AJP - Regu Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 288: R262-R269, 2005. First published September 2, 2004; doi:10.1152/ajpregu.00328.2004
0363-6119/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/1/R262    most recent
00328.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boscan, P.
Right arrow Articles by Paton, J. F. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boscan, P.
Right arrow Articles by Paton, J. F. R.

NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Excitatory convergence of periaqueductal gray and somatic afferents in the solitary tract nucleus: role for neurokinin 1 receptors

Pedro Boscan and Julian F. R. Paton

Department of Physiology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom

Submitted 18 May 2004 ; accepted in final form 28 August 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our previous studies (Boscan P, Kasparov S, and Paton JF. Eur J Neurosci 16: 907–920, 2002) showed that activation of somatic afferents attenuated the baroreceptor reflex via neurokinin type 1 (NK1) and GABAA receptors within the nucleus of the solitary tract (NTS). The periaqueductal gray matter (PAG) can also depress baroreceptor reflex function and project to the NTS. In the present study, we have tested the possibility that the dorsolateral (dl)-PAG projects to the NTS neurons that also respond to somatic afferent input. In an in situ, arterially perfused, unanesthetized decerebrate rat preparation, somatic afferents (brachial plexus), cervical spinal cord, and dl-PAG were stimulated electrically, whereas NTS neurons were recorded extracellularly. From 45 NTS neurons excited by either brachial plexus or dl-PAG stimulation, 41 received convergence excitatory inputs from both afferents. Onset latency and evoked peak discharge frequency from brachial plexus afferents were 39.4 ± 4.7 ms and 10.7 ± 1.1 Hz, whereas this was 43.9 ± 6.4 ms and 7.9 ± 1 Hz, respectively, following dl-PAG stimulation. As revealed by using a paired pulse stimulation protocol, monosynaptic connections were found in 9 of 36 neurons tested from both spinal cord and dl-PAG. We tested NK1-receptor sensitivity in 38 neurons that received convergent inputs from brachial plexus/PAG. Fifteen neurons were sensitive to selective antagonism of NK1 receptors. CP-99994, the NK1 antagonist, failed to alter ongoing firing activity but reduced the evoked peak discharge frequency following stimulation of both brachial plexus (from 12.3 ± 1.8 to 7.2 ± 1.3 Hz; P < 0.01) and PAG (from 7.8 ± 1.5 to 4.5 ± 1 Hz; P < 0.01). We conclude that 1) somatic brachial and PAG afferents can converge onto single NTS neurons; 2) this convergence occurs via either direct or indirect pathways; and 3) NK1 receptors are activated by some of these inputs.

nociception; defense response; substance P; baroreflex

ALTERATIONS IN THE BEHAVIOR of a mammal in response to somatic afferent activation are associated with the appropriate and concomitant cardiovascular responses. However, the central nervous mechanisms that underlie the coordination of somatic afferent inputs with the adjustment of cardiovascular autonomic motor outflows remain poorly defined.

Within the brain, two sites known to integrate somatic information and mediate powerful cardiovascular responses are the periaqueductal gray matter (PAG) and nucleus of the solitary tract (NTS). The PAG, especially its dorsolateral (dl) region, receives abundant somatic afferent information from the dorsal horn and also mediates profound cardiorespiratory changes comprising tachycardia and hypertension (3, 4, 13, 18, 20, 28, 53). Turning to the NTS, although it is a major site for integrating multiple visceral afferent inputs and mediating their reflex responses (e.g., Refs. 7, 39), it also processes somatic afferent information (9, 41, 49–51) and receives direct and indirect projections from both the dorsal horn (13, 14, 26, 32) and dl-PAG (5, 15). Interestingly, there are regions of the NTS that can evoke tachycardia and a pressor response, a pattern mimicking that produced both from the dl-PAG and following somatic afferent stimulation, such as that observed during exercise or nociception (30). However, it remains unknown whether somatic afferent-evoked tachycardia-pressor responses depend on either dl-PAG and/or NTS or the pathways that exist between these structures.

With the similarity in cardiovascular responses that can be evoked from the dl-PAG, NTS, and somatic afferents, and their intimate connectivity, the present study was designed to examine whether single NTS neurons that receive somatic afferent inputs from the brachial plexus are also excited following stimulation of dl-PAG. Because our previous data support a role for neurokinin type 1 (NK1) receptors in transmitting somatic afferent input into the NTS (8, 42), we also tested the hypothesis that any convergent inputs from the dl-PAG and somatic afferents to NTS neurons may involve activation of NK1 receptors.

A preliminary report of this work was communicated previously (10).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A working heart-brain stem preparation (WHBP; see Ref. 38) of Sprague-Dawley rats (75–150 g) of either sex was employed with home office animal care and use approval. This preparation was chosen because 1) much of the background work that led to this project was performed by using this preparation; 2) the pattern of cardiorespiratory reflex response during somatic afferent stimulation is consistent and comparable to that reported in lightly anesthetized and conscious animals (2, 6, 9, 35); 3) stable cellular recordings can be maintained because there is minimal arterial pulse pressure and no respiratory related movements; and 4) there is good visual and pharmacological access to both the brain stem and midbrain for simultaneous single-unit recording and stimulation.

