Chronotropic and Dromotropic Responses to Localized Glutamate Microinjections in the Rat $ Nucleus Ambiguus

brain research 1542 (2014) 93–103

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Research Report Chronotropic and dromotropic responses to localized glutamate microinjections in the rat $ nucleus ambiguus

Karla N. Sampaio1,He´lder Mauad2, K. Michael Spyer, Timothy W. Fordn

Division of Biosciences, Faculty of Life Sciences, University College London, Gower Street, London WC1E 6BT, UK article info abstract

Article history: The cardioinhibitory effects of cardiac vagal motoneurons (CVMs) are mediated by Accepted 18 October 2013 activation of postganglionic neurons in the epicardial ganglia which have been shown to Available online 24 October 2013 exert functionally selective effects on heart rate and atrioventricular conduction in the rat. Here we investigate whether CVMs producing these responses may occupy different Keywords: rostrocaudal positions within the nucleus ambiguus. Excitation of CVMs was attempted Autonomic nervous system by microinjections of glutamate into the nucleus ambiguus of an arterially perfused Vagus nerve preparation in a grid extending over 2 mm in the rostrocaudal plane using the obex as a Nucleus ambiguus reference point. Microinjections were paired, one made during pacing to measure changes Heart rate in atrioventricular conduction (P-R interval) independent of changes in heart rate and the Glutamate other looking for changes in heart period (P-P interval) un-paced. Although evidence of a differential distribution was found in 7 cases, in the majority (13/20), sites producing maximal effects on both variables coincided. Maximal changes in atrioventricular conduction resulted from more rostral sites in 6 cases and from a more caudal site in only one. Overall, the ratio of the change in atrioventricular conduction to the change in heart rate for a given site was signiﬁcantly greater 1 mm rostral to the obex than at either end of the test grid. We conclude that while CVMs controlling atrioventricular conduction are distributed with a peak somewhat rostral to those controlling heart rate in a number of animals, there is a signiﬁcant overlap and much greater variability in this distribution in the rat than in cats and dogs. & 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Abbreviation: CVM, cardiac vagal motoneuron ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. n Corresponding author. Present address: University of Nottingham School of Health Sciences, South Block Link, Queens Medical Centre, Nottinghamshire NG7 2HA, UK. Fax: þ44 115 823 1211. E-mail address: [email protected] (T.W. Ford). 1Present address: Department of Pharmaceutical Sciences, Federal University of Espírito Santo, Av. Marechal Campos, 1468, Maruípe, Vitória, ES, Brazil. 2Present address: Department of Physiological Sciences, Federal University of Espírito Santo, Av. Marechal Campos, 1468, Maruípe, Vitória, ES, Brazil.

