicholson, 1985).sing larger infusion volumes (100 nl) in freely moving rats, Dattand co-workers determined that the minimum concentration oflutamate administered to the PPT that produced increased rapidye movement sleep was ∼2 mM, whereas greater than 5 mM glu-amate was required to elicit increased wakefulness (Datta et al.,001b). Here, we employed 10 mM glutamate, suggesting that con-entrations would fall below 2 mM at a distance of no more than00 �m from the pipette tip.

Thus, it is possible that diffusion of glutamate to active sites up to300 �m distant from the injection might account for the variable

esponse latencies observed. This consideration limits the practi-al spatial resolution of our response mapping to ±150 �m. Thus,e expect that injections spaced at 100 �m increments provided

ee text for details). Unfilled circles represent injections that elicited no responseall black filled circles mark the location of cholinergic neurons. RRF—retrorubral

neiform nucleus; MiTg—microcellular tegmental nucleus; rs—rubrospinal tract.

a series of partially overlapping areas of glutamate stimulation.From this viewpoint it is not surprising that advancing the pipette100 �m could sometimes produce complex responses in transitionzones.

Thus, we observed a combination of apnea with HTN in 4 cases,apnea with EMG activation in 2 cases and tachypnea with HTN in2 cases. Based on the discussion above, we speculate that in thesecases, the diffusion distance of the glutamate allowed a single injec-tion to affect functionally distinct areas in close proximity to oneanother.

Additionally, a broad dendritic arborization could influence thelocation and latency of the responses. It was shown that neuronsof the PPT have radiating dendrites with terminals extended up to500 �m from the soma (Takakusaki et al., 1996). This could cause abroadening or even displacement of apparent response areas basedon our injection mapping technique. However, it was shown thatPPT dendrites extend primarily into adjacent sensory pathways(Rye et al., 1987). Additionally, it is possible, that glutamate recep-tors of PPT neurons are heavily located on their soma (Honda andSemba, 1995).

In our view, the functional topology of the PPT demonstratedin this study in not surprising. Like other pontine regions, thePPT participates in a wide variety of sensory and autonomicfunctions, in addition to behavioral state regulation. In terms ofcardiorespiratory functions, the PPT receives significant inputsfrom the medullary NTS (Gilbert and Lydic, 1991); another struc-ture with significant subnuclear functional differentiation (Herbertet al., 1990). Herbert et al. showed that NTS connections to theparabrachial complex of the pons demonstrate a high degree offunctional specificity (Herbert et al., 1990). The present findingssuggest that such functionally specific connections also may pertainto the PPT, but this determination will require future anatomicalstudies.

Virtually all glutamate-induced responses were elicited from

sites at which cholinergic neurons were located. The distributionof cholinergic neurons within the PPT has been descriptively sub-divided into the more dense pars compacta located in the posteriorpart of the PPT (PPTc) and the less dense pars dissipata (PPTd) inthe anterior part of the PPT (Olszewski and Baxter, 1954). It was

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hown that cholinergic neurons in rat are cytochemically and con-ectionally distinct from non-cholinergic cells with which they aredmixed (Rye et al., 1987). The cellular structure and arborizationf the cholinergic neurons within PPT pars compacta (PPTc) differsrom that of PPT pars dissipata (PPTd). The cholinergic neurons inhe PPTc are smaller than those in the PPTd. According to Lavoie andarent (1994a,b) “the neuronal processes are shorter and poorlyranched in the PPTc, whereas they are longer and more profuselyrborized in the PPTd”. Thus, one can expect more local responsesithin the PPTc. This anatomic difference could also explain the

ariety of latencies of the PPT responses.The anatomical pathways by which the PPT activation impacts

espiratory, cardiovascular or electroencephalographic processesannot be directly identified from the present data, although pre-ious neuroanatomical and neurophysiological studies allow uso make some suggestions. The PPT pars compacta is the majorholinergic input to the cardiorespiratory regions of the rostralentro-lateral medulla (RVLM) (Yasui et al., 1990) and individ-al cholinergic axons arborise extensively within separate nucleif their termination (Rye 1997). Anatomical tracing studies haveonfirmed that choline-acetyltransferase positive neurons directlynnervate BP regulating areas of the RVLM (Padley et al., 2007).urther, Padley et al. (2007) reported that bilateral injection oficuculline or excitatory dl-homocysteic acid into the PPT evokedn increase in BP and altered the baroreflex. Therefore, it is possi-le that the cardiovascular and respiratory responses we observedollowing PPT glutamate injections resulted from activation of