Preparation of the WHBP

Animals were anesthetized deeply with halothane until there was no sign of withdrawal reflexes following a pinch to a paw or the tail. Rats were decerebrated precollicularly, via a partial parietal bone craniotomy (so sparing part of the bregma suture line), bisected subdiaphragmatically, and exsanguinated. The halothane anesthesia was withdrawn at this time, and the head and thorax were immersed in ice-cold Ringer. The descending aorta and one phrenic nerve were surgically isolated. The brain stem and midbrain were exposed following a cerebellectomy. The preparation was transferred to a recording chamber where the head was fixed with the use of ear bars and a nasal clamp. The angle of the head was preset to a precise level in every experiment to allow for consistent positioning of the stimulating electrode (see below). The descending aorta was cannulated with a double-lumen catheter to perfuse the upper body with a Ringer solution that was prewarmed to 31°C, contained 1.25% ficoll, and was continually gassed with carbogen (i.e., 95% oxygen and 5% carbon dioxide). With the use of a roller pump, perfusion was maintained at a constant flow (range 28–32 ml/min; Watson Marlow 505S). The second lumen of the catheter was used to monitor aortic perfusion pressure via a transducer. The previously isolated phrenic nerve was drawn up into a suction electrode, and its activity was recorded as an index of central inspiratory activity. This returned 5–10 min after the onset of perfusion, and, once present, the preparation was paralyzed by the addition of vecuronium bromide (Norcuron; 1.5 µg/ml) to the perfusion solution. After adjustment of perfusion flow rate, the respiratory motor pattern consisted of an incrementing discharge indicative of adequate oxygenation of the brain stem and hence preparation viability. ECG was recorded simultaneously with the activity of the phrenic nerve as the suction electrode was within close vicinity of the left ventricle. The R wave of the ECG was discriminated by using a window discriminator to triggered transistor-transistor logic pulses used to determine heart rate (HR). Phrenic nerve activity and ECG signals were amplified and filtered (8 Hz to 3 kHz; Neurolog modules 104 and 125; Digitimer) and displayed on an oscilloscope.

Stimulation Methods

dl-PAG. A bipolar concentric stimulating electrode (Clarke; diameter 0.12 mm) was aimed toward the dl area of the left PAG. The coordinates used were –8.2 to –8.6 mm caudal to bregma, 0.5–1.0 mm lateral to the midline, and 2.3–2.9 mm down from the dorsal surface of the intercolliculi junction (see Fig. 1). The bipolar concentric electrode was connected to an isolated stimulator (Digitimer DS2A) controlled by a pulse generator (Digitimer 4030). All sites in the dl-PAG evoked an increase in respiratory drive, HR, and perfusion pressure during high-frequency stimulation (1–15 V, 0.1–0.2 ms, 50 Hz). At the end of each experiment, constant current was passed to mark stimulation sites (50 µA; 30 s). Brains were fixed (4% paraformaldehyde) and cryoprotected (20% sucrose), and histological sections (50 µm thick) were cut of the midbrain to anatomically identify electrode placement. During extracellular recording in the NTS, the dl-PAG was stimulated with the following parameters: 1–15 V, 0.1 ms, and 0.2–1 Hz.



View larger version (83K):
[in this window]
[in a new window]
  		Fig. 1. A: photomicrograph of a coronal section through the periaqueductal gray matter (PAG). The photomicrograph shows the stimulation site in the dorsolateral (dl)-PAG (see asterisk). Aq, aqueduct sylvius; DR, dorsal raphe nucleus; DRVL, dorsal raphe nucleus ventrolateral part; IC, inferior colliculi. B: to determine the right placement of the stimulating electrode for each experiment, the dl-PAG was stimulated at high frequency to evoke a cardiorespiratory response. The electrical stimulation evoked an increase in respiratory drive, heart rate (HR), and perfusion pressure (PP) and was similar in pattern to that evoked from the brachial plexus. Brachial plexus stimulation was 20 V, 0.2 ms, and 50 Hz; PAG stimulation was 10 V, 0.2 ms, and 50 Hz. {delta}PNA, integrated phrenic nerve activity; bpm, beats per minute.

 
Brachial plexus. The left brachial plexus was isolated close to the spine, and two silver wires (100 µm in diameter) were wrapped around the entire nerve bundle. Each wire was insulated except for their ends that were bared. Both the exposed silver wires and surrounding plexus were embedded in low-melting-point paraffin wax for electrical insulation. With the use of a second isolated stimulator (Digitimer DSA) driven by the same pulse generator, two types of stimuli were delivered. To evoke a systemic cardiorespiratory reflex response, 10–30 V, 0.2 ms, and 50 Hz were delivered for 3–6 s. In contrast, during extracellular recording, a stimulus consisting of 10–30 V, 0.5 ms, and 0.2–1 Hz was used.

Cervical spinal cord. To bypass any neuronal processing within the spinal cord and to assess monosynaptic connections from spinal afferents to the NTS, the cervical spinal cord was stimulated electrically. A bipolar electrode made from silver wire with tip diameters of 100 µm was positioned at the level of C3 (~5 mm caudal to calamus scriptorius). In neurons that did not receive monosynaptic spinal afferents from the C3 level but were activated, the latency of responses allowed some assessment of the degree of synaptic processing within the upper cord/NTS. Each pole of this electrode was in direct contact with the lateral edge of the spinal cord and connected to a third isolated electrical stimulator that was also driven by the pulse generator. The stimulation parameters were 3–10 V, 0.1 ms, and 0.2–1 Hz.

