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Activation of hypothalamic arcuate but not paraventricular neurons following carotid body chemoreceptor stimulation in the rat
0306-4522/88 $3.00 + 0.00 Pergamon Press plc
Neuroscience Vol. 24, No. 3, pp. 967-976, 1988
Printedin Great Britain
0 1988 IBRO
ACTIVATION OF HYPOTHALAMIC ARCUATE BUT NOT PARAVENTRICULAR NEURONS FOLLOWING CAROTID BODY CHEMORECEPTOR STIMULATION IN THE RAT D. BANKS* and M. C. HARRIS? Department of Physiology, The Medical School, Vincent Drive, Birmingham Bl5 2TJ, U.K. Ah&act--The effect of carotid body chemoreceptor stimulation on 292 neurons in midline hypothalamic nuclei has been examined electrophysiologically in ethyl carbamate/sodium pentobarbitone anaesthetized rats. Experiments demonstrated that specific stimulation of carotid body chemoreceptors activates a small group (16) of neurons in the mediobasal hypothalamic arcuate nucleus, but has no effect on neurons (157) in the hypothalamic paraventricular nucleus or the anterior hypothalamus. Of 16 arcuate neurons activated by the stimulus, six projected to the median eminence and three projected to the dorsal medulla, as defined by antidromic invasion. Three of the neurons activated from the carotid body also showed a resting discharge that was linked with ventilation rate, suggesting that the arcuate nucleus may have some involvement with respiratory processing. The activation of neurons projecting to the median eminence implies that the release of adenohypophyseal hormones may also be influenced by carotid body chemoreceptors.
Stimulation of carotid body chemoreceptors is usually followed by hyperventilation accompanied by variable changes in heart rate and blood pressure.*’ Moreover, it is well known that the dorsal medulla oblongata and pons are intimately involved in these changes and that this is reflected in the neuronal act&ity in these areas. 22 Much less is known of the influence of this stimulus on neurons in the forebrain. However, in both cats and rats it has been shown that carotid body chemoreceptor stimulation can evoke all the autonomic components of the defence reaction,6*2sand since, in cats, the same components are evoked by electrical stimulation within the amygdala and the hypothalamus, ‘*I7by implication it may be assumed that chemoreceptor stimulation will activate at least these regions. Share and Levy” also demonstrated that carotid chemoreceptor stimulation releases vasopressin, and the neuroendocrine neurons of the hypothalamic supraoptic nuclei (SO) that release vasopressin are known to be activated by the same stimulus.” Following this finding Harris et ~1.‘~ reported that chemoreceptor-induced activation of SO neurons was via an ipsilateral pathway only and
hypothalamic arcuate nucleus; CI, internal capsule; FX, fomix; LHA, lateral hypothalamic area; MFB, medial
that the activation was abolished by lesions which destroyed the preoptic area rostra1 and medial to SO, implying that the input had passed through rostra1 forebrain regions before descending to the medial hypothalamus. This, to some extent at least, agreed with an earlier report that destruction of the same region abolished part of the cardiovascular response to carotid occlusion in rats.23 Although the vasopressin neurons of the supraoptic nuclei are known to be activated by chemoreceptor stimulation, there has been no electrophysiological investigation of the influence of this stimulus on medial hypothalamic neurons. However, the extent of the carotid body chemoreceptor influence on metabolic activity within the forebrain was examined in a recent investigation using [L4C]2-deoxyglucose autoradiography in anaesthetized rats.5 This study showed that chemoreceptor stimulation resulted in widespread metabolic activation in the forebrain, mainly on the side ipsilateral to the stimulated carotid body. With the exception of the ipsilateral SO and the median eminence/arcuate region, however, the hypothalamus was not affected. This finding, which appeared to contradict much of the evidence from earlier studies mentioned above, led us to investigate the problem using electrophysiological methods, to see if, in the medial hypothalamus, activation by carotid body stimulation was confined to the median eminence/arcuate region. Part of this study has been presented as short communications.4J2
forebrain bundle; NT?$ tractus solitarius nuclei; OT optic tract; PVH, hypothalamic paraventricular nucleus; RE, thalamic reuniens nucleus; SM, stria medullaris thalami; SO, supraoptic nucleus; ST, stria terminalis; V, 3rd ventrical; VMH, hypothalamic ventromedial nucleus; ZI, zona inserta.
The experiments were performed on male SpragueDawley rats (300-52Og body wt) anaesthetized with an ethyl carbamate/sodium pentobarbitone mixture (Urethan
*Present address: Department of Physiology, The Medical School, University Walk, Bristol, BS8 ITD. tTo whom correspondence should be addressed. Abbreuiafions: AHA, anterior hypothalamic area; ARH,
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EXPERIMENTAL PROCEDURES
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D. BANKS and M. C. HARRIS
1.2-I .4 g/kg, Sagatal20 mg/kg, i.p.). Catheters were inserted into a femoral artery for the measurement of arterial blood pressure, and a femoral vein for injecting additional doses of anaesthetic (Sagatal 12 mg/ml saline). A cannula was placed into the trachea and attached to a “T” piece containing a glass-coated bead thermistor (GC32J1, Fenwal Electronics or U23UD, STC Electronic Services, U.K.) for recording respiration, and electrodes attached across the chest for recording ECG and heart rate. The respiration and ECG signals were passed through analogue discriminators and linear rate-meters for measurement of instantaneous respiration and heart rate. Body temperature was measured rectally using either a mercury-in-glass thermometer or thermoprobe (Gallenkamp temp stick, THV-150-G) and was maintained at 37 f 0.5”C with a water-heated radiator placed under the animal. Carotid body chemoreceptors on the side ipsilateral to the recording site were stimulated by the retrograde injection of 10-30~1 0.15 M NaCl equilibrated with 100% COr* via a catheter (o.d. 0.61 mm, i.d. 0.21 mm) placed in the external carotid artery (after removal of the posterior comu of the hyoid bone) such that the tip was at the level of the bifurcation of the internal and external carotid arteries. The superior and inferior thyroid arteries were ligated but the common carotid, internal carotid and occipital arteries remained patent at all times. Care was taken to ensure that central recording was performed only in those animals in which there was a clear and maintained reflex hyperventilation following stimulation of the carotid bodies. The ventral surface of the hypothalamus was exposed according to the technique of Dreifuss and Ruf.” The medulla was also exposed ventrally following a midline incision and removal of the stemohyoideus and longus colli muscles and trachea/larynx and oesophagus at the level of the thyroid isthmus. The atlanto-basioccipital membrane was removed and pairs of dorsal medullary stimulating electrodes lowered, 0.5 mm lateral to the midline bilaterally and 2.4-2.85 mm depth, into the tractus solitarius nuclei (NTS) using the confluence of the vertebral arteries with the caudal portion of the basilar artery as a reference point. Recording electrodes were inserted by eye under an operating microscope using the rostra1 edge of the median eminence and third ventricle as points of reference. Neuronal activity was recorded extracellulary in the medial hypothalamus using glass micropipettes (8-l 5 MfJ impedance at 1 kHz) filled with 2% (w/v) Pontamine Sky Blue 6BX in 0.5 M sodium acetate for localization of recording sites. Is Neuronal depth was calculated from the microdrive reading and used as an estimate for anatomical location during the experiment. Accurate reconstructions required the use of both the estimated depth reading and the localization of the blue spot either at the end of a track or else marking individual recording sites. All neurons were located by their spontaneous discharge and/or by evoked activity (antidromic or synaptic) following electrical stimulation of the pituitary stalk/median eminence junction or the NTS using concentric bipolar stainless steel electrodes (Rhodes Medical SNE-100, o.d. 0.25 mm, 120-300 kf2 impedance at I Hz) with the inner electrode ground flush with the outer to minimize current spread, and single stimuli applied at a maximum rate of 1 Hz (0.5 ms, 0.06-O. 17 mA). Each neuron located was tested for a response to chemoreeeptor stimulation regardless of whether or not it was influenced by the electrical stimuli. Stimulus-evoked changes in neuronal activity were analysed by either poststimulus or peristimulus histograms. The resting activity of spontaneously discharging neurons was cross-correlated with either respiration or cardiac cycle together with autocorrelation for rhythmicity. Neurons responding to electrical stimulation of the median eminence/ pituitary stalk junction of the NTS were classed as antidromically activated if they conformed to each of three criteria: (1) constant latency to suprathreshold stimuli, (2)