onosynaptic projections from the PPT to the RVLM, althougholysynaptic pathways cannot be ruled out. For example, thePT reciprocally innervates the parabrachial complex (Gilbert andydic, 1991; Quattrochi et al., 1998; Semba and Fibiger, 1992) andeurons of the parabrachical complex together with the Kölliker-use nucleus regulate respiratory phase switching. In addition,timulation of the medial parabrachial or Kölliker-Fuse areas pro-uces expiratory facilitation and apnea, whereas activation of the

ateral parabrachial region can produce tachypnea (Chamberlinnd Saper, 1994; Dick and Coles, 2000; Takayama and Miura,993). Thus, it is reasonable to speculate that activation of medialarabrachial neurons following PPT stimulation may account forpnea, whereas activation of the lateral parabrachial neurons mayccount for tachypnea. Definitive evaluation of these possibilitiesill require future investigation, and many other multisynapticathways may also account for or contribute to the observedesponses.

The present study also showed that neurochemical excitationt certain PPT injection sites evoked an activation of genioglossusMG. Consistent with this observation, Rukhadze and Kubin (2007)ave shown that cholinergic neurons of the PPT, originating in a dis-rete portion of its pars compacta, send bilateral projections to theotor nucleus of the hypoglossal nerve (Mo12). Thus, it is possible,

hat these projections contribute to both excitatory and inhibitoryodulation of the activity of hypoglossal motoneurons.The p-waves were registered in the medio-posterior part of

he PPT, dorsal to the respiratory “effective” sites. Pharmacologi-al and physiological studies have demonstrated a major role forholinergic neurons in the generation and transmission of p-wavesDatta et al., 1991; Datta, 1997; Henriksen et al., 1972). The influ-nce of glutamate on cholinergic cells of the PPT was shown tovoke either REM sleep and p-waves or wakefulness dependingn the dose (Datta and Siwek, 1997; Datta et al., 2001a,b), pro-iding support for the possibility that glutamatergic activation of

holinergic neurons evoked p-waves as we observed. Although wese animals, anesthetized by ketamine, a known NMDA receptorntagonist, we (current work; Saponjic et al., 2003) and othersDatta et al., 2001a,b) demonstrate the possibility to evoke p-wavesn ketamine-anesthetized animals by local application of small

& Neurobiology 173 (2010) 64–70 69

amounts of glutamate into the PPT. Thus, ketamine is permissiveof p-wave expression when administered systemically to achievesurgical anesthesia. However, additional local administration ofketamine directly to the PPT blocks subsequent glutamate-inducedp-waves. Thus, NMDA receptors within the PPT may play a role inevoked p-waves.

P-wave generation requires the participation of several differenttypes of neurons (Garcia-Rill, 1991; Datta, 1997; Rye, 1997), butespecially relies on bursting neurons. Although NMDA receptorsplay some role in the generation of bursting patterns, AMPA andmetabotropic glutamate receptors as well as GABA receptors alsoparticipate in generation of p-waves. As noted above, PPT glutamateinjections do not evoke p-waves under nembutal anesthesia.

In summary, the present study establishes a functional het-erogenity of responses elicited by glutamate within the PPT,including respiratory, cardiac, vascular, upper airway and EEGeffects. This supports a potentially broad role for the PPT indetermining state-depending autonomic functions. Moreover, ourfindings demonstrate that these functional responses to local PPTstimulation are anatomically mapped to partially distinct subre-gions. Most responses characterized in this study were elicited fromthe PPT regions associated with numerous cholinergic neurons, butelaboration of the synaptic processes and anatomical pathways bywhich the effects are mediated will require future investigation.

Conflict of interest

The authors: Irina Topchiy, Jonathan Waxman, Miodrag Radulo-vacki and David W. Carley do not have any actual or potentialconflict of interest including any financial, personal or other rela-tionships with other people or organizations within three (3) yearsof beginning the work submitted that could inappropriately influ-ence this work.

Acknowledgements

Authors acknowledge Milka Dokic for the technical support ofthe experiments. The work was supported by NIH grant AG016303.

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