Orthodromic and Antidromic Stimulation Protocols

To identify direct monosynaptic connections between either the spinal cord and NTS neurons or the PAG and NTS neurons, a paired pulse protocol, in which pulses were separated by 10 ms, was adopted as described previously (33, 47, 54). Neurons responding to each stimulus of this paired pulse were considered to receive monosynaptic inputs.

The antidromic test consisted of observing a constant latency-evoked action potential followed by a collision cancellation test of the antidromic spike with a spontaneous spike, as described before (e.g., Refs. 27, 37). Successful collision was used to distinguish between antidromic vs. short-latency orthodromic-evoked spikes.

Extracellular Recording from the NTS

Neurons were recorded using multibarreled glass microelectrodes, with an external tip diameter of 2–4 µm and resistance of 15–30 M{Omega}. Microelectrodes were driven in 3-µm steps by using a pizoelectric stepper motor (Burleigh inchworm), which provided z-axis positional feedback. NTS neurons were recorded bilaterally between 300 and 700 µm below the dorsal medullary surface. Under visual guidance of a binocular microscope, microelectrodes were aimed toward the NTS by placement onto the caudal region of the dorsal medulla relative to calamus scriptorius, a structure that is easily seen by the absence of the cerebellum. The rostrocaudal recording zone was between –13.7 and –14.4 mm relative to bregma. The microelectrodes were positioned between 0.4 and 1.5 mm lateral to the midline for more rostral sites and from midline to 1 mm lateral to midline more caudally. The cerebellectomy enhanced consistency of placement of recording microelectrodes within and between preparations. The three barrels of the microelectrodes were filled with 1) a mixture of 1.5 M NaCl and 100 mM glutamate (recording electrode); 2) a NK1 receptor antagonist, CP-99994 (10 mM; applied for 30–300 s); and 3) saline as control. The recording electrode signal was amplified and filtered (8 Hz to 3 kHz; Neurolog modules 104 and 125). The barrels were connected to a positive-pressure ejection system fitted with solenoids and valves that permitted fine control of applied pressure. The NaCl/glutamate recording solution was pressure ejected intermittently during cell searching to identify neurons that were either silent or had low-firing frequencies. This also maintained the patency of the microelectrode tip during cell searching. When a neuron was found, the positive pressure was nulled (or made slightly negative) to prevent NaCl/glutamate leakage. This allowed cells with ongoing firing to go back to baseline rates within seconds. The CP-99994 was pressure ejected onto neurons that responded to brachial plexus and/or PAG stimulation. As a pressure control, we applied equal pressure to the saline-filled barrel and assessed evoked activity responses. In all cases, saline affected neither ongoing firing nor the evoked firing responses.

Data Analysis

All data were relayed via a 1401 plus CED interface to a computer running Spike 2 software (Cambridge Electronic Design) with custom-written scripts for data acquisition and on- and off-line analysis. The ongoing activity of single units and the electrically evoked firing response frequency were both measured. The criteria used to consider an increase in firing frequency during stimulation was when neuronal activity increased by at least 25% relative to ongoing activity. Peristimulus time histograms (10-ms bin width) were plotted that showed the electrically evoked activity from single NTS neurons and allowed a measure of the onset latency. Changes in firing frequency and onset latencies after application of CP-99994 were compared by using a matched-paired nonparametric Wilcoxon signed-rank test. All values quoted are means ± SE, and n is the number of cells recorded. Differences were taken as significant at the 95% confidence limit.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cardiovascular and Respiratory Data

The data were obtained from a total of 17 preparations. The mean phrenic nerve activity cycle length, HR, and perfusion pressure were 3.7 ± 0.4 s, 268 ± 8 beats/min, and 92.3 ± 3.3 mmHg, respectively. High-frequency electrical stimulation (trains) applied to either the brachial plexus or the dl-PAG exhibited a similar pattern of response, including tachypnea, tachycardia, and a pressor effect (Fig. 1). Brachial plexus and dl-PAG stimulation decreased phrenic nerve activity cycle length to 0.7 ± 0.2 and 0.9 ± 0.2 s, increased HR to 313 ± 18 and 295 ± 16 beats/min, and increased perfusion pressure to 99 ± 5.4 and 94 ± 4.4 mmHg, respectively (all P < 0.01 except for dl-PAG stimulation changes in perfusion pressure; P = 0.1).

Patterns of NTS Neuronal Responses

This study is based on recordings from 205 NTS neurons. Of these, ~20% responded to stimulation of the brachial plexus and/or PAG. Thus single-pulse stimulation of the brachial plexus and the dl-PAG resulted in 41 NTS neurons being excited by both brachial plexus and dl-PAG (Fig. 2). In addition to these 41 neurons exhibiting convergent inputs, we found an additional four neurons that received only one of these inputs: three neurons were excited by the brachial plexus, and one by dl-PAG only. In a further five cells with ongoing activity and excited by brachial plexus stimulation, dl-PAG activation showed a trend of reducing the spontaneous firing (ongoing, 1.04 ± 0.4 Hz; brachial, 5.4 ± 1.7 Hz; PAG, 0.2 ± 0.2 Hz). However, these data were not significantly different (P = 0.1 for ongoing activity vs. activity during dl-PAG stimulation).