high frequency following (> 100 Hz), and where neurons were spontaneously active, (3) collision of the spontaneous orthodromic potential with an antidromic potential. Neurons that were not spontaneously active could only be tested by the first two criteria. At the end of each experiment the brain was fixed in 4% (W/V) form01 saline and the location of hypothalamic stimulation sites and medullary recording sites was verified histologically on 50-pm coronal sections stained with Neutral Red. The absolute position of each recording site was calculated by reference to Pontamine Blue spots and the readings on the micromanipulator taken during the experiment. The positions were then marked on diagrammatic sections of the rat brain taken from the atlas of Pellegrino et al.rs
WSLJLTS Neurons were sampled from the medial hypothalamus from the midline to 1.Omm lateral and up to 4.5 mm dorsal to the ventral surface which included the entire paraventricular nu&us (PVH) and the rostra1 half of the arcuate nucleus (ARH). The sample region shown in Fig. 1 corresponded to an area 0.2-1.0 mm rostra1 to bregma.28 Every neuron was tested for its response to carotid body chemoreceptor stimulation. The activity was recorded from a total of 292 neurons, of which 157 (53.8%) were localized within PVH, 87 (29.8%) within the ARH and 3 (1 .O%) in the anterior hypothalamic area. An additional 19, 6.5% of the total, were located within the medial hypothalamus but were not associated with welLdefined nuclei. This latter group were either situated rostrally between the optic tract and ARH or else just outside ARH or PVH. Figure 1 shows the recording sites on diagrammatic reconstructions of coronal sections from the atlas of Pellegrino et a1.*r A further 26 neurons were localized in the mediobasal thalamus within the reuniens nucleus, but these were not studied in detail other than for their responses to electrical and chemoreceptor stimuiation. As might be expected, the majority of neurons recorded within ARH and PVH were either synap tically or antidromically activated following median eminence/pituitary stalk stimulation or stimulationof the medulla. Of those neurons outside those nuclei, only one (in the thalamic reuniens nucleus) was activated, and that followed stimulation in the m&&a. The same neuron was also activated foIlowing chemoreceptor stimulation. A detailed breakdown of the various influences of electrical stimulation is given in Table 1. Of 292 neurons tested 18 (6%) were in&enced by chemoreceptor stimulation, and all but two of these were found to be within ARH. The other two were located at the hypothalamic/thalamic border within the thalamic reuniens nucleus (Fig. 1). Within the total of 16 ARH neurons intIuenced, three neurons were antidromically invaded from the medulla and each of these was activated by chemoreceptor stimulation. The mean latency-toonset of antidromic activation from NTS was 41.7 ms
Carotid chemoreceptor
d.
.
input to medial hypothalamus
x5
I
-I---++ x5
II
I 0
1
I 1
I
I 2
1
I 3mm
Fig. 1. Diagrammatic reconstructions of coronal sections from the medial-basal forebrain of the rat to show recording sites of neurons influenced by stimulation of the carotid body chemoreceptors (closed circles) or tested but uninfluenced by carotid body stimulation (open circles). These reconstructions have been made from all the experimental data but for the sake of clarity not all the uninfluenced neurons have been plotted. The majority of neurons excited by chemoreceptor stimulation are located in the arcuate nucleus (ARH), the two exceptions are found in the thalamic reuniens nucleus (RE, +0.4). The numbers to the left of each section correspond to the distances in millimeters rostra1 to bregma and are taken from the atlas of Pellegrino et af.= The major fibre pathways are shown stippled. Top left: Antidromic invasion of an arcuo-medullary projecting neuron. Each trace is an oscilloscope record, the top two showing five superimposed sweeps and the bottom trace a single sweep. The horizontal axis is time in milliseconds. In each case the stimulus artefact is shown at zero time. The antidromic potentials are at the arrows. The top record shows that stimulation of the NTS antidromically activates the neuron with a constant delay of 39 ms. The middle trace shows that the evoked potentials follow two closely placed stimuli separated by 5 ms (200 Hz), again with each stimulus evoking responses with constant delay. The fmal trace shows that when an orthodromic action potential occurs during the normal latency-to-onset of evoked activity the orthodromic action potential collides with the antidromic action potential, and there is no antidromic potential at the arrow.
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Table 1. Distribution of neurons and the effects of electrical stimulation of the pituitary stalk/median eminence junction or within the medulla Type of effect
Mean latency to onset (ms)
A S A
8.9 23.0 35.9 60.3 -
Site of stimulation
n
PMEJ PMEJ MED MED PMEJ/MED PMEJ/MED
27 5 9 6 2 11
As/S No effect
PVH
PMEJ PMEJ MED MED PMEJ/MED PMEJ/MED
52 8 7 9 3 17
A S A S A/S No effect
RE
MED PMEJ/MED
1 17
S No effect
AHA Others
PMEJ/MED PMEJ/MED
3 19
Noeffect No effect
Nucleus ARH
11.1 11.4 30.6 48.3 -
PMEJ, pituitary stalk/median eminence junction; medulla; A, antidromic; S, synaptic.
MED,
(range 34-63 ms) with a conduction velocity of 0.21-0.38 m/s assuming a distance of 13 mm between
the NTS-stimulating electrode and recording site. An example of an arcuomedullary neuron activated by chemoreceptor stimulation is shown in Fig. 1 (inset, top left). This illustrates the three antidromic criteria used for all the neurons. Using these criteria, six of the chemoreceptor-activated ARH neurons were shown to project to the median eminence. The projection of the remaining seven ARH neurons could not be determined since they could not be antidromically invaded from either the median eminence or the medulla. In 15 out of 18 neurons activated by chemoreceptor stimulation the response to the stimulus was an abrupt increase in discharge lasting l-10 S. The majority of the arcuate neurons (13 out of 16,8 1.3%) and both the thalamic reuniens neurons showed this short-lasting activation. Examples of an arcuate neuron (ARH) and thalamic reuniens neuron (RE) influenced in such a manner are shown in Fig. 2. The number of action potentials associated with each stimulus varied considerably regardless of the size of the respiratory response or blood pressure and heart rate responses. It is clear from all the traces that the activation following chemoreceptor stimulation occurs before there is any change in blood pressure or heart rate and that it coincides with the first ventilatory responses to the stimulus. Each trace was analysed in l-s epochs and the neuronal activity was found to remain elevated during the period of peak ventilation dropping rapidly to prestimulus levels of activity (Fig. 4). Despite this powerful activation, autocorrelation tests showed that the resting discharge showed no rhythm linked to inspiration or expir-
ation; indeed this was so for all neurons activated
via
the chemoreceptors.
