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. 2012 Nov 5;591(Pt 1):109–122. doi: 10.1113/jphysiol.2012.245217

Descending control of the respiratory neuronal network by the midbrain periaqueductal grey in the rat in vivo

Hari H Subramanian 1
PMCID: PMC3630775  PMID: 23129795

Abstract

Emotional reactions such as vocalization take place during expiration, and thus expression of emotional behaviour requires a switch from inspiration to expiration. I investigated how the midbrain periaqueductal grey (PAG), a known behavioural modulator of breathing, influences the inspiratory-to-expiratory phase transition. Contemporary models propose that late inspiratory (late-I) and post-inspiratory (post-I) neurones found in the medulla, which are active during the inspiratory-to-expiratory phase transition are involved in converting inspiration to expiration. I examined the effect of excitatory amino acid (d,l-homocysteic acid; DLH) stimulation of the PAG on the discharge function of late-I and post-I neurones. The data show a topographical organization of DLH-induced late-I and post-I neuronal modulation within the PAG. Dorsal PAG stimulation induced tachypnoea and caused excitation of both the late-I and post-I neurones. Lateral PAG induced inspiratory prolongation and caused an excitation of late-I neurones but inhibition of post-I neurones. Ventrolateral PAG induced expiratory prolongation and caused a persistent activation of post-I neurones. As well, PAG stimulation modulated both the late-I and post-I cells for least two–three breaths even prior to the change in respiratory motor pattern. This indicates that the PAG influences the late-I and post-I cells independent of pulmonary or other sensory afferent feedback. I conclude that the PAG modulates the activity of the medullary late-I and post-I neurones, and this modulation contributes to the conversion of eupnoea into a behavioural breathing pattern.

Key points

  • Basic respiratory rhythm is generated and maintained by neurones located in the medulla oblongata.

  • This basic respiratory rhythm is changed by neurones in the region called the periaqueductal grey (PAG) located in the midbrain, in order to modulate breathing for behaviour and emotional expressivity.

  • How the PAG converts basic respiratory rhythm into behavioural breathing is not known.

  • In this study I investigated the influence of the PAG on two important populations of medullary neurones, the late-inspiratory (late-I) and post-inspiratory (post-I) neurones, which are thought to be involved in mediating rhythmic inspiration to expiration phase transition.

  • I show that the PAG modulates the activity of the medullary late-I and post-I neurones, and this modulation contributes to the conversion of basic breathing into behavioural breathing.

Introduction

Brainstem respiratory neurones play a critical role in the generation of basic breathing rhythm or in other words eupnoea (Cohen, 1979; Richter, 1982; von Euler, 1983; Long & Duffin, 1986; Ezure, 1990; Bianchi et al. 1995; Feldman & del Negro, 2006; Smith et al. 2007). This eupneic rhythm is modified by neurones with behavioural roles to support development of emotional expressivity. Such modulatory neurones are hierarchically organized throughout the neuraxis from the cerebral cortex through to the brainstem (Holstege, 1991). A critical area within this hierarchy is the midbrain periaqueductal grey (PAG), which is involved in motor patterning of emotional reactions, such as flight and aggression (Bandler & Carrive, 1988), pain and anxiety (Lovick, 2000), fear and immobility (Zhang et al. 1990), and vocalization (Zhang et al. 1994; Subramanian et al. 2008). Thus, the PAG functions as the behavioural modulator of breathing (Subramanian et al. 2008; Subramanian & Holstege, 2009, 2010, 2011).

Behaviours such as vocalization take place during expiration. This means, initiation of behavioural expression would require switching from inspiration to expiration. Contemporary models (Rybak et al. 1997, 2004) propose that the inspiratory-to-expiratory phase transition during eupnoea is mediated by two types of neurones, the late-inspiratory (late-I) and post-inspiratory (post-I) cells located in the ventral respiratory group (VRG) of the caudal medulla (Cohen, 1979; von Euler, 1983; Long & Duffin, 1986; Ezure, 1990; Bianchi et al. 1995; Smith et al. 2007; Mörschel & Dutschmann, 2009). The peak discharge of the late-I cells is most intense when inspiration ends (Feldman & Cohen, 1978; Cohen & Feldman, 1984; Pierrefiche et al. 1998; Haji et al. 2002). Also, activation of pulmonary stretch receptor (PSR) afferents that convey information relating to tidal-volume increases the discharge rate of late-I neurones (Cohen et al., 1993; Hayashi et al., 1996). Thus, late-I cells are thought to initiate the inspiratory to expiratory phase transition. The phase transition is thought to be completed by the post-I neurones. The post-I neurones fire at maximum intensity at the beginning of stage-1 expiration, or in other words post-inspiration (Richter, 1982; Ballantyne & Richter, 1984; Richter et al. 1987; Dutschmann & Paton, 2002; Subramanian & Holstege, 2011), and has been suggested to hold the medullary neuronal network that generates inspiration inhibited throughout expiration (Richter, 1982; Ballantyne & Richter, 1984; Richter et al. 1987; Shannon et al. 2000). Many researchers have also classified the post-I cells as early expiratory-decrementing cells (Bianchi et al. 1988; Manabe & Ezure, 1988; Lindsey et al. 1989; Ezure et al. 1993; Hayashi et al., 1996; Shannon et al. 2000; Shen et al. 2003).