dromotropic responses with no effects on heart rate were 1. Introduction reported. The present experiments investigate whether differential cardiac effects can be elicited by focal application of The vagus carries the parasympathetic motor supply to the heart glutamate along the rostrocaudal extent of the rat nucleus where it exerts a predominantly inhibitory action. Increased ambiguus while monitoring the effects on heart rate and cardiac vagal activity thus results in a fall not only in the atrioventricular conduction. heart rate (negative chronotropic effect) but also in the rate of A preliminary report has been published in abstract form propagation of the cardiac action potential (negative dromo- (Sampaio et al., 2000). tropic effect) and in the force of myocardial contraction (negative inotropic effect) (see Jones (2001)). The vagal motor path- way comprises a preganglionic component, originating in the medulla oblongata, which in turn synapses with a postganglionic 2. Results component within the cardiac ganglia which are located predominantly in the atrial epicardium. Although axonal tracing 2.1. Effects of microinjections of glutamate studies have shown that mammalian preganglionic cardiac vagal motoneurons (CVMs) arise from the nucleus ambiguus, dorsal Glutamate microinjections commonly elicited changes in motor vagal nucleus and the intermediate reticular formation, heart rate and atrioventricular conduction when the elec- the nucleus ambiguus represents the principal site of origin of trode tip was between 1.5 and 1.8 mm lateral to the midline myelinated cardioinhibitory B-fibers (McAllen and Spyer, 1976, and at a depth of 1.6–1.9 mm depending on the rostrocaudal 1978; Nosaka et al., 1979) while the dorsal motor vagal nucleus level relative to the obex. A representative example of the gives rise to unmyelinated C-fibers which have a lesser effect on time course and extent of changes in P-P and P-R intervals at theheartandlittledemonstrableinputfromthebaroreceptors different rostrocaudal levels is illustrated in Fig. 1. (Ford and McWilliam, 1986; Ford et al., 1990; Jones et al., 1995, Responses typically occurred with an onset latency of less 1998; Nosaka et al., 1982). than 5 s, peaked some 5–30 s later and returned to base- Earlier studies from this laboratory have shown that it is line levels within 1–2 min. The responses were reproducible possible to produce excitation of discrete epicardial ganglia within any series of injections and could not be elicited by resulting in selective effects on the rat heart (Sampaio et al., injections of equivalent volumes of vehicle (20 nL Ringer's 2003). These data are consistent with the existence of func- solution). The sizes of the responses varied quite consider- tionally selective ganglia that have been demonstrated in ably however from one preparation to another. Sites produ- other species (Ardell and Randall, 1986; Gatti et al., 1995, 1997; cing maximal responses were well-localized; microinjections Randall et al., 1986a, 1986b). 0.5–1.0 mm medial or lateral to these sites were typically In the cat, the vagal preganglionic axons running from the ineffective or produced comparatively small responses at nucleus ambiguus to the heart appear to target specific longer onset latencies. Significant increases in both P-P and ganglionic clusters and therefore may also be functionally P-R interval were observed following microinjection of gluta- defined at the preganglionic level (Gatti et al., 1996; Massari mate at all levels of the nucleus ambiguus studied when et al., 1995). That preganglionic CVMs in the rat nucleus compared to pre-injection levels (Table 1). ambiguus may also be functionally specific is suggested by the results of preliminary tracing studies performed in this laboratory. Single discrete microinjections of anterograde 2.2. Rostrocaudal distribution tracers that label small numbers of vagal preganglionic motoneurons in the nucleus ambiguus result in a restricted For each injection level the overall change in P-P interval distribution of labeled axon terminals in the cardiac ganglia (unpaced) is illustrated in Fig. 2A. Comparing the effective- (Jones et al., 1999). While precise mapping of the injection ness of different sites in eliciting changes in P-P interval it sites to anatomically identified ganglia was not performed in can be seen that microinjection of glutamate at 0, þ0.5 and this study, labeled axons were traced running through gang- þ1 induced significantly greater changes in P-P interval lionated plexuses without making any apparent synaptic compared to the other two sites. Changes in P-R interval contact until reaching an apparent target zone where dense however (Fig. 2B) were observed to be significantly greater innervation of neighboring ganglion cells occurred. This only at the levels 0 and þ1.0 compared to the changes elicited suggests that single preganglionic CVMs may preferentially at the most rostral and most caudal sites. To compare the innervate ganglion cells projecting to one particular target relative effectiveness of microinjections on chronotropic and rather than multiple sites within the heart. That such a dromotropic responses, the ratio of the change in P-P interval targeting exists is supported by anterograde tracing experi- to the change in P-R interval (ΔP-R/ΔP-P) was calculated for ments which have shown that CVMs from the left and right all sites where both measurements were made (i.e. micro- sides of the brain stem appear to innervate different subdivi- injections during both unpaced and paced runs). The inten- sions within the cardiac ganglionic plexuses (Cheng et al., tion was to avoid loss of valuable paired data which would 2004). otherwise occur when data were pooled. If the CVMs con- In the study by Gatti et al. (1996), sites in the nucleus trolling P-P interval and those controlling P-R interval were ambiguus producing negative dromotropic effects to gluta- mixed homogeneously across the rostrocaudal extent of the mate tended to be located rostral to those producing negative nucleus then one would not expect to find peaks in this ratio chronotropic effects. In some rostral locations, sites producing at any particular rostrocaudal location. brain research 1542 (2014) 93–103 95

P-P interval P-R interval 300 95 P-P P-R (ms) (ms) 200 85 100 100 60 60 (mmHg) (mmHg) Pressure - 0.5 Pressure -0.5 L-Glu L-Glu

300 95 P-P P-R (ms) (ms) 200 85 100 100 60 60 (mmHg) (mmHg) Pressure Pressure Obex Obex L-Glu L-Glu

300 100 P-R P-P (ms) 200 (ms) 90 100 100 60 60 (mmHg) (mmHg) Pressure Pressure + 0.5 + 0.5 L-Glu L-Glu

300 90 P-R P-P (ms) (ms) 200 80 100 100 60 60 (mmHg) (mmHg) Pressure Pressure + 1.0 + 1.0 L-Glu L-Glu

300 90 P-P P-R (ms) 200 (ms) 80 100 100 60 60 (mmHg) (mmHg) Pressure + 1.5 Pressure + 1.5 L-Glu L-Glu

70 sec 70 sec

Fig. 1 – Examples of Spike2 traces showing the changes in P-P and P-R intervals following glutamate microinjections at 5 levels of the right nucleus ambiguus in one preparation. Recordings of P-R intervals were performed during cardiac pacing. Note that changes in heart rate (P-P interval) had relatively little effect on the perfusion pressure which was largely determined by the flow rate set by the perfusion pump less the flow through the bypass circuit. The pulse pressure arises through the addition to the perfusate of the contents of the left ventricle, the filling of which was not controlled for in these experiments i.e. no attempt was made to eliminate return from pulmonary veins. There was a relatively small effect on pulse pressure presumably attributable to greater diastolic filling and therefore greater ejection through the Frank–Starling mechanism.