None of the 157 PVH neurons recorded were affected by the stimulus regardless of whether they projected to the pituitary or to the medulla. An example of a PVH-pituitary neuron with the phasic pattern of discharge characteristic of vasopressinsecreting neurons is shown in Fig. 3. This figure shows that in marked contrast to vasopressinsecreting neurons of the hypothalamic supraoptic nucleus3*‘4 there is no effect of stimulating the ipsilateral carotid body during a period of quiescence, i.e. between bursts of phasic activity. In three out of six neurons shown to be projecting to the median eminence, the chemoreceptor stimulation was followed by prolonged activation of discharge lasting several minutes. This is illustrated in Fig. 4 which shows that not only was activation prolonged, but also that the absolute rate of discharge (up to 50 Hz) was much higher than one normally associates with arcuate neurons (l-2 Hz3*). Further analysis of neuronal activity revealed that in each of these neurons the sustained activity could be directly related to ventilation changes and for one neuron with the heart rate changes (Fig. 4, bottom plots). These neurons showed ventilation-linked modulation of their discharge even with occasional small oscillations in instantaneous respiration rate unconnected with changes in blood pressure or heart rate (Fig. 4). With these neurons the chemoreceptor stimulus was given during the period of lowered respiratory rate which coincided with the point of minimal neuronal activity. During a 20-s period before and 20 s after the stimulus the neurons were analysed statistically for the correlation of neuronal spike rate against changes in respiration rate, heart rate and blood pressure. In none of the three neurons analysed was there any correlation between spike rate and blood pressure. For one neuron only there was a significant correlation between spike rate and heart rate, but there was a strong correlation between neuronal activity and respiration rate for all three neurons. An example of scatter plots of spike rate against respiratory rate and heart rate is also illustrated in Fig. 4 showing a clear relationship between these variables. The Kendall’s rank correlation coefficient, T, was calculated for both the scatter plots shown and in each case the spike rate/respiration rate as well as the spike rate/heart rate correlation were highly significant (P -C0.1%). For all 18 neurons activated by chemoreceptor stimulation the initial activation was synchronized to the start of stimulation and not the consequent changes in blood pressure even during augmented breaths when there was a gasp and transient increase in ventilatory rate. The slow resting discharge rate and the brief activation following chemoreceptor stimulation made detailed statistical analysis impossible for most neurons. However, during the recording period for each neuron (often 30 min or more) the initial pattern
Carotid chemoreceptor
971
input to medial hypothalamus
A)
ARH
BP 120 (mmHg) 0 RR
100
(bpm)
6. [
1111I HR 410 (bpm)390 [
I
I
1 t
t TIME (mind
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BP 120 (mmHg) I O-
Spike rate (Hz)
10 O[
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HR 420 (bpm)qOO [ ,
4 t
t
TIME (mind
Fig. 2. Activation of neurons in the hypothalamic arcuate nucleus (ARH) and thalamic reuniens nucleus (RE) following carotid body chemoreceptor stimulation in anaesthetized rats. (A) Arcuate nucleus (ARH, top traces). The effect of chemoreceptor stimulation (arrows, CO,) on a neuron in the arcuate nucleus showing a transient response to the stimulus. From top to bottom is shown femoral artery blood pressure (BP mmHg), breath-by-breath respiratory rate (RR breaths per min), neuronal activity (spikes) and beat-to-beat rate (HR beats per min). The horizontal axis is time with l-min markers. (B) Thalamic reuniens (RE, bottom traces). Thalamic reuniens neuron activated by chemoreceptor stimulation showing a typical transient response to chemoreceptor stimulation (arrows, CO,). In the bottom panel is shown (from top to bottom) femoral artery blood pressure (BP mm Hg), neuronal activity (spikes), integrated neuronal activity in l-s epochs (spike rate, Hz) and beat-to-beat heart rate (HR beats per min).
of discharge did not vary, and neurons with a shortlasting activation were never seen to change to the prolonged form. DISCUSSION
The results of our study indicate that there is a subclass of arcuate neurons which is powerfully
activated by carotid body chemoreceptor stimulation. Included in this population are neurons projecting to the median eminence with a probable neuroendocrine role, and arcuo-medullary neurons which may have a modulating effect on the dorsal medulla. The activatinn of latter erou~ of m.av explain ____ . ______ __this _____ _-___.a---r -- neurons --- --the observation of Lopez et aL2’ that destruction of this portion of the hypothalamus abolished part of
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4
Fig. 3. Lack of activation of a phasicaily-firing magnocellular neuron in the hypothalamic paraventricular nucleus following stimulation of carotid body chemoreceptors in the anaesthetized rat. (A) Oscilloscope record of five superimposed sweeps showing antidromic response of the neuron with constant latency (mean latency of discharge of 7.0 ms (arrowed)) following electrical stimulation of the pituitary stalk. (B) Record showing the coihsion of a spontaneous orthodromic action potential (starred) with an antidromic action potential when the orthodromic action potential occurs within the normal latency-to-onset of evoked potential. (C) The lack of influence of chernoreceptor stimulation (arrows, CO,) on the phasically active PVH-pituitary neuron shown in A and R. From top to bottom is shown blood pressure (BP mm Hg), beat-to-beat heart rate (HR beats per min). neuronal activity integrated in 1-s epochs (spikes, Hz), clipped action potentials and breath-by-breath respiration rate (RR breaths per min). The horizontal axis is time with I-min markers. At the arrows the ipsilateral carotid body was stimulated between bursts of activity causing a slight rise followed by a prolonged fall in blood pressure, a slight rise in heart rate and a rise in respiration rate from 75 to 130 breaths per min. It is obvious from these results that there is no iduence of the stimulus on the neuron.
RR ‘*’ (bpm)70
(Hz)
0
410 [
sRpd,“s”
tbpm)
FIR 440
a
120
(mmHg)
BP
Cl
II
B)
914
D.
BANKS
and M. C.
nucleus, because of its proximity to the ventral surface of the brain, rather than the actual density of such neurons in the nucleus. Furthermore, although we have limited our investigation to arcuate neurons with median eminence or medullary projections, the possible influence that the arcuate nucleus may have over the forebrain and brainstems is extensive. The intrahypothalamic projections of arcuate neurons in the rat are well established,’ and the nucleus is also known to send axons as far as the septum, thalamus, midbrain and pons-medulla.‘8,29 Exactly which functional type of arcuate neuron is involved in the chemoreceptor response is not known at present since no study to date has correlated patterns of neuronal activity with type of arcuate cell and the projection. The arcuate nucleus contains large numbers of neurons synthesizing neuropeptides (see Palkovits for review2’), however neurons synthesizing adrenocorticotrophic hormone, growth hormone releasing factor, b-endorphin and GI-melanocyte stimulating hormone predominate. Various pharmacological interactions between arcuate peptides and monoamine and optiate systems have also been studied36.37 and there is electrophysiological evidence that ARH stimulation modulates the activity of locus coeruleus neurons via a naloxone reversible mechanism involving /?-endorphin.3s In our study most of the neurons activated by chemoreceptor stimulation projected to the median eminence. These neurons form part of the population of tubero-infundibular neurons located in the hypothalamus and basal forebrain and identified electrophysiologically by antidromic activation following stimulation of the median eminence.” Therefore it is probable that the chemoreceptor input is influencing the release of adenohypophyseal hormones, and if this is so, the length of the increased discharge suggests that hormone release might also be prolonged. There is no indication from our results of the hormones involved in this response and there is no published experimental data on release profiles of adenohypophyseal hormones following specific chemoreceptor stimulation. There is ample evidence, however, that chemoreceptor stimulation using carotid occlusion, hypoxia or following close glomus injection of CO* has a profound action on general endocrine status. For example, the renin-angiotensin system is inhibited in dogs during acute hypoxia*’ and the plasma levels of vasopressin, adrenocorticotrophic hormone and corticosteroids are elevated during hypercapnia and graded hypoxia in dogs.3’ The elevated cortisol response to hypoxia in dogs30 and the release of vasopressin in rats following bilateral carotid occlusion’3 have both been shown to be mainly the result of carotid body stimulation. In the light of the evidence from Raff et ~f.~‘*~’it seems likely that at least part of the neuronal activity seen in these experiments might also be linked to adrenocorticotrophic hormone release. However, the present results show no activation of either magno-
HARRIS
cellular or parvocellular neurons by the chemoreceptors, and the parvocellular PVH neurons are known to be the major source of corticotrophinreleasing factor. I9 Therefore, any adrenocorticotrophic hormone release must be due to corticotrophinreleasing factor from a different site, presumably the arcuate nucleus. So far as we are aware, this study is the first to demonstrate a physiological input to caudallyprojecting arcuate neurons, although the existence of these projections had already been established following the injection of horseradish peroxidase into the dorsal vagal complex and nucleus ambiguus.16 Such injections label cells in the arcuate and paraventricular nuclei but not the ventromedial nucleus. Unlike the paraventricular projection, however, the caudally-projecting arcuate population is rather sparse and although mostly ipsilateral does contain a high percentage of bilateral labelling, suggesting crossover either within the nucleus or on the way down to the dorsal medulla.26”3 In our experiments, no medullary-projecting neurons were seen in the ventromedial margin of the acellular area surrounding the ventromedial hypothalamic nucleus even though these cells have been described by Schwanzel-Fukuda et ~1.~~ The sparse distribution and lateral extent of the population may be a reason for the lack of electrophysiological confnmation.