I hypothesize that the PAG modulates the activity of medullary late-I and post-I neurones, and this modulation contributes to the conversion of eupnoea into a behavioural breathing pattern. Thus, in this study, I investigated the influence of PAG on the discharge pattern of late-I and post-I neurones, and their correlation to the PAG-induced respiratory motor pattern changes.

Methods

Experimental protocols and use of pentobarbiturate anaesthesia

Experiments were designed and performed at The University of Sydney, Australia. Approval for the study was obtained from the Institutional Animal Care Ethics Committee. Sprague–Dawley rats (n= 32) weighing 350–450 g of either sex were used for the study. Two series of experiments were conducted. First the ventrolateral medulla was mapped for late-I and post-I neurones and their extracellular potentials were recorded. The stereotaxic co-ordinates for this search were adapted to Sprague–Dawley rats based on Paxinos & Watson's (1997) rat atlas. The second series of experiments examined the effect of excitatory amino acid (EAA) stimulation of various regions of the PAG on the late-I and post-I neuronal function and on the respiratory motor output. The nucleus ambiguous (NA) is located in the same ventrolateral medullary region as the late-I and post-I neurones. Because NA contains pharyngeal and laryngeal motoneurones, NA also plays a role in respiration. The cyclic discharge of these NA motoneurones interferes with localizing in vivo the late-I and post-I neurones. However, barbiturate anaesthesia suppresses the spontaneous activity of the NA motoneurones (Merrill, 1970, 1974), for which reason I used pentobarbiturate-anaesthetized instead of decerebrate unanaesthetized preparations.

Surgery

The rats were initially anaesthetized with sodium pentobarbitone (nembutal, 70 mg kg−1; i.p.). Catheters were placed in the femoral artery and vein for monitoring blood pressure (BP) and administration of supplementary fluids, respectively. Additional doses of nembutal (5 mg kg−1) were delivered intravenously as required to maintain adequate and stable levels of anaesthesia. Depth of anaesthesia was assessed by checking for the absence of the withdrawal reflex and/or arterial BP changes after a hindpaw pinch. A tracheal cannula was introduced via tracheostomy to allow spontaneous breathing and also to connect a mechanical ventilator if required. The animal's body temperature was maintained between 36°C and 38°C using a feedback-controlled heating blanket and a rectal probe. Animals were then placed in a stereotaxic frame and a burr hole was drilled in the skull to allow access to the midbrain PAG. An occipital craniotomy was performed to allow access to the medulla. The brain was covered with paraffin oil.

Diaphragm electromyogram (EMG) recording

Two Teflon-coated stainless steel wires (0.0045 in/0.11 mm), stripped 2 mm at each end, were surgically implanted into the crural diaphragm (Subramanian & Holstege, 2011) to measure inspiratory motor output through diaphragm EMG activity. The same part of the muscle was chosen carefully to implant the EMG electrodes, in order to maintain similar electrode geometry and signal output. Electrode placements were verified at the conclusion of each experiment.

Extracellular recording of late-I and post-I neurones in the ventrolateral medulla

Micropipettes (in the double-barrel cluster) filled with 3 m NaCl (DC impedance approx. 8–10 MΩ) were used to localize the late-I and post-I neurones in the rostral ventrolateral medulla. All extracellular recordings were obtained from cell bodies and not from fibres of passage. This verification was done by microstimulation via an injection of 3–6 nl d,l-homocysteic acid (DLH) through the DLH barrel while simultaneously recording via the NaCl barrel from the same cell. This technique enabled to check whether or not this cell showed excitation as a result of DLH administration (Fries & Zieglgansberger, 1974; Goodchild et al. 1982; Lipski et al. 1988). Excitation of the cell verified that the recorded action potential was from the cell and not from fibres of passage.