Table 1 – Baseline and maximum responses to glutamate injection into the nucleus ambiguus at the levels shown.

A Level of injection 0.5 (n¼13) 0 (n¼19) þ0.5 (n¼10) þ1.0 (n¼19) þ1.5 (n¼16) Pre-injection P-P (ms) 23275.6 23276.0 20774.5 22875.4 23676.1 n nn nn nn nn Post-injection P-P (ms) 24476.8 289712 267714 268710 24776.1

B Level of injection 0.5 (n¼12) 0 (n¼19) þ0.5 (n¼10) þ1.0 (n¼19) þ1.5 (n¼16) Pre-injection P-R (ms) 7673.9 7673.0 7874.8 7873.4 7473.1 n nn nn nn n Post-injection P-R (ms) 7773.8 8373.5 8475.4 8774.5 7673.1

Values represent the mean7SEM. For the level of injection, 0 represents the level of the obex with þ being rostral and – being caudal relative to obex. Note that the availability of paired data allowed the detection of signiﬁcant differences despite the relatively small size of the responses and the high variability indicated by the SEMs. nn po0.01. n po0.05 signiﬁcantly different from control using a paired t-test, two-tailed. 96 brain research 1542 (2014) 93–103

80 15 0.40 ** **# **

60 0.30 #** 10 ** *

40 0.20

20 0.10

0 0 0.00 -0.5 0 +0.5 +1.0 +1.5 -0.5 0 +0.5 +1.0 +1.5 -0.50 +0.5 +1.0 +1.5 Rostro-Caudal Coordinates Rostro-CaudalCoordinates Rostro-Caudal Coordinates

Fig. 2 – Changes in P-P interval (A), P-R interval (B) and ΔP-R/ΔP-P (C) following microinjections of glutamate at 5 levels of the nn NA. In (A), po0.01 indicates statistically significant differences compared to the 0.5 and þ1.5 level and #po0.05 indicates a nn n statistically significant difference compared to the þ1.0 level. In (B), po0.01 and po0.05 indicate statistically significant n differences compared to 0.5 and þ1.5 levels. In (C), #po0.05 and po0.01 indicate statistically significant differences compared to the 0.5 and þ1.5 levels, respectively. The distribution of sites affecting P-P interval (heart rate) is unimodal, peaking between obex and 0.5 mm rostral (A). The distribution of sites affecting P-R interval are more skewed in the rostral direction with the largest peak at 1 mm rostral to obex (B). The site producing the greatest change in P-R interval per unit change in P-P interval is at 1 mm rostral to obex (C).

In the event, ΔP-R/ΔP-P peaked at 1 mm rostral to obex Stimulation of the peripheral cut ends of the sectioned vagi which reached signiﬁcance when compared to 0.5 and þ1.5 elicited signiﬁcant effects on both P-P and P-R interval and but not to 0 and þ0.5 (Fig. 2C). did not appear to show any right/left selectivity on either. The distribution of the peaks in these histograms is Sites eliciting rapid-onset responses that were marked skewed in the caudal direction in A compared to B and C with biocytin were shown to be localized within the area of suggesting a greater preponderance of chronotropic CVMs at the nucleus ambiguus when visualized under a conventional caudal than at rostral sites. The tendency for the more rostral light microscope. An example of biocytin labeled cells in the sites to have a greater effect on P-R than on P-P is also area of the right nucleus ambiguus following a microinjection apparent from the relative positions of the most effective at 0.5 mm relative to obex is reproduced in Fig. 6. In this sites in any set of unilateral microinjections in individual case, the biocytin was pressure injected to a volume of preparations: most (13/20) showed peak changes in both P-P approximately 20 nL and resulted in labeled cells within the and P-R interval following injections at the same level (see area of the nucleus ambiguus and the surrounding periambi- example in Fig. 3A). However in 6/20, peak changes in P-R gual regions but not in the adjacent lateral reticular nucleus were elicited from sites rostral to those eliciting peak changes situated about 0.5 mm ventrolateral to it (Fig. 6). in P-P (see example in Fig. 3B) and in only 1 was the reverse found. At 9 sites in 8 preparations atrioventricular conduction block was observed following glutamate microinjections 3. Discussion between obex and 1 mm rostral to obex but not below (see example in Fig. 4B). If preganglionic cardiac vagal motoneurons innervated their target neurons in the cardiac ganglia in a non-selective 2.3. Left versus right manner and irrespective of their location in the medulla oblongata, then one would expect to see homogeneity in the Microinjections of glutamate into the left nucleus ambiguus responses evoked by their excitation at different rostrocaudal were statistically no more or less effective than those on the levels. So preganglionic neurons in the rostral extent of their right at prolonging P-P or P-R interval (p40.05). However, it is distribution would be expected to elicit similar responses on worth noting that 6 out of the 9 sites producing atrioven- heart rate and the rate of atrioventricular conduction as tricular block were on the left. This is of relevance because neurons found toward the caudal pole. The results of the when second degree atrioventricular block intervened, the present study have shown that there is in fact a considerable preceding P-R interval was usually taken as the maximum degree of variation in the responses evoked at different size of the response. In two animals an ipsilateral vagotomy rostrocaudal levels. This is illustrated in Fig. 3 where a was performed which abolished the typical response induced comparatively homogeneous response in (A) is contrasted by glutamate injections into the nucleus ambiguus (Fig. 5). with that in (B) where a much greater effect was elicited on brain research 1542 (2014) 93–103 97