View larger version (46K):
[in this window]
[in a new window]
  		Fig. 2. Nucleus of the solitary tract (NTS) neuron excited by both brachial plexus and dl-PAG stimulation. The convergence property is shown as raw data traces with 2 different time scales (top and middle) and depicted in a peristimulus time histogram (bottom).

 
The 41 neurons with excitatory convergent inputs (brachial plexus and dl-PAG) were considered further. All 41 neurons displayed ongoing activity with a mean of 2.6 ± 0.4 Hz. The onset latency evoked from the brachial plexus and dl-PAG was 39.4 ± 4.7 and 43.9 ± 6.4 ms (ranging from 9–100 and 4–120 ms), respectively. The firing response evoked from stimulation of either the brachial plexus or dl-PAG was a burst of spikes lasting up to 2 s in some neurons. The evoked firing frequency following stimulation of the brachial plexus and dl-PAG was 10.7 ± 1.1 and 7.9 ± 1 Hz, respectively, which was significantly higher than the ongoing activity during control (P < 0.01 in both cases).

Role of NK1 Receptors

Of the 41 neurons receiving convergent excitatory inputs from brachial plexus and dl-PAG, 38 were tested with the NK1-receptor antagonist CP-99994. In all 38 neurons, there was no change in ongoing activity, suggesting an absence of endogenous substance P release or NK1-receptor activity. Of these 38 neurons, 15 exhibited a significant reduction in the firing response evoked from both brachial plexus and dl-PAG when CP-99994 was administered; in all cases, the effect was reversible (Fig. 3 and Table 1). Thus the evoked discharge frequency was attenuated from 12.3 ± 1.8 to 7.2 ± 1.3 Hz (P < 0.01) during brachial plexus stimulation and from 7.8 ± 1.5 to 4.5 ± 1 Hz (P < 0.01) during dl-PAG stimulation. There was a reversible trend for an increased onset latency in the presence of CP-99994 in these 15 neurons (brachial plexus: 46.2 ± 9.7 ms for control vs. 97.2 ± 37.7 ms during CP-99994, P = 0.011; dl-PAG: 73.5 ± 28.7 ms for control vs. 121.8 ± 43.1 ms during CP-99994, P = 0.001). Following a 3- to 10-min washout period, the effect of CP-99994 was reversed in the 15 neurons: the evoked response for brachial plexus stimulation was 11.6 ± 2.3 Hz and 66.7 ± 12.9 ms, and for dl-PAG stimulation was 7.2 ± 1.4 Hz and 87.5 ± 33.6 ms. These values in washout were not different to control.



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 3. Convergent excitatory inputs from brachial plexus and dl-PAG to some NTS neurons is mediated by neurokinin type 1 (NK1) receptors. Application of the selective NK1-receptor antagonist CP-99994 reduced the firing frequency response evoked following stimulation of both brachial plexus and dl-PAG. This effect was reversible.

 

View this table:
[in this window]
[in a new window]
  		Table 1. NTS neuron activity during NK1-receptor blockade

 
To control for nonspecific effects of CP-99994, such as sodium channel inactivation (12), we coapplied glutamate (100 mM) from the adjoining barrel before and during CP-99994 application to 8 of the 15 neurons inhibited with CP-99994. The NK1 antagonist did not change the glutamate-evoked firing frequency response (control: 19.8 ± 2.9 vs. 18.6 ± 2.6 Hz; P = 0.7). Additionally, this antagonist did not alter the shape of the action potentials recorded in all neurons tested.

Paired Pulse Stimulation and a Role for NK1 Receptors

A paired pulse protocol (10-ms interpulse interval) from both the spinal cord and dl-PAG was tested in 36 of the 41 neurons exhibiting convergent inputs following stimulation of the brachial plexus and dl-PAG. It was noteworthy that spinal cord stimulation evoked a firing response pattern and peak frequency reminiscent of that evoked from the brachial plexus. This consisted of a peak firing frequency of 7.9 ± 1 Hz with an onset latency of 34 ± 6.8 ms (range 5–160 ms). Of the 36 neurons tested, 9 were capable of following the paired pulse stimuli such that firing responses were faithfully evoked without failures from both spinal cord and dl-PAG (Fig. 4). Thus convergence from spinal cord and dl-PAG in these NTS neurons was monosynaptic. In addition, five neurons were orthodromically activated only from the spinal cord and three only from dl-PAG. In all cells that followed the paired pulse protocol (17 neurons in total), the evoked response was sensitive to NK1-receptor antagonist in six neurons (3 monosynpatic from both spinal cord and dl-PAG, 2 monosynaptic only from spinal cord, and 1 monosynaptic only from dl-PAG). The latter suggests a role for NK1 receptors in mediating 33% of the direct afferent inputs from the spinal cord and/or the dl-PAG.



View larger version (29K):
[in this window]
[in a new window]
  		Fig. 4. Some NTS neurons received direct monosynaptic inputs from both spinal cord and dl-PAG stimulation as determined by using a paired pulse stimulation protocol. Top: the peristimulus time histograms show that this NTS neuron was excited by both brachial plexus and dl-PAG stimulation. Bottom: the same neuron following the pair pulse (10-ms) protocol from both the spinal cord and dl-PAG.

 
As a result, six neurons with monosynaptic inputs represent 40% of the total neurons sensitive to the NK1 antagonist. The remaining 60% (9 of 15 neurons) received polysynaptic inputs.