EAA microinjections into the PAG

In order to stimulate various parts of the PAG intracerebral microinjection of the glutamate agonist, DLH was employed. Microinjecting DLH is a standard method for selective stimulation of neuronal cell bodies within the CNS (Goodchild et al. 1982). For stimulation of the PAG, single barrel micropipettes (tip diameters 10–30 μm; filled with 50 mm DLH) were inserted into its dorsal, lateral and ventrolateral parts. A pressure system (Picospritzer II, Parker Instrumentation, USA) delivered the microinjections. The injected volume was determined using a pre-calibrated scale and observing the movement of the meniscus on the scale. To eliminate the pharmacological effect of a previous injection, a 25 min interval was provided between two microinjections. Rhodamine-B fluorescent microspheres (Sigma, Australia) beads were added to the DLH solution in order to later determine the precise location of the injection sites. Isotonic saline injections were used as control.

Histology

At the end of each experiment, the animal was deeply anaesthetized and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in phosphate buffer, pH 7.2. After perfusion the brain was removed and stored in 4% formaldehyde for 2 h. It was then transferred to a 30% sucrose/formaldehyde mixture for at least 48 h to prevent formation of ice crystals. The midbrain and brainstem were cut on a freezing microtome into 50 μm coronal sections. The injection sites, marked by rhodamine microspheres, were identified using fluorescence microscopy and were represented on standard drawings according to the stereotaxic atlas of Paxinos & Watson (1997). Marked sites were reconstructed on standard sections by first identifying the bottom of each tract and then reconstructing the injection sites based on the depth of each injection as noted from the stereotaxic micro-manipulator record.

Data analyses

The PCM Vetter-Maclab-Macintosh (SDR Scientific, Sydney, Australia; ADI Instruments, Sydney, Australia) data acquisition system was used for collecting data. Using MacLab software (AD Instruments), ensemble averages were derived from amplified (×1000), low-pass-filtered signals using sampling rates of 20 000 per second. Respiratory frequency (RF), inspiratory duration (Ti) and expiratory duration (Te) were calculated from the diaphragm EMG. Late-I and post-I neuronal activity was amplified, recorded and displayed on an analogue as well as on a digital storage oscilloscope (Scope Software, Maclab Systems, Sydney, Australia) to ensure that the unit under study was unambiguously discriminated throughout the experiment. Signals were also fed into a window discriminator (chart software) for continuous assessment of the configuration, shape and amplitude of the recorded action potentials. Once a late-I or post-I neurone was identified from its background activity, the spike size was optimized prior to PAG stimulation. The cell-unit firing rate (spikes s−1) was measured for 10 breaths for both pre-stimulation (control) and post-stimulation periods. The latency of the response was measured from the time of injection until the time of onset of significantly increased firing rate (mean ± SE). The duration of the response was measured from the time when the discharge rate reached resting mean ± SE until the time when the rate returned to the pre-injection control values. During the peak effect following PAG stimulation, the bursting characteristics of the late-I and post-I cells (spikes s−1) were computed. This study only includes neurones whose spike configuration remained constant and could clearly be discriminated from background activity throughout the entire experiment. This approach allowed determining the activity characteristics of the same late-I/post-I cell before and after PAG stimulation. Spike discharge (spikes s−1), power density and frequency spread of both late-I and post-I neurones were computed. Statistical comparisons for RF, Ti and Te, and DLH effects were carried out using analysis of variance (ANOVA). Statistical comparisons between control and DLH effect on the late-I and post-I cells utilized two-tailed, paired t tests. Computer chart records were transferred into a drawing program (Adobe Illustrator, Australia) for data presentation.

Power density spectral analysis

The spectrum module of the chart software (AD Instruments) was used to compute the power density spectra of the diaphragm EMG and that of late-I and post-I neurones. The power density spectrum uses a discrete Fast Fourier Transform (FFT) algorithm to convert data from time to frequency domains. The spectrum divides both the diaphragm EMG and the extracellular neurone discharge data into segments, with the number of data points in each segment equal to a power of two as determined by the FFT algorithm. I selected a FFT size of 1 kHz for representation of the spectrum in the frequency domain. For each signal the power density spectral windows were computed for 10 successive breaths. At the end of computation, all individual power spectral windows were averaged and the spectra (FFTs) presented as connected points. The frequency domain (0–1 kHz) is converted to time-spread (0–300 ms) of the inspiratory cycle and represented on the x-axis, while the y-axis represents arbitrary units.

Results

Spontaneously breathing vagi-intact pentobarbiturate-anaesthetized adult rats (n= 32) had the following respiratory and cardiovascular parameters (control values expressed as mean ± SEM): Ti, 0.30 ± 0.05 s; Te, 0.50 ± 0.10 s; RF, 75 ± 10 breaths min−1; BP, 100 ± 5 mmHg; heart rate, 380 ± 5 beats min−1, arterial blood pH, 7.4 ± 0.01; Inline graphic, 35.2 ± 3.0 Torr; Inline graphic, 120.5 ± 5.0 Torr.