110 10 100 9 90 8 80 7 70 6 60 5 50 4 40 3

P-Pinterval (ms) 30 20 2 1 10 10 ms 0 0 0.5 caudal Obex 1.0 rostral 1.5 rostral

120 40

35 100 30 80 25

60 20

15 40

P-Pinterval (ms) 10 20 5

0 0 0.5 caudal Obex 1.0 rostral 1.5 rostral

Fig. 3 – Responses to glutamate microinjections into the 10 ms right nucleus ambiguus on changes in P-P interval (left axis, squares with solid lines) and P-R interval (right axis, circles Fig. 4 – Waterfall plots showing beat-by-beat changes in P-R with broken lines) in two preparations. In (A), the size of the interval following microinjections into the nucleus response on one variable follows that on the other, both ambiguus (A) at the level of the obex and (B) 1.0 mm rostral peaking 1 mm rostral to obex. In (B), there is a clear to the obex. In both traces, the atria were paced at a rate separation of the peaks with a maximal change in P-R slightly above the resting heart rate. Glutamate was injected interval 1 mm rostral to obex while the peak change in P-P just prior to the first sweep which is at the bottom in both interval occurs at obex. This suggests that in this plots with subsequent sweeps displaced by a small preparation (B) CVMs affecting atrioventricular conduction increment in the Y-axis. The time between the start of each were localized at þ1 compared to obex where there was a sweep, which corresponds to the P wave and the start of the preponderance of CVMs affecting heart rate. Note the QRS complex (i.e. the PR interval) can be seen to lengthen in differences in the PR axis scales in (A) and (B) illustrating the both cases. In (B) however, second degree atrioventricular variability in the sizes of the responses obtained in different block can be seen to develop from the sweep indicated by preparations. the dark arrowhead on the left. In this condition, one or more of the cardiac action potentials fails to be conducted across the atrioventricular node. This condition remains heart rate (as reflected in the P-P interval) than on atrioven- until the sweep indicated by the clear arrowhead at which tricular conduction (as reflected by the P-R interval) at the point all action potentials begin once again to reach the level of the obex and was reversed just 1 mm rostral. This ventricles. Between the arrows, a variable number of action suggests that at least in some animals, there is a spatial potentials are successfully conducted to the ventricles, separation of preganglionic motoneurones innervating the typically at a reduced P-R interval presumably attributable to different cardiac ganglia which have been shown to exert the removal of the refractory effects of the non-conducted such differential effects in this species (Sampaio et al., 2003). action potential. Where a differential effect was observed, the sites eliciting maximal effects on atrioventricular conduction were rostral to those eliciting maximal effects on heart rate in all but one case (6/7). Such a “cardiotopic” organization of the nucleus degree of apparent overlap in the present study may have ambiguus has been previously described in the cat and dog resulted to an extent from methodological limitations since (Blinder et al., 1998, 2007; Gatti et al., 1996; Massari et al., the absolute size of the nucleus containing CVMs is smaller in 1995). However, it is clear that a degree of variation exists in the rat in relation to the size of the microinjection. Central the topographical organization of the preganglionic neurons recording studies in the cat have shown that excitation of in the rat since in most cases (13/20) the maximal effects both CVMs using ionophoresis of excitatory amino acids can on atrioventricular conduction and heart rate were obtained produce measurable changes in heart rate (McAllen and by microinjections at the same rostrocaudal level. The greater Spyer, 1978), so it is possible that more discrete, low volume 98 brain research 1542 (2014) 93–103