CONCLUSIONS The results con&m the tindings, obtained with [“‘C12-deoxyghmose,5 that much of the hypothalamus is unaffected by carotid body chemoreceptor stimulation. This negative result is itself of some importance, particularly because of the lack of effect on PVH. Both PVH and the SO contain magnocellular neurosecretory neurons which secrete vasopressin, and have apparently identical eiectrophysiological characteristics. Moreover, both nuclei receive baroreceptor input9+11+‘9and there is evidence for an influence of PVH on descending baroreceptormediated effects.8~MThe lack of any chemoreceptor input to either the magnocellular or the parvocellular neurons of PVH was unexpected therefore, and indicates that this nucleus can have nothing to do with the descending e&cts of this reflex. We believe that this is the first demonstration of a clear division of the two descending influences of baroreceptor and chemoreceptor reflexes in the bypothaiamus. Further studies to investigate the action of descending arcuomeduhary neurons are necessary to elucidate a role for these extra-hypothalamic projections.
authors would like to thank the MRC, the Smith Kline Foundation and the N&&i Foundation for their support, and Mrs J. S. Dean for her
Acknowfe&emem-The
technical assistance.
Carotid chemoreceptor
input to medial hypothalamus
915
REFERENCES
1. Abrahams V. C., Hilton S. M. and Zbroiyna A. W. (1960) Active muscle vasodilatation produced by stimulation in the brain stem. Its significance in the defence reaction. J. Physic/., Land. 154, 491-513. 2. Band D. M., Cameron I. R. and Semple S. J. G. (1970) The effect on respiration of abrupt changes in carotid artery pH and pC0, in the cat. J. Physiol., Land. 211, 479494. 3. Banks D. and Harris M. C. (1984) Lesions of the locus coeruleus abolish baroreceptor-induced depression of supraoptic neurones in the rat. J. Physiol., Land. 355, 383-398. 4. Banks D. and Harris M. C. (1986) An electrophysiological study of arcuate neurones activated by carotid body chemoreceptor stimulation in the anaesthetised rat. J. Physiol., Land. 378, 71P. 5. Banks D., Harris M. C. and Stokes W. N. (1986) Carotid body chemoreceptor stimulation in rats activates large areas of the forebrain but not the hypothalamus. J. Physiol., Land. 371, 114P. 6. Bizzi E., Libretti A., Malliani A. and Zanchetti A. (1961) Reflex chemoreceptive excitation of diencephalic behaviour. Am. J. Physiol. 200, 923-926. 7. Bodoky M. and Rethelyi M. (1977) Dendritic arborization and axon trajectory of neurons in the hypothalamic arcuate nucleus of the rat. Expl Brain Res. 28, 543-555. 8. Ciriello J. and Calaresu F. R. (1980) Role of paraventricular and supraoptic nuclei in central cardiovascular regulation in the cat. Am. J. Physiol. 239, Rl37-R142.
9. Day T., Ferguson A. V. and Renaud L. P. (1984) Facilitatory influence of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells. J. Physiol., L.ond. 355, 237-250. 10. Dreifuss J. J. and Ruf K. B. (1972) A transpharyngeal approach to the rat hypothalamus. In Experiments in Physiology and Biochemistry, Vol. 5 (ed. Kerkut G. A.), pp. 213-228. Academic Press, London. 11. Harris M. C. (1979) Effects of chemoreceptor and baroreceptor stimulation on the discharge of hypothalamic supraoptic neurones in rats. J. Endow. 82, 115-125. 12. Harris M. C., Banks D., Stokes W. N. and Jamieson S. M. (1987) Carotid body chemoreceptors and forebrain activation. In Organisation of the Autonomic Nervous System: Central and Peripheral Mechanisms (eds Polosa C. and Calaresu F. R.). Alan R. Liss, New York. 13. Harris M. C., Dreifuss J. J. and Legros J. J. (1975) Excitation of phasically-firing supraoptic neurones during vasopressin release. Nature 258, 80-82. 14. Harris M. C., Ferguson A. V. and Banks D. (1984) The afferent pathway for carotid body chemoreceptor input to the hypothalamic supraoptic nucleus in the rat. Pfltigers Arch. ges. Physiol. 400, 80-87. 15. Hellon R. F. (1971) The marking of electrode tip position in nervous tissue. J. Physiol., Land. 214, 12P. 16. Horst G. J., Luiten P. G. M. and Kuipers F. (1984) Descending pathways from hypothalamus to dorsal motor vagus and ambiguus nuclei in the rat. J. auton. nerv. Syst. 11, 59-75. 17. Hilton S. M. and Zbroiyna A. W. (1963) Amygdaloid region for defence reactions and its efferent pathways to the brainstem. J. Physiol., Lond. 165, 160-173. 18. Joseph S. A. (1980) Immunoreactive adrenocorticotrophin in rat brain: a neuroanatomical study using antiserum generated against synthetic ACTH’-39. Am. J. Anal. 15s, 533-548. 19. Kannan H. and Yagi K. (1978) Supraoptic neurosecretory neurones: evidence for the existence of converging inputs from both carotid baroreceptors and osmoreceptors. Brain Req. 14, 385390. 20. Kannan H. and Yamashita H. (1983) Electrophysiological study of paraventricular nucleus neurons projecting to the dorsomedial medulla and their response to baroreceptor stimulation in rats. Brain Res. 279, 31-40. 21. Liang C. S. and Gavras H. (1978) Renin-angiotensin system inhibition in conscious dogs during acute hypoxia. J. clin. Invest. 62, 961-970. 22. Lipski J., McAllen R. M. and Trzebski A. (1976) Carotid baroreceptor and chemoreceptor inputs onto single medullary neurones. Brain Res. 107, 132-136. 23. Lopez 0. U., Cipola-Neto J. and Rocha e Silva M. (1977) Hypothalamic component in pressor response to carotid occlusion in the rat. Am. J. Physiol. 233, 240-247. 24. Makara G. B., Harris M.C. and Spyer K. M. (1972) Identification and distribution of tubero-infundibular neurones. Brain Res. 40, 283-290. 25. Marshall J. M. (1981) Interaction between the responses to stimulation of peripheral chemoreceptors and baroreceptors: the importance of chemoreceptor activation of the defence areas. J. auton. nerv. Sysr. 3, 389-w.
26. Morrell J. I., Schwanzel-Fukuda M., Fahrback S. E. and Pfaff D. W. (1984) Axonal projections and peptide content of steroid hormone concentrating neurons. Peptides 5, Suppl. 1, 227-239. 27. Palkovits M. (1984) Distribution of neuropeptides in the central nervous system: a review of biochemical mapping studies. Prog. Neurobiol. 23, 151-189. 28. Pellegrino L. J., Pellegrino A. S. and Cushman A. J. (1979) A Stereotaxic Atlas of the Rat Brain, 2nd edn. Plenum Press, New York. 29. Pelletier G., Leclerc R., Saavedra J. M., Brownstein M. J., Vaudry H., Ferland L. and Labrie F. (1980) Distribution of p-lipotropin, adrenocorticotropin and melanocyte-stimulating hormone in the rat brain. I. Origin of melanocytestimulating hormone in the rat brain. II. Origin of the extrahypothalamic fibers. Brain Res. 192, 43%440. 30. Raff H., Tzankoff S. P. and Fitzgerald R. S. (1982) Chemoreceptor involvement in cortisol responses to hypoxia in ventilated dogs. J. appl. Physiol. 52, 1092-1096. 31. Raff H., Shinsako L., Keil C. and Calhnan M. F. (1983) Vasopressin, ACTH and corticosteroid during hypercapnia and graded hypoxia in dogs. Am. J. Physiol. 244, E453-458. 32. Sanghera M. K., Harris M. C. and Morgan R. M. (1978) Effects of microiontophoretic and intravenous application of gonadotrophic hormones on the discharge of medial-basal hypothalamic neurones in rats. Brain Res. 140, 63-74. 33. Schwanzel-Fukuda M., Morrell J. I. and Pfaff D. W. (1984) Localisation of forebrain neurones which project directly to the medulla and spinal cord of the rat by retrograde tracing with wheat germ agglutinin. J. camp. Neurol. 226, l-20. 34. Share L. and Levy M. N. (1966) Effect of carotid chemoreceptor Am. J. Physiol. 210, 157-161.
stimulation on plasma antidiuretic hormone titre.