Late-I and post-I neurones in the ventrolateral medulla

A total of 52 late-I and 41 post-I neurones were recorded in the VRG. The action potential discharge (spike discharge) of the neurones was temporally correlated to the central respiratory rhythm measured via the crural diaphragm EMG. The late-I cells (Fig. 1A) commenced their firing after the start of inspiration indicated by the diaphragm EMG. In most cases, they commenced firing around mid-inspiration and ceased firing at the end of inspiration. The late-I cells were predominantly circumscribed between 0.1 and 0.6 mm rostral to the obex, 1.5 and 2.5 mm lateral to the midline, and 2.0 and 2.6 mm below the dorsal surface of the medulla oblongata (Fig. 1C). During eupnoea the late-I cells possessed a discharge rate of 10 ± 2 spikes s−1 (computed from n= 26). The late-I neurones exhibited an augmenting discharge pattern, with their peak frequency discharge occurring at the end of inspiration. Their peak frequency ranged from 10 to 100 Hz (Fig. 1D). The peak power density of late-I neurones (Fig. 1E) occurred at the end of inspiration (or just prior to the start of post-inspiration) as measured from the power density spectrum of the crural diaphragm EMG (see Subramanian & Holstege, 2011 for detailed analysis of inspiratory and post-I components of the crural diaphragm).

Figure 1. late-I and post-I neurones in the VRG of the caudal medulla.

Figure 1

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A and B, extracellular recording of late-inspiratory (late-I; A) and post-inspiratory (post-I; B) neurone in the ventral respiratory group (VRG) combined with diaphragm electromyogram (EMG). C, spatial distribution of late-I and post-I neurones in the VRG. D, peak frequency discharge spread of late-I (n= 26) and post-I (n= 25) neurones. E, power density spectrum of the late-I and post-I neurones (computed from n= 10 each type).

The post-I cells (Fig. 1B) showed abrupt onset at the end of inspiration, i.e. time-locked to the offset of the inspiratory component of the crural diaphragm and just prior to the commencement of the post-inspiratory firing of the crural diaphragm. The post-I cells continued to discharge into the stage-1 expiration, ceasing firing just before the start of stage-2 expiratory period (see Richter, 1982 for stage-1 and stage-2 expiratory period definitions). The post-I neurones were circumscribed at 1.0–1.8 mm rostral to the obex, 1.5–2.6 mm lateral to the midline and 2.2–2.8 mm below the dorsal surface of the medulla (Fig. 1C). During eupnoea the post-I cells possessed a discharge rate of 8 ± 2 spikes s−1 (computed from n= 25). The post-I neurones exhibited a decrementing discharge pattern, with their peak frequency discharge occurring at the start of post-I activity of the crural diaphragm. Their peak frequency ranged from 50 to 100 Hz (Fig. 1D). The peak power density of post-I cells (Fig. 1E) occurred at the start of post-inspiration as measured from the power density spectrum of the crural diaphragm EMG.

Effect of EAA stimulation of the PAG on the late-I and post-I neuronal activity

In this study design DLH stimulation was undertaken at least twice into a single location of the PAG, and recorded at least one late-I and one post-I neurone during reproducible PAG-evoked breathing changes.

Dorsal PAG stimulation

Stimulation (n= 20; DLH, 20 nl) of the dorsal PAG (−7.0 mm bregma; Fig. 2C) evoked tachypnoea. The Ti decreased from 0.30 ± 0.05 s to 0.15 ± 0.05 s (P < 0.05), whilst the Te decreased from 0.50 ± 0.15 s to 0.25 ± 0.05 s (P < 0.05), thus evoking an increase in the RF from 75 ± 5 breaths min−1 to 150 ± 10 breaths min−1 (P < 0.05). Dorsal PAG stimulation also resulted in a twofold increase of the diaphragm EMG amplitude. The late-I (Fig. 2A) and the post-I neurones (Fig. 2B) increased their firing rates for 2–3 breaths even prior to the onset of tachypnoea. During tachypnoea, late-I cells firing increased from 10 ± 2 spikes s−1 (control) to 19 ± 8 spikes s−1 (n= 15, P < 0.01), while post-I cells firing increased from 8 ± 2 spikes s−1 (control) to 15 ± 6 spikes s−1 (n= 15, P < 0.01; Fig. 2D). For both the cells the peak discharge frequency was seen during the tachypnoea. The spike discharge average of the late-I was 21 spikes s−1, and for the post-I cell was 15 spikes s−1 following dPAG stimulation (Fig. 2D). Both the late-I and post-I neurones maintained their respective phase relationship with the crural diaphragm discharge during the dorsal PAG-induced tachypnoea.