NTS Neuronal Antidromic Responses from the dl-PAG and Spinal Cord

None of the 36 NTS neurons orthodromically excited from either the spinal cord or dl-PAG could be antidromically activated from the spinal cord. In contrast, 3 of these 36 NTS neurons were activated antidromically from dl-PAG (Fig. 5). An additional group of three NTS neurons orthodromically activated from spinal cord but not from dl-PAG were activated antidromically from dl-PAG.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5. Antidromic response from NTS neuron during dl-PAG stimulation. Top: antidromic test with 4 superimposed traces following the criteria of short onset latency (1–3 ms), consistent onset latency, and only 1 action potential per stimuli (see evoked action potential with solid arrow). Bottom: confirmation of the antidromic property with a collision test. Four superimposed traces show that, if the stimulus is given immediately after a spontaneous action potential (see dashed arrow), there was no antidromically evoked action potential. The solid arrow in the bottom panel indicates the expected timing for the antidromic spike.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
To our knowledge, this is the first study showing convergence of somatic and dl-PAG afferents to single NTS neurons. The majority of the NTS neurons that were excited by brachial nerve stimulation were also excited by dl-PAG stimulation (i.e., 41 of 45 neurons). Thus a NTS neuron that responds to brachial nerve stimulation has a 91% chance of responding to dl-PAG. NK1 receptors appear to mediate ~39% of the somatic and dl-PAG afferent inputs to NTS neurons. All neurons receiving brachial nerve inputs were also activated by ascending spinal afferents, with some of these being mediated by a monosynaptic pathway.

Somatic Afferent Inputs to NTS

Previous studies have shown that stimulation of somatic nerves that include nociceptive fibers excite NTS neurons (11, 49, 50). Both the present study and a previous publication from our laboratory (8) indicate that the brachial plexus-evoked NTS responses occur over a large range of onset latencies in a subpopulation of NTS neurons. These studies support the presence of both direct and polysynaptic pathways. One possibility is that brachial plexus inputs arrive via direct spinal projections from the dorsal horn. This is supported by both anatomic and electrophysiological evidence showing direct projections of dorsal horn neurons to caudal regions of the NTS that overlap with our recording sites (see Refs. 13, 14, 17, 24, 26, 32, 45). While we show monosynaptic spinal inputs to NTS neurons also activated from the brachial plexus, this does not necessarily imply that these direct spinal inputs mediated the inputs evoked from the brachial plexus. Indeed, indirect pathway(s) from somatic afferents to NTS-mediating nociceptive information cannot be ruled out and may include spinal, medullary, as well as midbrain relays. Certainly the latter would explain the longer latency-evoked responses from the brachial plexus and account for the failure of many spinal-evoked responses to follow the paired-pulse stimulation protocol.

There was no difference in the overall mean latencies between inputs from the brachial plexus and the dl-PAG to the NTS. However, with the finding that 51% of NTS neurons received longer latency inputs from the brachial plexus compared with dl-PAG, the possibility that this midbrain region acts as a relay for somatic afferents exists. This is supported by the following previously published evidence: first, the PAG receives dense projections from the spinal cord (4, 13, 20); second, the PAG projects directly to the NTS (5, 15); third, NTS neurons were shown to respond to stimulation of the dl-PAG (19); and, fourth, activation of either somatic nociceptive afferents or skeletal muscle contraction-sensitive afferents increases c-Fos expression in the lateral region of the PAG (21–23, 25).

NTS Projections to dl-PAG and Spinal Cord

The finding of only six neurons antidromically activated from the dl-PAG suggests that this is a minority target for NTS neurons recorded in the present study. Furthermore, no NTS neurons could be antidromically activated from the spinal cord. One potential pitfall in the present study regarding the antidromic activation is that the stimulus artifact may have camouflaged antidromic spikes of short latency. Thus the data may underrepresent the number of NTS neurons projecting to dl-PAG and/or spinal cord. Nevertheless, the data as presented suggest that the majority of NTS neurons excited from both the brachial plexus and dl-PAG may be either intrinsic interneurons or neurons with projections to brain sites that do not include the dl-PAG and spinal cord.

New Role for Substance P in the NTS

Our data support a role for substance P in mediating the firing responses of NTS neurons responding to either brachial plexus or dl-PAG inputs. In this study, we showed that a percentage of the neurons excited by somatic brachial afferents and dl-PAG were sensitive to NK1-receptor blockade. Axon terminals from dorsal horn into NTS have been shown to contain substance P-like immunoreactivity (16). Moreover, activation of skeletal muscle receptors from the hindlimb releases substance P in the NTS (44). We are unaware of studies that have specifically examined whether a direct substance P-containing pathway from the dl-PAG to NTS exists. The paired pulse stimulation protocol with CP-99994 administration is suggestive that a subset of directly projecting fibers from dl-PAG could contain substance P. Clearly, this needs to be substantiated, as electrical stimulation as used here does not ensure against activation of fibers of passage that originate outside of dl-PAG. Indeed, we acknowledge this as a caveat of the present study. Alternatively, activation of the dl-PAG releases substance P in the NTS via indirect projections.

Roles for SP in the NTS remain controversial (see Ref. 46 for a review). Substance P has produced variable and inconsistent responses in respiration, HR, and arterial blood pressure. Substance P is reported to be released in the NTS during baroreceptor activation (34, 43). Some authors have indicated that substance P in NTS potentiates the vascular response of the baroreceptor reflex (48). In contrast, other data indicate that the peptide attenuates carotid sinus nerve-evoked responses (40) and mediates somatic nociception-induced inhibition of the cardiac baroreceptor reflex via an NK1-GABAA receptor mechanism in the NTS (8, 42).