35. Strahlendorf H. F., Strahlendorf J. C. and Barnes C. D. (1981) Stimulation of hypothalamic arcuate inhibits
locus
916
D. BANKSand M. C. HARRIS
coeruleus unit activity: evidence for endorphin mediation. In The Role of Peprides and Amino Acids as Neurotransmitters. A.R. Liss, New York. 36. Versteeg D. H. G. (1980) Interactions on peptides related to ACTH, MSH and jI-LPH with neurotransmitters in the brain. Pharmac. Ther. 11, 535-557. 37. Watson S. J., Richard C. W., Ciaranello R. D. and Barchas J. D. (1980) Interaction of opiate peptide and noradrenalin systems: light microscopic studies. Peprides 1, 2330. (Accepted
30 July 1987)
Neuroscience Vol. 24, No. 3, pp. 967-976, 1988
Printedin Great Britain
0 1988 IBRO
ACTIVATION OF HYPOTHALAMIC ARCUATE BUT NOT PARAVENTRICULAR NEURONS FOLLOWING CAROTID BODY CHEMORECEPTOR STIMULATION IN THE RAT D. BANKS* and M. C. HARRIS? Department of Physiology, The Medical School, Vincent Drive, Birmingham Bl5 2TJ, U.K. Ah&act--The effect of carotid body chemoreceptor stimulation on 292 neurons in midline hypothalamic nuclei has been examined electrophysiologically in ethyl carbamate/sodium pentobarbitone anaesthetized rats. Experiments demonstrated that specific stimulation of carotid body chemoreceptors activates a small group (16) of neurons in the mediobasal hypothalamic arcuate nucleus, but has no effect on neurons (157) in the hypothalamic paraventricular nucleus or the anterior hypothalamus. Of 16 arcuate neurons activated by the stimulus, six projected to the median eminence and three projected to the dorsal medulla, as defined by antidromic invasion. Three of the neurons activated from the carotid body also showed a resting discharge that was linked with ventilation rate, suggesting that the arcuate nucleus may have some involvement with respiratory processing. The activation of neurons projecting to the median eminence implies that the release of adenohypophyseal hormones may also be influenced by carotid body chemoreceptors.
Stimulation of carotid body chemoreceptors is usually followed by hyperventilation accompanied by variable changes in heart rate and blood pressure.*’ Moreover, it is well known that the dorsal medulla oblongata and pons are intimately involved in these changes and that this is reflected in the neuronal act&ity in these areas. 22 Much less is known of the influence of this stimulus on neurons in the forebrain. However, in both cats and rats it has been shown that carotid body chemoreceptor stimulation can evoke all the autonomic components of the defence reaction,6*2sand since, in cats, the same components are evoked by electrical stimulation within the amygdala and the hypothalamus, ‘*I7by implication it may be assumed that chemoreceptor stimulation will activate at least these regions. Share and Levy” also demonstrated that carotid chemoreceptor stimulation releases vasopressin, and the neuroendocrine neurons of the hypothalamic supraoptic nuclei (SO) that release vasopressin are known to be activated by the same stimulus.” Following this finding Harris et ~1.‘~ reported that chemoreceptor-induced activation of SO neurons was via an ipsilateral pathway only and
hypothalamic arcuate nucleus; CI, internal capsule; FX, fomix; LHA, lateral hypothalamic area; MFB, medial
that the activation was abolished by lesions which destroyed the preoptic area rostra1 and medial to SO, implying that the input had passed through rostra1 forebrain regions before descending to the medial hypothalamus. This, to some extent at least, agreed with an earlier report that destruction of the same region abolished part of the cardiovascular response to carotid occlusion in rats.23 Although the vasopressin neurons of the supraoptic nuclei are known to be activated by chemoreceptor stimulation, there has been no electrophysiological investigation of the influence of this stimulus on medial hypothalamic neurons. However, the extent of the carotid body chemoreceptor influence on metabolic activity within the forebrain was examined in a recent investigation using [L4C]2-deoxyglucose autoradiography in anaesthetized rats.5 This study showed that chemoreceptor stimulation resulted in widespread metabolic activation in the forebrain, mainly on the side ipsilateral to the stimulated carotid body. With the exception of the ipsilateral SO and the median eminence/arcuate region, however, the hypothalamus was not affected. This finding, which appeared to contradict much of the evidence from earlier studies mentioned above, led us to investigate the problem using electrophysiological methods, to see if, in the medial hypothalamus, activation by carotid body stimulation was confined to the median eminence/arcuate region. Part of this study has been presented as short communications.4J2
forebrain bundle; NT?$ tractus solitarius nuclei; OT optic tract; PVH, hypothalamic paraventricular nucleus; RE, thalamic reuniens nucleus; SM, stria medullaris thalami; SO, supraoptic nucleus; ST, stria terminalis; V, 3rd ventrical; VMH, hypothalamic ventromedial nucleus; ZI, zona inserta.
The experiments were performed on male SpragueDawley rats (300-52Og body wt) anaesthetized with an ethyl carbamate/sodium pentobarbitone mixture (Urethan
*Present address: Department of Physiology, The Medical School, University Walk, Bristol, BS8 ITD. tTo whom correspondence should be addressed. Abbreuiafions: AHA, anterior hypothalamic area; ARH,
967
EXPERIMENTAL PROCEDURES
968
D. BANKS and M. C. HARRIS
1.2-I .4 g/kg, Sagatal20 mg/kg, i.p.). Catheters were inserted into a femoral artery for the measurement of arterial blood pressure, and a femoral vein for injecting additional doses of anaesthetic (Sagatal 12 mg/ml saline). A cannula was placed into the trachea and attached to a “T” piece containing a glass-coated bead thermistor (GC32J1, Fenwal Electronics or U23UD, STC Electronic Services, U.K.) for recording respiration, and electrodes attached across the chest for recording ECG and heart rate. The respiration and ECG signals were passed through analogue discriminators and linear rate-meters for measurement of instantaneous respiration and heart rate. Body temperature was measured rectally using either a mercury-in-glass thermometer or thermoprobe (Gallenkamp temp stick, THV-150-G) and was maintained at 37 f 0.5”C with a water-heated radiator placed under the animal. Carotid body chemoreceptors on the side ipsilateral to the recording site were stimulated by the retrograde injection of 10-30~1 0.15 M NaCl equilibrated with 100% COr* via a catheter (o.d. 0.61 mm, i.d. 0.21 mm) placed in the external carotid artery (after removal of the posterior comu of the hyoid bone) such that the tip was at the level of the bifurcation of the internal and external carotid arteries. The superior and inferior thyroid arteries were ligated but the common carotid, internal carotid and occipital arteries remained patent at all times. Care was taken to ensure that central recording was performed only in those animals in which there was a clear and maintained reflex hyperventilation following stimulation of the carotid bodies. The ventral surface of the hypothalamus was exposed according to the technique of Dreifuss and Ruf.” The medulla was also exposed ventrally following a midline incision and removal of the stemohyoideus and longus colli muscles and trachea/larynx and oesophagus at the level of the thyroid isthmus. The atlanto-basioccipital membrane was removed and pairs of dorsal medullary stimulating electrodes lowered, 0.5 mm lateral to the midline bilaterally and 2.4-2.85 mm depth, into the tractus solitarius nuclei (NTS) using the confluence of the vertebral arteries with the caudal portion of the basilar artery as a reference point. Recording electrodes were inserted by eye under an operating microscope using the rostra1 edge of the median eminence and third ventricle as points of reference. Neuronal activity was recorded extracellulary in the medial hypothalamus using glass micropipettes (8-l 5 MfJ impedance at 1 kHz) filled with 2% (w/v) Pontamine Sky Blue 6BX in 0.5 M sodium acetate for localization of recording sites. Is Neuronal depth was calculated from the microdrive reading and used as an estimate for anatomical location during the experiment. Accurate reconstructions required the use of both the estimated depth reading and the localization of the blue spot either at the end of a track or else marking individual recording sites. All neurons were located by their spontaneous discharge and/or by evoked activity (antidromic or synaptic) following electrical stimulation of the pituitary stalk/median eminence junction or the NTS using concentric bipolar stainless steel electrodes (Rhodes Medical SNE-100, o.d. 0.25 mm, 120-300 kf2 impedance at I Hz) with the inner electrode ground flush with the outer to minimize current spread, and single stimuli applied at a maximum rate of 1 Hz (0.5 ms, 0.06-O. 17 mA). Each neuron located was tested for a response to chemoreeeptor stimulation regardless of whether or not it was influenced by the electrical stimuli. Stimulus-evoked changes in neuronal activity were analysed by either poststimulus or peristimulus histograms. The resting activity of spontaneously discharging neurons was cross-correlated with either respiration or cardiac cycle together with autocorrelation for rhythmicity. Neurons responding to electrical stimulation of the median eminence/ pituitary stalk junction of the NTS were classed as antidromically activated if they conformed to each of three criteria: (1) constant latency to suprathreshold stimuli, (2)