Figure 2. Dorsal PAG induced tachypnoea and phasic excitation of late-I and post-I neurones.

Figure 2

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A and B, phasic activation of late-inspiratory (late-I; A) and post-inspiratory (post-I; B) neurone following stimulation in the dorsolateral periaqueductal grey (PAG). Dorsal PAG induced tachypnoea with reduction in both Ti and Te, and an increase in the diaphragm amplitude. C, site of PAG stimulation. D, histogram illustrating dose-dependent phasic excitation of late-I (n= 20) and post-I (n= 20) cells. A higher d,l-homocysteic acid (DLH) dosage (40 nl) induced a greater increase in spiking of the late-I and post-I cells. Spike-interval spreads for the late-I (averaged from n= 20) and post-I neurones (averaged from n= 20) were plotted against the peak frequency percentage attained during a bin width of 50 ms. Amb, nucleus ambiguus; AP, area prostrema; aq, aqueduct; dl, dorsolateral; dm, dorsomedial; EMG, electromyogram; IO, inferior olive; lat, lateral; LRt, lateral reticular nucleus; mlf, medial longitudinal fasciculus; NTS, nucleus tractus solitarius; py, pyramidal tract; sp5, spinal trigeminal tract; sp5I, spinal 5 nucleus interpolar; vlat, ventrolateral; VRG, ventral respiratory group; XII, hypoglossal motonuclei.

Dorsal part of the lateral PAG stimulation

Stimulation (n= 20; DLH, 20 nl) of the dorsal part of the lateral PAG (−7.00 mm bregma; Fig. 3C) triggered an increase in the Ti from 0.30 ± 0.05 s to 0.50 ± 0.10 s (P < 0.05), whilst the Te decreased from 0.50 ± 0.15 s to 0.30 ± 0.05 s (P < 0.05). This balanced increase and decrease in the respiratory phases maintained the RF at the control level of 75 ± 5 breaths min−1. The amplitude of the diaphragm EMG increased twofold. The dorsal part of the lateral PAG stimulation evoked an increase in the late-I neurones discharge (Fig. 3A) and advanced their firing onset by 50–100 ms. The late-I cells increased firing rate from 10 ± 2 spikes s−1 (control) to 34 ± 5 spikes s−1 (n= 15, P < 0.01; Fig. 3D). The cells maintained phase-lock with the diaphragm. On the contrary, stimulation of the dorsal part of the lateral PAG inhibited the firing of post-I neurones (Fig. 3B; n= 15, P < 0.001).

Figure 3. Stimulation in the dorsal part of the lateral periaqueductal grey (PAG) produced an increase in Ti and a decrease in Te.

Figure 3

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The frequency of breathing did not alter significantly. The stimulation caused an increase in the late-inspiratory (late-I; A) cell activity but inhibited the post-inspiratory (post-I) cell (B). C, site of PAG stimulation. D, quantitative changes to neuronal spike discharge following central PAG stimulation. For abbreviations, see Fig. 2.

Ventral part of the lateral PAG stimulation

Stimulation (n= 20; DLH, 20 nl) of the ventral part of the lateral PAG (−7.6 mm bregma; Fig. 4B) also induced tachypnoea, with a pronounced increase in RF from 75 ± 5 breaths min−1 to 140 ± 10 breaths min−1 (P < 0.05). The Ti increased from 0.30 ± 0.05 s to 0.40 ± 0.05 s (P < 0.05), while the Te decreased from 0.50 ± 0.15 s to 0.20 ± 0.05 s (P < 0.05). The amplitude of the diaphragm EMG also increased twofold. Lateral PAG induced a transient tonic firing of the late-I cells (Fig. 4A) at the peak of tachypnoea, with the cells firing continuously for the 6–10 breaths. During this effect the late-I cells (n= 15) expressed a discharge of 43 ± 4 spikes s−1 (from 9 ± 3 spikes s−1 control; P < 0.01) with a peak frequency of 100 Hz, representing a 400% increase in their discharge rate (Fig. 4C).

Figure 4. Stimulation in the lateral periaqueductal grey (PAG) induced tachypnoea with an increase in Ti and diaphragm amplitude.

Figure 4

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During this form of tachypnoea, the late-inspiratory (late-I) neurones (A) increased their firing rate and exhibited tonic activity spanning several breaths. B, site of PAG stimulation. C, quantitative changes to neuronal spike discharge following lateral PAG stimulation. For abbreviations, see Fig. 2.