DVn

CC 12n

LRn

Fig. 6 – Photomontage of a transverse section from the brain stem stained with neutral red showing biocytin-labeled neurons (blue, arrowed) following a microinjection in the right nucleus ambiguus of one animal (kp11). 20 nL biocytin 2% in 0.5 M NaCl was injected at a point 0.5 mm caudal to obex, 1.6 mm lateral to the midline and at a depth of Fig. 5 – Examples of Spike2 traces showing the changes in 1.6 mm. Scale bar 500 lm. The nucleus ambiguus at this P-P interval following glutamate microinjections into the level is indistinct and CVMs are found outside the main nucleus ambiguus (A) before and (B) after ipsilateral vagal column (Corbett et al., 2007; Kalia and Sullivan, 1982). Note section in one preparation. Vagal section abolished the the absence of labeling in the lateral reticular nucleus (LRn). characteristic short-latency bradycardia evoked by nA – nucleus ambiguus, DVn – dorsal vagal nucleus, 12n – glutamate. The small, transient bradycardia seen at about hypoglossal nucleus. 20 s in (B) was not reproducible and most likely represents a spontaneous minor variation in the heart rate in this case. Since the ipsilateral vagus was cut and the spinal cord sectioned, activation of pathways to the contralateral vagal Mesulam, 1980; Kalia and Sullivan, 1982). Glutamate is known motor nuclei would be the only remaining possibility. to excite neurons while leaving axons of passage unaffected (Goodchild et al., 1982; Zieglgansberger and Puil, 1973)and CVMs express glutamate receptors of both AMPA and NMDA microinjections (Shaw et al., 2002) or ionophoretic application subtypes (Corbettetal.,2003) but it is possible that local circuits of glutamate would have revealed a clearer topographical wereactivatedaswellasCVMs.However,supportforthe localization. However, the fact that quite distinct differential assumption is provided by the observation that effects were effects could be elicited in individual preparations suggests rapid in onset and strongly localized. Injections outside the that methodology was not the primary reason for the varia- immediate area of the nucleus were smaller and had much bility in the relative positions of chronotropic and dromo- longer onset latencies. Furthermore the rapid onset cardiac tropic maxima. Rather it seems more likely that there exists a effects were abolished by ipsilateral vagal section (Fig. 5). Since significant variability from animal to animal in the extent of the spinal cord was sectioned at C2, sympathetically mediated the localization and the degree of overlap. This may also effects are unlikely to be operational, leaving synaptic activa- reflect an imperfect segregation apparent in the functional tion of contralateral CVMs as the only likely remaining neu- characteristics of the cardiac vagal ganglia in individual rats roeffector mechanism. While the small, brief bradycardia seen where selective electrical stimulation of the ganglia was in Fig. 5B about 20 s post-injection may be attributable to such a attempted (Sampaio et al., 2003). mechanism rather than being a random occurrence, it is The conclusion is based upon the assumption that the considered unlikely that this would have made a significant microinjections of glutamate produced their effects primarily contribution to the measured responses. Attempts to block through direct activation of CVMs in the area of the nucleus synaptically-evoked responses in the brain stem using a low- ambiguus and that the same subset of CVMs were excited by calcium, high-magnesium Ringer's solution were unsuccessful repeat microinjections into the same area. The technique was since synaptic blockade also occurred at the ganglia despite chosen in preference to electrical stimulation to avoid activa- efforts to selectively perfuse the coronary arteries with the tion of axons of passage which are known to pass through normal Ringer's solution. This was attributed to a significant this area from other regions of the brain stem e.g. dorsal vascular supply to the cardiac ganglia via an extracoronary motor vagal nucleus and nucleus tractus solitarius, (Kalia and source (Halpern, 1957; Jones, 2001). brain research 1542 (2014) 93–103 99