high frequency following (> 100 Hz), and where neurons were spontaneously active, (3) collision of the spontaneous orthodromic potential with an antidromic potential. Neurons that were not spontaneously active could only be tested by the first two criteria. At the end of each experiment the brain was fixed in 4% (W/V) form01 saline and the location of hypothalamic stimulation sites and medullary recording sites was verified histologically on 50-pm coronal sections stained with Neutral Red. The absolute position of each recording site was calculated by reference to Pontamine Blue spots and the readings on the micromanipulator taken during the experiment. The positions were then marked on diagrammatic sections of the rat brain taken from the atlas of Pellegrino et al.rs
WSLJLTS Neurons were sampled from the medial hypothalamus from the midline to 1.Omm lateral and up to 4.5 mm dorsal to the ventral surface which included the entire paraventricular nu&us (PVH) and the rostra1 half of the arcuate nucleus (ARH). The sample region shown in Fig. 1 corresponded to an area 0.2-1.0 mm rostra1 to bregma.28 Every neuron was tested for its response to carotid body chemoreceptor stimulation. The activity was recorded from a total of 292 neurons, of which 157 (53.8%) were localized within PVH, 87 (29.8%) within the ARH and 3 (1 .O%) in the anterior hypothalamic area. An additional 19, 6.5% of the total, were located within the medial hypothalamus but were not associated with welLdefined nuclei. This latter group were either situated rostrally between the optic tract and ARH or else just outside ARH or PVH. Figure 1 shows the recording sites on diagrammatic reconstructions of coronal sections from the atlas of Pellegrino et a1.*r A further 26 neurons were localized in the mediobasal thalamus within the reuniens nucleus, but these were not studied in detail other than for their responses to electrical and chemoreceptor stimuiation. As might be expected, the majority of neurons recorded within ARH and PVH were either synap tically or antidromically activated following median eminence/pituitary stalk stimulation or stimulationof the medulla. Of those neurons outside those nuclei, only one (in the thalamic reuniens nucleus) was activated, and that followed stimulation in the m&&a. The same neuron was also activated foIlowing chemoreceptor stimulation. A detailed breakdown of the various influences of electrical stimulation is given in Table 1. Of 292 neurons tested 18 (6%) were in&enced by chemoreceptor stimulation, and all but two of these were found to be within ARH. The other two were located at the hypothalamic/thalamic border within the thalamic reuniens nucleus (Fig. 1). Within the total of 16 ARH neurons intIuenced, three neurons were antidromically invaded from the medulla and each of these was activated by chemoreceptor stimulation. The mean latency-toonset of antidromic activation from NTS was 41.7 ms
Carotid chemoreceptor
d.
.
input to medial hypothalamus
x5
I
-I---++ x5
II
I 0
1
I 1
I
I 2
1
I 3mm
Fig. 1. Diagrammatic reconstructions of coronal sections from the medial-basal forebrain of the rat to show recording sites of neurons influenced by stimulation of the carotid body chemoreceptors (closed circles) or tested but uninfluenced by carotid body stimulation (open circles). These reconstructions have been made from all the experimental data but for the sake of clarity not all the uninfluenced neurons have been plotted. The majority of neurons excited by chemoreceptor stimulation are located in the arcuate nucleus (ARH), the two exceptions are found in the thalamic reuniens nucleus (RE, +0.4). The numbers to the left of each section correspond to the distances in millimeters rostra1 to bregma and are taken from the atlas of Pellegrino et af.= The major fibre pathways are shown stippled. Top left: Antidromic invasion of an arcuo-medullary projecting neuron. Each trace is an oscilloscope record, the top two showing five superimposed sweeps and the bottom trace a single sweep. The horizontal axis is time in milliseconds. In each case the stimulus artefact is shown at zero time. The antidromic potentials are at the arrows. The top record shows that stimulation of the NTS antidromically activates the neuron with a constant delay of 39 ms. The middle trace shows that the evoked potentials follow two closely placed stimuli separated by 5 ms (200 Hz), again with each stimulus evoking responses with constant delay. The fmal trace shows that when an orthodromic action potential occurs during the normal latency-to-onset of evoked activity the orthodromic action potential collides with the antidromic action potential, and there is no antidromic potential at the arrow.
969
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Table 1. Distribution of neurons and the effects of electrical stimulation of the pituitary stalk/median eminence junction or within the medulla Type of effect
Mean latency to onset (ms)
A S A
8.9 23.0 35.9 60.3 -
Site of stimulation
n
PMEJ PMEJ MED MED PMEJ/MED PMEJ/MED
27 5 9 6 2 11
As/S No effect
PVH
PMEJ PMEJ MED MED PMEJ/MED PMEJ/MED
52 8 7 9 3 17
A S A S A/S No effect
RE
MED PMEJ/MED
1 17
S No effect
AHA Others
PMEJ/MED PMEJ/MED
3 19
Noeffect No effect
Nucleus ARH
11.1 11.4 30.6 48.3 -
PMEJ, pituitary stalk/median eminence junction; medulla; A, antidromic; S, synaptic.
MED,
(range 34-63 ms) with a conduction velocity of 0.21-0.38 m/s assuming a distance of 13 mm between
the NTS-stimulating electrode and recording site. An example of an arcuomedullary neuron activated by chemoreceptor stimulation is shown in Fig. 1 (inset, top left). This illustrates the three antidromic criteria used for all the neurons. Using these criteria, six of the chemoreceptor-activated ARH neurons were shown to project to the median eminence. The projection of the remaining seven ARH neurons could not be determined since they could not be antidromically invaded from either the median eminence or the medulla. In 15 out of 18 neurons activated by chemoreceptor stimulation the response to the stimulus was an abrupt increase in discharge lasting l-10 S. The majority of the arcuate neurons (13 out of 16,8 1.3%) and both the thalamic reuniens neurons showed this short-lasting activation. Examples of an arcuate neuron (ARH) and thalamic reuniens neuron (RE) influenced in such a manner are shown in Fig. 2. The number of action potentials associated with each stimulus varied considerably regardless of the size of the respiratory response or blood pressure and heart rate responses. It is clear from all the traces that the activation following chemoreceptor stimulation occurs before there is any change in blood pressure or heart rate and that it coincides with the first ventilatory responses to the stimulus. Each trace was analysed in l-s epochs and the neuronal activity was found to remain elevated during the period of peak ventilation dropping rapidly to prestimulus levels of activity (Fig. 4). Despite this powerful activation, autocorrelation tests showed that the resting discharge showed no rhythm linked to inspiration or expir-
ation; indeed this was so for all neurons activated
via
the chemoreceptors.