Ventrolateral PAG stimulation

The ventrolateral PAG (−8.0 mm bregma; Fig. 5B) stimulation (n= 13; DLH, 20 nl) triggered Te prolongation. This Te prolongation was characterized by a tonic activation of the crural diaphragm with reduced amplitude in the absence of phasic inspiratory discharge. During this effect the post-I neurones fired continuously (Fig. 5A). The cells increased their firing rate from 8 ± 2 spikes s−1 to 47 ± 5 spikes s−1 (n= 11, P < 0.01), signifying an increase in their discharge rate of over 400% (Fig. 5C).

Figure 5. Stimulation in the ventrolateral periaqueductal grey (PAG) induced short Te prolongation characterized by tonic activation of the crural diaphragm at low amplitude.

Figure 5

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The post-inspiratory (post-I) neurones (A) showed continuous firing during the prolonged expiratory interval. B, site of PAG stimulation. C, quantitative changes to post-I neuronal spike discharge during prolonged Te. For abbreviations, see Fig. 2.

Caudal part of the ventrolateral PAG stimulation

Stimulation (n= 20; DLH, 20 nl) of the caudal part of the ventrolateral PAG (−8.3 mm bregma; Fig. 6C) induced Te prolongation for 8–10 s. During this Te prolongation the late-I neurones were inhibited (Fig. 6A). Prior to inhibition, the late-I cells (n= 16) were tonically activated firing continuously for 2–3 breaths, exhibiting a discharge of 29 ± 3 spikes s−1 (from 10 ± 2 spikes s−1 control; P < 0.01). During this initial tonic activation the late-I neurones increased their firing rate by 200% from control value with a peak frequency of 100 Hz (Fig. 6D). Following the return of normal breathing the late-I neurones did not exhibit any tonic activity, but returned to normal phasic function and pre-stimulation values. The post-I neurones (Fig. 6B) on the contrary fired continuously during this ventrolateral PAG-triggered Te prolongation exhibiting a discharge of 14 ± 3 spikes s−1 (from 8 ± 2 spikes s−1 control; P < 0.01). In addition, post-I neurones (n= 15) were also tonically activated for 1–2 breaths prior to the Te prolongation. During the initial tonic activation, the post-I cells discharge increased from 8 ± 2 spikes s−1 to 19 ± 3 spikes s−1 (P < 0.01), which represented a 250% increase in their firing rate with a peak frequency of 100 Hz (Fig. 6D).

Figure 6. Stimulation in the caudal portion of the ventrolateral periaqueductal grey (PAG) induced Te prolongation.

Figure 6

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During Te prolongation the late-inspiratory (late-I) neurones (A) remained inhibited. Prior to their inhibition the late-I cells were tonically activated for 2–3 breaths. Following the return of eupneic breathing the late-I neurones showed normal phasic activation. The post-inspiratory (post-I) neurones (B) on the contrary showed continuous firing during Te prolongation. Post-I cells were also tonically activated for 1–2 breaths prior to the onset of prolonged Te. Following the return of eupneic breathing, the post-I neurones showed normal phasic activation. C, site of PAG stimulation. D, quantitative changes to neuronal spike discharge during tonic activation prior to prolonged Te. For abbreviations, see Fig. 2.

Discussion

Significant findings

This study reports the changes to the discharge of late-I and post-I neurones located in the VRG following EAA stimulation of various subdivisions of the midbrain PAG. The major conclusions from this study are: (1) the increased excitatory drive to the late-I/post-I neuronal cascades following dorsal PAG stimulation contributes to the development of tachypnoea; (2) during lateral PAG-induced inspiratory prolongation, other neurones in addition to late-I/post-I cells may be recruited to mediate inspiratory-to-expiratory phase transition; (3) ventrolateral PAG-induced breath-hold is characterized by persistent activation of post-I neurones.

Late-I and post-I neurones of the VRG

The discharge pattern of the VRG late-I neurones with spiking onset in mid inspiration and incrementing frequency towards the end of the inspiratory phase is similar to the previous recordings in the cat and the rat (Feldman & Cohen, 1978; Baker & Remmers, 1980; Cohen et al. 1993; Paton, 1996; Pierrefiche et al. 1998; Haji et al. 2002; Mörschel & Dutschmann, 2009). The activity pattern of post-I cells that shows abrupt and maximal discharge in coincidence with termination of inspiration and ceased their activity during mid expiration is also well reported (Richter et al. 1987; Zheng et al. 1991a,b; Ezure, 1990; Paton, 1996; Dutschmann & Paton, 2002; Subramanian & Holstege, 2011). Cross-correlational evidence for these two cells to be involved in inspiratory to expiratory conversion was also provided by Shannon et al. (2000), who showed that increased post-I neurone activity correlated with inspiratory phase termination together with inhibition of other inspiratory neurones. In line with previous studies (Zheng et al. 1991a,b), I found a greater proportion of post-I cells at the rostral parts of the VRG.