Why Does the dl-PAG Input to the NTS?

The answer to the question posed is that, despite being well established as a major visceral sensory nucleus within the brain, the NTS also integrates somatic afferent activity. Our previous data have shown that 50% of the tachycardia evoked by somatic noxious stimulation in the rat are mediated by the NTS (9). Moreover, noxious stimulation attenuated the cardiac baroreceptor reflex that was dependent on the integrity of GABAAergic synapses in the NTS (8). Thus the NTS plays a major role as an interface for somatic afferent-evoked modulation of autonomic motor activity as well as homeostatic reflexes. This is the first time that we show, in the WHBP of rat, activation of the dl-PAG region. The cardiorespiratory response from dl-PAG is tachypnea, tachycardia, and a pressor response, similar to that shown in anesthetized or decerebrated animals (1, 18, 28, 53). Thus the response evoked from dl-PAG is similar to those following somatic afferent stimulation (2, 6, 9, 31, 36). One possibility is that the NTS may mediate a component(s) of this common pattern of cardiovascular response. Additionally, the pathways from the dl-PAG to the rostral ventrolateral medulla, which are known to increase bulbospinal rostral ventrolateral medulla presympathetic motoneuron activity (29, 52), may also play a role. A caveat with the present study is that the NTS neurons studied were not identified as "cardiovascular," and this is a limitation, with any speculation concerning the functional significance for the dl-PAG-NTS pathway identified here. Nevertheless, not all NTS neurons affecting central cardiovascular (or respiratory) function need to be activated by cardiovascular afferents. Whether the neurons recorded in this study fall within this subgroup remains an open question.