These data support the results of preliminary anterograde Effects on cardiac inotropy were not investigated in the tracing studies following discrete injections into the nucleus present study and it has not yet been determined whether ambiguus which suggest targeting by CVMs of distinct groups reports of a similar functional separation at the preganglionic of cardiac ganglion cells (Jones et al., 1999). Anterograde level in the nucleus ambiguus of the cat and dog (Blinder tracing studies in the rat (Ai et al., 2007; Cheng et al., 1999, et al., 1998; Blinder et al., 2007) are also to be found in the rat. 2004; Cheng and Powley, 2000) have shown in exquisite detail A study looking at the release of acetylcholine in the left that both the nucleus ambiguus and the dorsal motor vagal ventricular myocardium following vagal stimulation in the nucleus project to all the cardiac ganglia in this species. Since cat suggests that acetylcholine levels in the left ventricle are CVMs from the two sites appear to target separate subpopu- affected by both right and left vagal nerves and show little lations of principal neurons (Cheng et al., 2004) there is an evidence of interactions at the level of the cardiac ganglia anatomical basis for differential control of cardiac function by (Akiyama and Yamazaki, 2001). There was a great deal of location. The injections of anterograde tracers in the above variability between preparations on the relative effects of left studies were made with the aim of labeling each nucleus versus right vagal nerve stimulation in the current experi- in its entirety rather than targeting discrete regions of the ments with the result that no statistically significant differ- nucleus ambiguus or dorsal motor vagal nucleus so it is ence was found between the two sides with regard to effects difficult to speculate about differential organization within on heart rate and atrioventricular conduction. The fact that any one nucleus. However, neurons in the dorsal motor atrioventricular block was more commonly obtained from the vagal nucleus did not appear to converge on the same left nucleus ambiguus may suggest a greater involvement of population of principal cells in the rat cardiac ganglia that the left vagus in atrioventricular conduction, as has been were innervated by neurons of the nucleus ambiguus reported by others (Hamlin and Smith, 1968; Parker et al., (Cheng et al., 2004). 1984). Retrograde labeling of CVMs in the rat nucleus ambi- Recordings made in a reduced preparation similar to that guus from the cardiac ganglion nearest to the sinoatrial node described here provide compelling evidence that a typical has been reported to be greater on the right than the left active principal cardiac ganglion cell receives its input from a (Corbett et al., 2007) though the authors are cautious about single active CVM (McAllen et al., 2011). Furthermore, the attributing this to a functional separation rather than a same active CVM appears to drive reflex responses from simple topographical arrangement. Similarly, anterograde diverse sources such as the peripheral chemoreceptors, the labeling of CVMs in the left and right sides of the brain stem arterial baroreceptors and the diving response. The authors has shown that their axons enter the ganglia via different do not exclude the possibility of convergence from different routes and project to different neuronal subdivisions within CVMs. Indeed they show that convergence from silent fibers the cardiac ganglia (Cheng et al., 2004). While these findings is revealed during direct electrical stimulation of the vagus lend support to the notion of a functional topographical but the source of the CVMs remains unknown. Silent CVMs arrangement of CVMs in this species, localized micro- unresponsive to reflex inputs such as those above have been injections of anterograde tracers into different regions of described in the nucleus ambiguus and dorsal motor vagal the nucleus ambiguus would be required to confirm that an nucleus in cats (Ford et al., 1990; McAllen and Spyer, 1978) anatomical substrate exists for the functional organization and in rats (Jones, 2001; Rentero et al., 2002) and while they within this nucleus. Such studies and others using retrograde remain a source of speculation with regard to the circum- tracing techniques which lay beyond the scope of the present stances under which they might be normally be activated, experiments may determine whether differences exist in they provide little insight into whether their projections the mediolateral and dorsoventral localization within this might be functionally specific. The reflex inputs described nucleus in addition to those demonstrated in the rostrocau- above are known to produce both chronotropic and dromo- dal plane in the present experiments. tropic responses as are those resulting from stimulation of cardiopulmonary afferent fibers so the reasons for the devel- opment of separate populations of CVMs controlling heart 4. Experimental procedures rate and atrioventricular conduction are not immediately apparent. The suggestion that CVMs from the dorsal motor 4.1. Preparation vagal nucleus might be selectively activated by cardiopulmonary inputs (Daly and Kirkman, 1989; Jones and Daly, Experiments were performed using a rat preparation per- 1997) no longer appears tenable (Jones, 2001; Wang et al., fused with oxygenated Ringer's solution (Ford and Spyer, 2000). The bradycardia evoked by stimulation of cardiopul- 1999; Paton, 1996). All the experiments were performed in monary afferents in the rat is attenuated after bilateral accordance with the European Commission Directive 86/609/ microinjection of P2X antagonist in the nucleus ambiguus EEC (European Convention for the Protection of Vertebrate suggesting CVMs in this region are also activated by this Animals used for Experimental and Other Scientific Purposes) input via release of purines (Passamani et al., 2011). Interest- and the UK Home Office (Scientific Procedures) Act (1986) ingly, an essential role for CVMs in the dorsal motor vagal under institutional guidelines and every effort was made to nucleus has recently been proposed for the cardioprotective minimize animal discomfort and numbers used. Twenty- effects of remote ischemic preconditioning in this species eight juvenile Sprague-Dawley rats weighing between 100 (Mastitskaya et al., 2012) which appears to be independent of and 150 g were deeply anaesthetized with isoflurane (5% in effects on heart rate. The specific groups of ganglion cells oxygen) delivered via a vaporizer (Fluotec 3) in a custom mediating these effects remain to be established. designed induction chamber until breathing ceased and a 100 brain research 1542 (2014) 93–103