The PAG-evoked modulation of the late-I and post-I neuronal activity

The PAG neurones mediating respiratory responses are topographically organized. Typically the PAG stimulations produced three types of respiratory patterns: (1) tachypnoea; (2) lengthening of the Ti; and (3) prolongation of the Te. The discharge pattern of the late-I and post-I neurones following PAG-evoked breathing modulation is in agreement with their proposed role in inspiratory-to-expiratory phase transition. Synaptic modelling of eupneic respiratory rhythm proposes that late-I neurones initiate the termination of inspiration. The subsequent activation of the post-I neurones inhibits late-I neurones and all other neurones of the medullary respiratory Central Pattern Generator (rCPG), and thus completes the inspiratory-to-expiratory phase transition (see Richter, 1982; von Euler, 1983; Bianchi et al. 1995; Rybak et al. 1997, 2004; Shannon et al. 2000; Smith et al. 2007; Mörschel & Dutschmann, 2009). During eupnoea the inspiratory-to-expiratory phase transition is under the control of two convergent mechanisms: (1) the sensory afferent feedback (Hering–Breuer reflex) of PSRs during lung inflation (Hayashi et al. 1996; see Kubin et al. 2006); and (2) the intrinsic rCPG mechanism governed by the parabrachial and Kölliker–Fuse nuclei of the dorsolateral pons (Dutschmann & Herbert, 2006). However, under eupnoea it has been shown that the PSR feedback habituates (Siniaia et al. 2000), suggesting that normal tidal-volume breathing is controlled by the rCPG intrinsic mechanisms (Dutschmann et al. 2009a).

Following PAG stimulation, the late-I and post-I neurones are modulated even prior to any acute change in the breathing pattern. This suggests that descending inputs target intrinsic inspiratory-to-expiratory phase transition mechanism of the rCPG involving ponto-medullary synaptic interactions. This suggestion is supported by a previous finding that PAG-induced respiratory responses are mediated via the pontine parabrachial areas (Hayward et al. 2004). Nevertheless, the role of neurones located in the dorsolateral pons, which are critical for inspiratory-to-expiratory phase transition and potentially also important for PAG-evoked breathing modulation, require future investigation.

Inspiratory-to-expiratory phase transition during dorsal PAG-evoked tachypnoea

The dorsal PAG-induced tachypnoea produces a balanced increase in the discharge of both the late-I and post-I neurones. During this tachypnoea both Ti and Te were shortened. The shortening of Ti is a clear indication that increased excitatory drive to the phase transition sequence is reflected in the respiratory motor pattern. The parallel decrease in Te and thus the overall increase in the RF suggest that descending inputs from the PAG also affect the inspiratory rhythmogenic neurones within the rCPG as evidenced by pre-inspiratory neurone excitation (Subramanian & Holstege, under review). In summary, the dorsal PAG apparently can provide general tonic excitatory drive for a variety of respiratory neurones.

Inspiratory-to-expiratory phase transition during the dorsal part of the lateral PAG-evoked inspiratory prolongation

Stimulation of the dorsal part of the lateral PAG caused inspiratory prolongation. This inspiratory prolongation was accompanied by strong phasic excitation of late-I cells while activity of post-I neurones was supressed. The observed cellular activities correlate with the general model of inspiratory-to-expiratory phase transition mechanisms. The supressed discharge of post-I neurones causes a delay in the inspiratory-to-expiratory phase transition and thus inspiratory prolongation. Nevertheless, during inspiratory prolongation the phase transition has to be mediated via the activation of compensatory mechanisms. A prime candidate for compensation of suppressed post-I activity could be a two-phase rCPG oscillation between inspiratory and stage-2 expiratory (abdominal expiratory activity) neurones, as suggested in a dual oscillator hypothesis for inspiration and abdominal expiration (see Duffin, 2004; Feldman & Del Negro, 2006).

A possible explanation for the selective suppression of post-I neuronal activity following stimulation of the dorsal part of the lateral PAG derives from its anatomical connectivity. The descending projections of the dorsal part of the lateral PAG target cell populations medial to the primary rCPG including the medullary raphe nuclei (Holstege, 1991). The release of serotonin from medullary raphé neurones was recently shown to have a strong inhibitory effect on the post-I neurones via the activation of serotonin 1A receptors (Manzke et al. 2009, 2010; see modelling by Shevtsova et al. 2011). This is further substantiated by the dense expression of serotonin 1A receptors in the brain structures governing the inspiratory-to-expiratory phase transition, such as the Kölliker–Fuse nuclei and rostral VRG (Dutschmann et al. 2009b), and the observation that medullary raphé nuclei specifically modulate ponto-medullary respiratory synaptic interactions (Nuding et al. 2009; Morris et al. 2010).