None of the 157 PVH neurons recorded were affected by the stimulus regardless of whether they projected to the pituitary or to the medulla. An example of a PVH-pituitary neuron with the phasic pattern of discharge characteristic of vasopressinsecreting neurons is shown in Fig. 3. This figure shows that in marked contrast to vasopressinsecreting neurons of the hypothalamic supraoptic nucleus3*‘4 there is no effect of stimulating the ipsilateral carotid body during a period of quiescence, i.e. between bursts of phasic activity. In three out of six neurons shown to be projecting to the median eminence, the chemoreceptor stimulation was followed by prolonged activation of discharge lasting several minutes. This is illustrated in Fig. 4 which shows that not only was activation prolonged, but also that the absolute rate of discharge (up to 50 Hz) was much higher than one normally associates with arcuate neurons (l-2 Hz3*). Further analysis of neuronal activity revealed that in each of these neurons the sustained activity could be directly related to ventilation changes and for one neuron with the heart rate changes (Fig. 4, bottom plots). These neurons showed ventilation-linked modulation of their discharge even with occasional small oscillations in instantaneous respiration rate unconnected with changes in blood pressure or heart rate (Fig. 4). With these neurons the chemoreceptor stimulus was given during the period of lowered respiratory rate which coincided with the point of minimal neuronal activity. During a 20-s period before and 20 s after the stimulus the neurons were analysed statistically for the correlation of neuronal spike rate against changes in respiration rate, heart rate and blood pressure. In none of the three neurons analysed was there any correlation between spike rate and blood pressure. For one neuron only there was a significant correlation between spike rate and heart rate, but there was a strong correlation between neuronal activity and respiration rate for all three neurons. An example of scatter plots of spike rate against respiratory rate and heart rate is also illustrated in Fig. 4 showing a clear relationship between these variables. The Kendall’s rank correlation coefficient, T, was calculated for both the scatter plots shown and in each case the spike rate/respiration rate as well as the spike rate/heart rate correlation were highly significant (P -C0.1%). For all 18 neurons activated by chemoreceptor stimulation the initial activation was synchronized to the start of stimulation and not the consequent changes in blood pressure even during augmented breaths when there was a gasp and transient increase in ventilatory rate. The slow resting discharge rate and the brief activation following chemoreceptor stimulation made detailed statistical analysis impossible for most neurons. However, during the recording period for each neuron (often 30 min or more) the initial pattern
Carotid chemoreceptor
971
input to medial hypothalamus
A)
ARH
BP 120 (mmHg) 0 RR
100
(bpm)
6. [
1111I HR 410 (bpm)390 [
I
I
1 t
t TIME (mind
co (32
2
BP 120 (mmHg) I O-
Spike rate (Hz)
10 O[
-
HR 420 (bpm)qOO [ ,
4 t
t
TIME (mind
Fig. 2. Activation of neurons in the hypothalamic arcuate nucleus (ARH) and thalamic reuniens nucleus (RE) following carotid body chemoreceptor stimulation in anaesthetized rats. (A) Arcuate nucleus (ARH, top traces). The effect of chemoreceptor stimulation (arrows, CO,) on a neuron in the arcuate nucleus showing a transient response to the stimulus. From top to bottom is shown femoral artery blood pressure (BP mmHg), breath-by-breath respiratory rate (RR breaths per min), neuronal activity (spikes) and beat-to-beat rate (HR beats per min). The horizontal axis is time with l-min markers. (B) Thalamic reuniens (RE, bottom traces). Thalamic reuniens neuron activated by chemoreceptor stimulation showing a typical transient response to chemoreceptor stimulation (arrows, CO,). In the bottom panel is shown (from top to bottom) femoral artery blood pressure (BP mm Hg), neuronal activity (spikes), integrated neuronal activity in l-s epochs (spike rate, Hz) and beat-to-beat heart rate (HR beats per min).
of discharge did not vary, and neurons with a shortlasting activation were never seen to change to the prolonged form. DISCUSSION
The results of our study indicate that there is a subclass of arcuate neurons which is powerfully
activated by carotid body chemoreceptor stimulation. Included in this population are neurons projecting to the median eminence with a probable neuroendocrine role, and arcuo-medullary neurons which may have a modulating effect on the dorsal medulla. The activatinn of latter erou~ of m.av explain ____ . ______ __this _____ _-___.a---r -- neurons --- --the observation of Lopez et aL2’ that destruction of this portion of the hypothalamus abolished part of
[
c
4 10 ms
-
I
4 c02
co2
TIME (mid
----rL_l_
I
I
I I
4
I
I
I
I
I
114
4
Fig. 3. Lack of activation of a phasicaily-firing magnocellular neuron in the hypothalamic paraventricular nucleus following stimulation of carotid body chemoreceptors in the anaesthetized rat. (A) Oscilloscope record of five superimposed sweeps showing antidromic response of the neuron with constant latency (mean latency of discharge of 7.0 ms (arrowed)) following electrical stimulation of the pituitary stalk. (B) Record showing the coihsion of a spontaneous orthodromic action potential (starred) with an antidromic action potential when the orthodromic action potential occurs within the normal latency-to-onset of evoked potential. (C) The lack of influence of chernoreceptor stimulation (arrows, CO,) on the phasically active PVH-pituitary neuron shown in A and R. From top to bottom is shown blood pressure (BP mm Hg), beat-to-beat heart rate (HR beats per min). neuronal activity integrated in 1-s epochs (spikes, Hz), clipped action potentials and breath-by-breath respiration rate (RR breaths per min). The horizontal axis is time with I-min markers. At the arrows the ipsilateral carotid body was stimulated between bursts of activity causing a slight rise followed by a prolonged fall in blood pressure, a slight rise in heart rate and a rise in respiration rate from 75 to 130 breaths per min. It is obvious from these results that there is no iduence of the stimulus on the neuron.
RR ‘*’ (bpm)70
(Hz)
0
410 [
sRpd,“s”
tbpm)
FIR 440
a
120
(mmHg)
BP
Cl
II
B)
914
D.
BANKS
and M. C.
nucleus, because of its proximity to the ventral surface of the brain, rather than the actual density of such neurons in the nucleus. Furthermore, although we have limited our investigation to arcuate neurons with median eminence or medullary projections, the possible influence that the arcuate nucleus may have over the forebrain and brainstems is extensive. The intrahypothalamic projections of arcuate neurons in the rat are well established,’ and the nucleus is also known to send axons as far as the septum, thalamus, midbrain and pons-medulla.‘8,29 Exactly which functional type of arcuate neuron is involved in the chemoreceptor response is not known at present since no study to date has correlated patterns of neuronal activity with type of arcuate cell and the projection. The arcuate nucleus contains large numbers of neurons synthesizing neuropeptides (see Palkovits for review2’), however neurons synthesizing adrenocorticotrophic hormone, growth hormone releasing factor, b-endorphin and GI-melanocyte stimulating hormone predominate. Various pharmacological interactions between arcuate peptides and monoamine and optiate systems have also been studied36.37 and there is electrophysiological evidence that ARH stimulation modulates the activity of locus coeruleus neurons via a naloxone reversible mechanism involving /?-endorphin.3s In our study most of the neurons activated by chemoreceptor stimulation projected to the median eminence. These neurons form part of the population of tubero-infundibular neurons located in the hypothalamus and basal forebrain and identified electrophysiologically by antidromic activation following stimulation of the median eminence.” Therefore it is probable that the chemoreceptor input is influencing the release of adenohypophyseal hormones, and if this is so, the length of the increased discharge suggests that hormone release might also be prolonged. There is no indication from our results of the hormones involved in this response and there is no published experimental data on release profiles of adenohypophyseal hormones following specific chemoreceptor stimulation. There is ample evidence, however, that chemoreceptor stimulation using carotid occlusion, hypoxia or following close glomus injection of CO* has a profound action on general endocrine status. For example, the renin-angiotensin system is inhibited in dogs during acute hypoxia*’ and the plasma levels of vasopressin, adrenocorticotrophic hormone and corticosteroids are elevated during hypercapnia and graded hypoxia in dogs.3’ The elevated cortisol response to hypoxia in dogs30 and the release of vasopressin in rats following bilateral carotid occlusion’3 have both been shown to be mainly the result of carotid body stimulation. In the light of the evidence from Raff et ~f.~‘*~’it seems likely that at least part of the neuronal activity seen in these experiments might also be linked to adrenocorticotrophic hormone release. However, the present results show no activation of either magno-
HARRIS
cellular or parvocellular neurons by the chemoreceptors, and the parvocellular PVH neurons are known to be the major source of corticotrophinreleasing factor. I9 Therefore, any adrenocorticotrophic hormone release must be due to corticotrophinreleasing factor from a different site, presumably the arcuate nucleus. So far as we are aware, this study is the first to demonstrate a physiological input to caudallyprojecting arcuate neurons, although the existence of these projections had already been established following the injection of horseradish peroxidase into the dorsal vagal complex and nucleus ambiguus.16 Such injections label cells in the arcuate and paraventricular nuclei but not the ventromedial nucleus. Unlike the paraventricular projection, however, the caudally-projecting arcuate population is rather sparse and although mostly ipsilateral does contain a high percentage of bilateral labelling, suggesting crossover either within the nucleus or on the way down to the dorsal medulla.26”3 In our experiments, no medullary-projecting neurons were seen in the ventromedial margin of the acellular area surrounding the ventromedial hypothalamic nucleus even though these cells have been described by Schwanzel-Fukuda et ~1.~~ The sparse distribution and lateral extent of the population may be a reason for the lack of electrophysiological confnmation.