Conclusion

Somatic afferents from the brachial plexus and inputs from the dl-PAG can show excitatory convergence onto single NTS neurons. Furthermore, our data suggest that, if a NTS neuron were excited by dl-PAG stimulation, it is likely that it would also be excited by brachial nerve stimulation. This convergence appears to be mediated by both monosynaptic and indirect pathways and stimulates, in part, NK1 receptors in NTS. Our data support a role for the NTS as a major interface for somatic afferent integration with descending inputs from the midbrain. The possibility remains that these connections form the substrate for modulation of visceral function, whether it be cardiovascular, respiratory or gastrointestinal.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The work was supported by the British Heart Foundation (BS/93003 and PG /99055).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Boscan, VMTH, Univ. of California, Davis, CA 95616–8747 (E-mail: plboscan{at}ucdavis.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abrahams VC, Hilton SM, and Zbrozyna AW. Active muscle vasodilatation produced by stimulation of the brain stem: its significance in the defence reaction. J Physiol 154: 491–513, 1960.[Free Full Text]
  2. Abram SE, Kostreva DR, Hopp FA, and Kampine JP. Cardiovascular responses to noxious radiant heat in anesthetized cats. Am J Physiol Regul Integr Comp Physiol 245: R576–R580, 1983.[Abstract/Free Full Text]
  3. Bandler R, Carrive P, and Zhang SP. Integration of somatic and autonomic reactions within the midbrain periaqueductal grey: viscerotopic, somatotopic and functional organization. Prog Brain Res 87: 269–305, 1991.[ISI][Medline]
  4. Bandler R and Shipley MT. Columnar organization in the midbrain periaqueductal gray: modules for emotional expression? Trends Neurosci 17: 379–389, 1994.[CrossRef][ISI][Medline]
  5. Bandler R and Tork I. Midbrain periaqueductal grey region in the cat has afferent and efferent connections with solitary tract nuclei. Neurosci Lett 74: 1–6, 1987.[CrossRef][ISI][Medline]
  6. Bang OU and Lund A. Inhibition of glutamate antagonists, MK-801 and NBQX, of cutaneo-cardiovascular pain reflex in rats. Eur J Pharmacol 203: 133–135, 1991.[CrossRef][ISI][Medline]
  7. Blessing WW. Anatomy of the lower brainstem. In: The Lower Brainstem and Bodily Homeostasis. Oxford, UK: Oxford University Press, 1997, p. 29–101.
  8. Boscan P, Kasparov S, and Paton JF. Somatic nociception activates NK1 receptors in the nucleus tractus solitarii to attenuate the baroreceptor cardiac reflex. Eur J Neurosci 16: 907–920, 2002.[CrossRef][ISI][Medline]
  9. Boscan P and Paton JFR. Role of the solitary tract nucleus in mediating nociceptive evoked cardiorespiratory responses. Auton Neurosci 86: 170–182, 2001.[CrossRef][ISI][Medline]
  10. Boscan P and Paton JFR. NK1 receptors are involved in the convergence of somatic and periaqueductal grey afferents to the solitary tract. Proc Experimental Biology Annual Meeting, Board LB143, San Diego, CA 2003.
  11. Boscan P, Pickering AE, and Paton JF. The nucleus of the solitary tract: an integrating station for nociceptive and cardiorespiratory afferents. Exp Physiol 87: 259–266, 2002.[Abstract]
  12. Caeser M, Seabrook GR, and Kemp JA. Block of voltage-dependent sodium currents by the substance P receptor antagonist (+/–)-CP-96,345 in neurones cultured from rat cortex. Br J Pharmacol 109: 918–924, 1993.[ISI]
  13. Craig AD. Distribution of brainstem projections from spinal lamina I neurons in the cat and the monkey. J Comp Neurol 361: 225–248, 1995.[CrossRef][ISI][Medline]
  14. Esteves F, Lima D, and Coimbra A. Structural types of spinal cord marginal (lamina I) neurons projecting to the nucleus of the tractus solitarius in the rat. Somatosens Mot Res 10: 203–216, 1993.[ISI][Medline]
  15. Farkas E, Jansen AS, and Loewy AD. Periaqueductal gray matter projection to vagal preganglionic neurons and the nucleus tractus solitarius. Brain Res 764: 257–61, 1997.[CrossRef][ISI][Medline]
  16. Gamboa-Esteves FO, Kaye JC, McWilliam PN, Lima D, and Batten TF. Immunohistochemical profiles of spinal lamina I neurones retrogradely labelled from the nucleus tractus solitarii in rat suggest excitatory projections. Neuroscience 104: 523–538, 2001.[CrossRef][ISI][Medline]
  17. Gamboa-Esteves FO, Tavares I, Almeida A, Batten TF, McWilliam PN, and Lima D. Projection sites of superficial and deep spinal dorsal horn cells in the nucleus tractus solitarii of the rat. Brain Res 921: 195–205, 2001.[CrossRef][ISI][Medline]
  18. Hilton SM and Redfern WS. A search for brain stem cell groups integrating the defence reaction in the rat. J Physiol 378: 213–228, 1986.[Abstract/Free Full Text]
  19. Huang ZG, Subramanian SH, Balnave RJ, Turman AB, and Moi CC. Roles of periaqueductal gray and nucleus tractus solitarius in cardiorespiratory function in the rat brainstem. Respir Physiol 120: 185–195, 2000.[CrossRef][ISI][Medline]
  20. Hylden JL, Hayashi H, Dubner R, and Bennett GJ. Physiology and morphology of the lamina I spinomesencephalic projection. J Comp Neurol 247: 505–515, 1986.[CrossRef][ISI][Medline]
  21. Iwamoto GA, Wappel SM, Fox GM, Buetow KA, and Waldrop TG. Identification of diencephalic and brainstem cardiorespiratory areas activated during exercise. Brain Res 726: 109–122, 1996.[CrossRef][ISI][Medline]
  22. Keay KA and Bandler R. Distinct central representations of inescapable and escapable pain: observations and speculation. Exp Physiol 87: 275–279, 2002.[Abstract]
  23. Lanteri-Minet M, Weil-Fugazza J, Pommery J, and Menetrey D. Hindbrain structures involved in pain processing as revealed by the expression of c-Fos and other immediate early gene proteins. Neuroscience 58: 287–298, 1994.[CrossRef][ISI][Medline]
  24. Leah J, Menetrey D, and De Pommery J. Neuropeptides in long ascending spinal tract cells in the rat: evidence for parallel processing of ascending information. Neuroscience 24: 195–207, 1988.[CrossRef][ISI][Medline]
  25. Li J and Mitchell JH. c-Fos expression in the midbrain periaqueductal gray during static muscle contraction. Am J Physiol Heart Circ Physiol 279: H2986–H2993, 2000.[Abstract/Free Full Text]
  26. Lima D, Esteves F, and Coimbra A. C-fos activation by noxious input of spinal neurons projecting to the nucleus of the tractus solitarius in the rat. In: Proc 7th World Pain Congress Paris. Seattle, WA: IASP, 1994, p. 423–434.