Inspiratory-to-expiratory phase transition during lateral PAG-evoked tachypnoea

The respiratory effect following lateral PAG stimulation was a combination of tachypnoea and inspiratory prolongation while the cellular response followed the pattern observed after dorsal part of the lateral PAG stimulation (e.g. late-I excitation, post-I inhibition). A specific interpretation of this effect is difficult, but it is not unlikely that DLH injection may have activated overlapping circuits of the PAG.

Inspiratory-to-expiratory phase transition during ventrolateral and caudal-ventrolateral PAG-evoked expiratory prolongation

Chemical stimulation of the ventrolateral and caudal-ventrolateral PAG consistently triggered expiratory prolongation. The expiratory prolongation was always associated with a tonic post-I neuronal discharge and, in the case of ventrolateral PAG stimulation, also with a low amplitude post-I activity of the diaphragm. Due to the persistent activation of post-I neurones, which are known to drive the laryngeal constrictor muscles (Dutschmann & Herbert, 2006), I classify the ventrolateral PAG-evoked expiratory prolongation as a ‘breath-hold’.

The rCPG oscillation is clamped in the post-I phase during the breath-hold. Hence, all other neurones of the CPG are inhibited (see Richter, 1982). This was reflected in the absence of late-I neuronal activity in this study. However, late-I neurones were excited after PAG stimulation just prior to the breath-hold and may still initiate the inspiratory-to-expiratory phase transition. The pronounced effect on the post-I neurones implies that the ventrolateral PAG targets the pontine Kölliker–Fuse nucleus, which is known to trigger similar breath-holds following its excitation. (Dutschmann & Herbert, 2006).

Considerations

I did not investigate the neurochemical profile of the late-I and post-I cells. It is known that late-I neurones are predominantly excitatory, while post-I cells are both excitatory and inhibitory (Ezure, 1990). Nevertheless, my recordings validate their role as the principal cascades of the inspiratory-to-expiratory phase transition mechanism because modulation of their discharge pattern following PAG stimulation is reflected in the respiratory motor output and therefore complies with both their inhibitory or excitatory roles. The anatomical connectivity of the PAG with limbic forebrain structures and sensory relays (Holstege, 1991) and its critical role in emotional behaviours such as vocalization (Zhang et al. 1994; Subramanian et al. 2008) clearly indicate that the PAG provides a higher command to the neurones of the rCPG to convert eupnoea into a behavioural breathing pattern (Subramanian et al. 2008; Subramanian & Holstege, 2009, 2010, 2011). The PAG-evoked responses such as tachypnoea could be very well linked to exercise, flight or fight response or panic attacks (Bandler & Carrive, 1988; Zhang et al. 1990; Basnayake et al. 2011). The breath-hold responses elicited from the ventrolateral PAG could be associated with a freezing response (it takes your breath away!). Nevertheless, an extended breath-hold spanning over 10–15 breathing cycles (from caudal ventrolateral PAG) can trigger hypoxia, while an extended inspiratory prolongation can cause hypercapnia because CO2 is not sufficiently expired due to extremely short expiratory intervals. What behaviour corresponds to such atypical breathing patterns is not clear, but could also be linked to activation of overlapping but functionally different PAG circuitry or by depolarizing block and disturbances in the ionic composition of the extracellular space following microinjections of EAAs (Lipski et al. 1988).

Acknowledgments

The support of Dr. Ron Balnave (Discipline of Biomedical Science, The University of Sydney, Australia) and Dr. Gert Holstege (University Medical Center, Groningen, The Netherlands) is gratefully acknowledged. I am grateful to Dr. Mathias Dutschmann (The Florey Institute, Melbourne, Australia) for comments on a draft of the manuscript and suggestions about data interpretation.

Glossary

BP

blood pressure

DLH

d,l-homocysteic acid

EAA

excitatory amino acid

EMG

electromyogram

FFT

Fast Fourier Transform

late-I

late-inspiratory

NA

nucleus ambiguous

PAG

periaqueductal grey

post-I

post-inspiratory

PSR

pulmonary stretch receptor

rCPG

respiratory Central Pattern Generator

RF

respiratory frequency

Te

expiratory duration

Ti

inspiratory duration

VRG

ventral respiratory group

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