complete loss of withdrawal to forepaw pinch was observed. a Swan-Ganz 123F6 double lumen catheter, Baxter Health- They were immediately removed from the chamber, bisected care, Deerfield, IL, USA) and maintained between 70 and at level of the diaphragm and immersed in oxygenated 120 mmHg by dividing the flow appropriately through the Ringer's solution at 2–4 1C. The preparation was decerebrated cannula and a bypass circuit that returned perfusate directly by removal of all brain tissue above the inferior border of the back to the reservoir via a variable roller clamp or Hoffman colliculus using a suction tube and most of the skin removed clip. The preparation was held in the prone position with the from the neck down. The forelimbs were excised below the head somewhat ventroflexed using an acrylic holder with ear shoulder, following ligation of the brachial arteries, to facil- bars and the spinal segments and ribs below the level of T4 itate access to the vagi and the dorsal upper thoracic cavity. were removed to allow access to the heart. Both cervical vagi The descending aorta was then cannulated and perfused were identified and loose threads were passed behind them retrogradely with Ringer's solution at 31-32 1C using a peri- to aid later placement on stimulating electrodes if required. staltic pump (Watson Marlow, Cornwall, UK; flow rate, The spinal cord was sectioned at C2 to eliminate bulbospinal 40-60 ml min 1) so that the remaining tissue, including the sympathetic pathways (Fig. 7). brain stem remained viable. The composition of the Ringer's solution was as described 4.2. ECG recording and electrical pacing by Paton (1996) and contained (mM): NaCl 125, NaHCO3 24,

KCl 5, CaCl2 2.5, MgSO4 1.25, KH2PO4 1.25 and D-glucose 20; the The ECG was recorded using local platinum electrodes posi- solution was bubbled with a mixture of 95% oxygen and 5% tioned close to the atria so as to give a prominent P wave as carbon dioxide (carbogen). Ficoll 70 (5%, Sigma) was added to well as an identifiable QRS complex. An intelligent computer the vascular perfusate to increase colloid osmotic pressure interface (CED1401plus, Cambridge Electronic Design, Cam- and the pH was adjusted to 7.3-7.4. Perfusion pressure was bridge, UK) was used to digitize the recordings of the perfusion monitored using a pressure transducer (Neurolog NL108T1, pressure, ECG and stimulus event timings. The ECG signal Digitimer Limited, UK) attached to the minor lumen of a was band-pass filtered from 10 Hz to 2 kHz and sampled at double lumen cannula used for the perfusion (modified from 5 kHz. Data were transferred to a PC for on-line display and held on the computer hard disk for later analysis and backup to CD-ROM. Preliminary on-line analysis of heart period (P-P interval) was achieved by level triggering from the P wave of the ECG using a window discriminator (Neurolog Glutamate 0.1 M NL201, Digitimer Limited) which fed event time data into Medulla 20 nL the 1401plus for continuous display using Spike2 software oblongata (Cambridge Electronic Design). A falling heart rate was indicated by an increase in the P-P interval of the ECG. Atrioventricular conduction time was taken as an index of dromotropy and was derived from the P-R interval. To avoid indirect effects of Obex changes in heart rate on the P-R interval, standard measurements were undertaken during atrial pacing at constant rate. Section C2 Pacing electrodes made from fine lacquered copper wire were attached to the right atrial appendage by piercing its lateral L. Vagus R. Vagus border with the bare copper wire and forming a loop. P-R interval during atrial pacing was monitored directly on an oscillo- scope triggered from a timing device (Digitimer 4030) which was also used to trigger the stimulators driving the pacing and stimulating electrodes (Digitimer DS2 constant voltage isolated stimulators). The stimulus interval for pacing was set between Pacing electrodes 90% and 99% of the minimum unpaced P-P interval in the period prior to stimulation. ECG 4.3. Microinjections into the NA

Ringer’s solution Pressure injections of 0.1 M L-Glutamate (20 nL in Ringer's solution) were made into the nucleus ambiguus from one Fig. 7 – Schematic of experimental preparation. barrel of a triple-barreled micropipette (20–25 mm tip) at sites Microinjections were made according to a grid which used from 0.5 mm caudal to 1.5 mm rostral to the obex (0.5, 0, as its reference point the obex as illustrated in Paxinos and þ0.5, þ1, þ1.5) using compressed nitrogen controlled by a Watson (2007). The nucleus ambiguus moves laterally as it solenoid valve (Neurophore system, Digitimer Limited). The ascends in the rostral direction and this was taken into remaining two barrels contained vehicle (Ringer's solution account in the penetrations which were all perpendicular to alone) and a biotinylated marker respectively. At each level, the dorsal surface. The ventroﬂexion of the head was such sites were sought that elicited maximal effects at the shortest that the dorsal surface of the brain stem over the area latencies (Fig. 7). All measurements were made relative to the explored was close to the horizontal. obex as it is described in Paxinos and Watson (2007).Itis brain research 1542 (2014) 93–103 101