  27. Lipski J. Antidromic activation of neurones as an analytic tool in the study of the central nervous system. J Neurosci Methods 4: 1–32, 1981.[CrossRef][ISI][Medline]
  28. Lovick TA. Inhibitory modulation of the cardiovascular defence response by the ventrolateral periaqueductal grey matter in rats. Exp Brain Res 89: 133–139, 1992.[ISI][Medline]
  29. Lovick TA. Midbrain influences on ventrolateral medullo-spinal neurones in the rat. Exp Brain Res 90: 147–152, 1992.[ISI][Medline]
  30. Marchenko V and Sapru HN. Different patterns of respiratory and cardiovascular responses elicited by chemical stimulation of dorsal medulla in the rat. Brain Res 857: 99–109, 2000.[CrossRef][ISI][Medline]
  31. McCloskey DI and Mitchell JH. Reflex cardiovascular and respiratory responses originating in exercising muscle. J Physiol 224: 173–186, 1972.[Abstract/Free Full Text]
  32. Menetrey D and Basbaum AI. Spinal and trigeminal projections to the nucleus of the solitary tract: a possible substrate for somatovisceral and viscerovisceral reflex activation. J Comp Neurol 255: 439–450, 1987.[CrossRef][ISI][Medline]
  33. Miles R. Frequency dependence of synaptic transmission in nucleus of the solitary tract in vitro. J Neurophysiol 55: 1076–1090, 1986.[Abstract/Free Full Text]
  34. Morilak DA, Morris M, and Chalmers J. Release of substance P in the nucleus tractus solitarius measured by in vivo mirodialysis: response to stimulation of the aortic depressor nerves in rabbit. Neurosci Lett 94: 131–137, 1988.[CrossRef][ISI][Medline]
  35. Ness TJ and Gebhart GF. Visceral pain: a review of experimental studies. Pain 41: 167–234, 1990.[CrossRef][ISI][Medline]
  36. Oberlander TF, Grunau RE, Pitfield S, Whitfield MF, and Saul JP. The developmental character of cardiac autonomic responses to an acute noxious event in 4-and 8-month-old healthy infants. Pediatr Res 45: 519–525, 1999.[ISI][Medline]
  37. Papas S and Ferguson AV. Electrophysiological characterization of reciprocal connections between the parabrachial nucleus and the area postrema in the rat. Brain Res Bull 24: 577–582, 1990.[CrossRef][ISI][Medline]
  38. Paton JFR. A working heart-brainstem preparation of the mouse. J Neurosci Methods 65: 63–68, 1996.[CrossRef][ISI][Medline]
  39. Paton JFR. The Sharpey-Schafer Prize Lecture: nucleus tractus solitarii—integrating structures. Exp Physiol 84: 815–833, 1999.[Abstract]
  40. Perez H, Ruiz S, Inostroza H, and Perretta M. Substance P depresses bioelectrical responses evoked in the nucleus tractus solitarii: interaction with gamma-aminobutyric acid-ergic neurons. Eur J Pharmacol 213: 435–437, 1992.[CrossRef][ISI][Medline]
  41. Pezzone MA, Lee WS, Hoffman GE, Pezzone MA, and Rabin BS. Activation of brainstem catecholaminergic neurons by conditioned and unconditioned aversive stimuli as revealed by c-Fos immunoreactivity. Brain Res 608: 310–318, 1993.[CrossRef][ISI][Medline]
  42. Pickering AE, Boscan P, and Paton JF. Nociception attenuates parasympathetic but not sympathetic baroreflex via NK1 receptors in the rat nucleus tractus solitarii. J Physiol 551: 589–599, 2003.[Abstract/Free Full Text]
  43. Potts JT and Fuchs IE. Naturalistic activation of barosensitive afferents release substance P in the nucleus tractus solitarius of the cat. Brain Res 893: 155–164, 2001.[CrossRef][ISI][Medline]
  44. Potts JT, Fuchs IE, Li J, Leshnower B, and Mitchell JH. Skeletal muscle afferent fibres release substance P in the nucleus tractus solitarii of anaesthetized cats. J Physiol 514: 829–841, 1999.[Abstract/Free Full Text]
  45. Potts JT, Paton JF, Mitchell JH, Garry MG, Kline G, Anguelov PT, and Lee SM. Contraction-sensitive skeletal muscle afferents inhibit arterial baroreceptor signalling in the nucleus of the solitary tract: role of intrinsic GABA interneurons. Neuroscience 119: 201–214, 2003.[CrossRef][ISI][Medline]
  46. Sapru HN. Transmitter/receptor mechanisms in cardiovascular control by the NTS: excitatory amino acids, acetylcholine, and substance P. In: Nucleus of the Solitary Tract, edited by Barraco IRA. Boca Raton, FL: CRC, 1994, p. 267–281.
  47. Scheuer DA, Zhang J, Toney GM, and Mifflin SW. Temporal processing of aortic nerve evoked activity in the nucleus of the solitary tract. J Neurophysiol 76: 3750–3757, 1996.[Abstract/Free Full Text]
  48. Seagard JL, Dean C, and Hopp FA. Modulation of the carotid baroreceptor reflex by substance P in the nucleus tractus solitarius. J Auton Nerv Syst 78: 77–85, 2000.[CrossRef][ISI][Medline]
  49. Toney GM and Mifflin SW. Sensory modalities conveyed in the hindlimb somatic afferent input to nucleus tractus solitarius. J Appl Physiol 88: 2062–2073, 2000.[Abstract/Free Full Text]
  50. Toney GM and Mifflin SW. Time-dependent inhibition of hindlimb somatic afferent transmission within nucleus tractus solitarius: an in vivo intracellular recording study. Neuroscience 68: 445–453, 1995.[CrossRef][ISI][Medline]
  51. Traub RJ, Sengupta JN, and Gebhart GF. Differential c-fos expression in the nucleus of the solitary tract and spinal cord following noxious gastric distention in the rat. Neuroscience 74: 873–884, 1996.[CrossRef][ISI][Medline]
  52. Verberne AJM and Struyker Boudier HAJ. Midbrain central grey: regional haemodynamic control and excitatory amino acidergic mechanisms. Brain Res 550: 86–94, 1991.[CrossRef][ISI][Medline]
  53. Yardley CP and Hilton SM. The hypothalamic and brainstem areas from which the cardiovascular and behavioural components of the defence reaction are elicited in the rat. J Auton Nerv Syst 15: 227–244, 1986.[CrossRef][ISI][Medline]
  54. Zhang J and Mifflin SW. Responses of aortic depressor nerve-evoked neurones in rat nucleus of the solitary tract to changes in blood pressure. J Physiol 529: 431–443, 2000.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Neurophysiol.Home page
S.-i. Sekizawa and A. C. Bonham
Group I Metabotropic Glutamate Receptors on Second-Order Baroreceptor Neurons Are Tonically Activated and Induce a Na+-Ca2+ Exchange Current
J Neurophysiol, February 1, 2006; 95(2): 882 - 892.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/1/R262    most recent
00328.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boscan, P.
Right arrow Articles by Paton, J. F. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boscan, P.
Right arrow Articles by Paton, J. F. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.