worth noting that these authors designate the obex as the injectate spread (e.g. Fig. 6). The brain stem was removed and caudal end of the area postrema in the rat, corresponding to fixed in fresh phosphate-buffered formal saline (4% parafor- the tip of the calamus scriptorius. In this preparation, this lay maldehyde in 0.1 M phosphate buffer, 0.9% NaCl, pH 7.4) for approximately 0.6 mm caudal to the rostral border of the area 24-72 h. Brain stems were later transferred to a sucrose postrema at the midline which some authors identify as the solution (sucrose 10% in 0.1 M phosphate buffer) at 4 1Cat obex (e.g. Kalia and Sullivan, 1982). least 24 h prior to sectioning. Sections were cut at 50 mmona Microinjections commonly elicited changes in heart rate freezing microtome, washed twice in phosphate buffered when the electrode tip was between 1.5 and 1.8 mm lateral to saline (0.01 M) and incubated in the avidin–biotin–HRP com- the midline and at a depth of 1.6–1.9 mm from the dorsal plex (ABC, Vector Labs, UK). The sections were then washed surface as measured using micromanipulators (Narishige, and processed using diaminobenzidine (DAB, Sigma, UK) with Japan). The mediolateral and dorsoventral coordinates of nickel intensification. Sections were mounted on gelatin the target injection sites were initially based on histological coated slides and counterstained with neutral red (1%, Sigma, material from an earlier series of studies but were modi- UK) to visualize the marked sites. The positions of the injection fied on the basis of the responses obtained. Typically tracks sites revealed by the characteristic blue/black staining were within 0.5 mm rostral and 0.5 mm caudal to obex were captured by photomicrographs obtained using a Leica DMR selected for the first injection site and were assessed by the microscope under conventional bright field illumination with a latency and size of the response. If a site did not elicit short Nikon Coolpix 4500 digital camera. latency changes in heart rate or atrioventricular conduction then the electrode was moved to a different depth or if necessary medially or laterally until optimal responses were 4.5. Data analysis obtained. The lateral excursion and depth increased as the brain stem was explored from caudal to rostral. The order in The ECG data were analyzed off-line using Spike2 program which the sites were explored varied but injections at the scripts to rewrite the P-P and P-R intervals as waveform data level of the obex were generally the first to be made; one side following identification of ECG waves using the template (left or right) was usually chosen for completion first. Only matching functions of the program. All template-generated responses with an onset latency of 10 s or less were included event identities were verified by visual inspection of the in the data. After advancing the electrode to the injection site, record. Event times were not adjusted to correspond exactly at least two microinjections were made at each locus: one with the start of the respective waves but the trigger point for without pacing and the other during atrial pacing, to measure each wave was maintained during the period of measure- chronotropic and dromotropic effects, respectively. Periods of ment. In most cases during pacing, the pacing stimulus event at least 2 min were allowed between microinjections to time was used as the trigger point for the P wave. If second enable full recovery. All measurements of P-R interval used degree atrioventricular block developed this was noted but to assess atrioventricular conduction were made during the P-R interval was not updated so that only fully conducted pacing to eliminate heart rate dependent changes in this cardiac action potentials were used to derive the maximum variable. The volume injected (20 nL) was measured by direct interval. Values are expressed as the mean7standard error of observation of the drop of the meniscus against a known the mean (S.E.M.). Statistical comparisons between baseline scale fabricated from nylon mesh glued to the micropipette, and maximal values of the P-P and P-R interval data at the as observed under a dissecting microscope. Control micro- various sites were made using Student's t test for paired data injections of Ringer's solution into selected sites were also (two-tailed). Comparisons between the responses at the made. If time permitted and the preparation remained in various levels were by one-way analysis of variance (ANOVA) good condition, the contralateral brain stem was explored at followed by a Fisher test for multiple comparisons. A prob- the same levels. There was a tendency for edema to develop ability level of less than 0.05 was taken to indicate statistical in these preparation, despite the presence of the oncotic significance. agents in the perfusate so the condition of the preparation was tested at intervals by observing the characteristic bradycardia in response to baroreceptor stimulation (brief eleva- tions in perfusion pressure brought about by restricting the Acknowledgments bypass flow) or chemoreceptor stimulation (10–50 mL bolus of 0.05% sodium cyanide injected into the perfusion cannula). The support of the British Heart Foundation and CAPES Reproducible responses could be maintained for at least 4 h Foundation is gratefully acknowledged. after the initial preparation but typically measurements were references completed within 3 h of the first microinjection.

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