CONCLUSIONS The results con&m the tindings, obtained with [“‘C12-deoxyghmose,5 that much of the hypothalamus is unaffected by carotid body chemoreceptor stimulation. This negative result is itself of some importance, particularly because of the lack of effect on PVH. Both PVH and the SO contain magnocellular neurosecretory neurons which secrete vasopressin, and have apparently identical eiectrophysiological characteristics. Moreover, both nuclei receive baroreceptor input9+11+‘9and there is evidence for an influence of PVH on descending baroreceptormediated effects.8~MThe lack of any chemoreceptor input to either the magnocellular or the parvocellular neurons of PVH was unexpected therefore, and indicates that this nucleus can have nothing to do with the descending e&cts of this reflex. We believe that this is the first demonstration of a clear division of the two descending influences of baroreceptor and chemoreceptor reflexes in the bypothaiamus. Further studies to investigate the action of descending arcuomeduhary neurons are necessary to elucidate a role for these extra-hypothalamic projections.
authors would like to thank the MRC, the Smith Kline Foundation and the N&&i Foundation for their support, and Mrs J. S. Dean for her
Acknowfe&emem-The
technical assistance.
Carotid chemoreceptor
input to medial hypothalamus
915
REFERENCES
1. Abrahams V. C., Hilton S. M. and Zbroiyna A. W. (1960) Active muscle vasodilatation produced by stimulation in the brain stem. Its significance in the defence reaction. J. Physic/., Land. 154, 491-513. 2. Band D. M., Cameron I. R. and Semple S. J. G. (1970) The effect on respiration of abrupt changes in carotid artery pH and pC0, in the cat. J. Physiol., Land. 211, 479494. 3. Banks D. and Harris M. C. (1984) Lesions of the locus coeruleus abolish baroreceptor-induced depression of supraoptic neurones in the rat. J. Physiol., Land. 355, 383-398. 4. Banks D. and Harris M. C. (1986) An electrophysiological study of arcuate neurones activated by carotid body chemoreceptor stimulation in the anaesthetised rat. J. Physiol., Land. 378, 71P. 5. Banks D., Harris M. C. and Stokes W. N. (1986) Carotid body chemoreceptor stimulation in rats activates large areas of the forebrain but not the hypothalamus. J. Physiol., Land. 371, 114P. 6. Bizzi E., Libretti A., Malliani A. and Zanchetti A. (1961) Reflex chemoreceptive excitation of diencephalic behaviour. Am. J. Physiol. 200, 923-926. 7. Bodoky M. and Rethelyi M. (1977) Dendritic arborization and axon trajectory of neurons in the hypothalamic arcuate nucleus of the rat. Expl Brain Res. 28, 543-555. 8. Ciriello J. and Calaresu F. R. (1980) Role of paraventricular and supraoptic nuclei in central cardiovascular regulation in the cat. Am. J. Physiol. 239, Rl37-R142.
9. Day T., Ferguson A. V. and Renaud L. P. (1984) Facilitatory influence of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells. J. Physiol., L.ond. 355, 237-250. 10. Dreifuss J. J. and Ruf K. B. (1972) A transpharyngeal approach to the rat hypothalamus. In Experiments in Physiology and Biochemistry, Vol. 5 (ed. Kerkut G. A.), pp. 213-228. Academic Press, London. 11. Harris M. C. (1979) Effects of chemoreceptor and baroreceptor stimulation on the discharge of hypothalamic supraoptic neurones in rats. J. Endow. 82, 115-125. 12. Harris M. C., Banks D., Stokes W. N. and Jamieson S. M. (1987) Carotid body chemoreceptors and forebrain activation. In Organisation of the Autonomic Nervous System: Central and Peripheral Mechanisms (eds Polosa C. and Calaresu F. R.). Alan R. Liss, New York. 13. Harris M. C., Dreifuss J. J. and Legros J. J. (1975) Excitation of phasically-firing supraoptic neurones during vasopressin release. Nature 258, 80-82. 14. Harris M. C., Ferguson A. V. and Banks D. (1984) The afferent pathway for carotid body chemoreceptor input to the hypothalamic supraoptic nucleus in the rat. Pfltigers Arch. ges. Physiol. 400, 80-87. 15. Hellon R. F. (1971) The marking of electrode tip position in nervous tissue. J. Physiol., Land. 214, 12P. 16. Horst G. J., Luiten P. G. M. and Kuipers F. (1984) Descending pathways from hypothalamus to dorsal motor vagus and ambiguus nuclei in the rat. J. auton. nerv. Syst. 11, 59-75. 17. Hilton S. M. and Zbroiyna A. W. (1963) Amygdaloid region for defence reactions and its efferent pathways to the brainstem. J. Physiol., Lond. 165, 160-173. 18. Joseph S. A. (1980) Immunoreactive adrenocorticotrophin in rat brain: a neuroanatomical study using antiserum generated against synthetic ACTH’-39. Am. J. Anal. 15s, 533-548. 19. Kannan H. and Yagi K. (1978) Supraoptic neurosecretory neurones: evidence for the existence of converging inputs from both carotid baroreceptors and osmoreceptors. Brain Req. 14, 385390. 20. Kannan H. and Yamashita H. (1983) Electrophysiological study of paraventricular nucleus neurons projecting to the dorsomedial medulla and their response to baroreceptor stimulation in rats. Brain Res. 279, 31-40. 21. Liang C. S. and Gavras H. (1978) Renin-angiotensin system inhibition in conscious dogs during acute hypoxia. J. clin. Invest. 62, 961-970. 22. Lipski J., McAllen R. M. and Trzebski A. (1976) Carotid baroreceptor and chemoreceptor inputs onto single medullary neurones. Brain Res. 107, 132-136. 23. Lopez 0. U., Cipola-Neto J. and Rocha e Silva M. (1977) Hypothalamic component in pressor response to carotid occlusion in the rat. Am. J. Physiol. 233, 240-247. 24. Makara G. B., Harris M.C. and Spyer K. M. (1972) Identification and distribution of tubero-infundibular neurones. Brain Res. 40, 283-290. 25. Marshall J. M. (1981) Interaction between the responses to stimulation of peripheral chemoreceptors and baroreceptors: the importance of chemoreceptor activation of the defence areas. J. auton. nerv. Sysr. 3, 389-w.
26. Morrell J. I., Schwanzel-Fukuda M., Fahrback S. E. and Pfaff D. W. (1984) Axonal projections and peptide content of steroid hormone concentrating neurons. Peptides 5, Suppl. 1, 227-239. 27. Palkovits M. (1984) Distribution of neuropeptides in the central nervous system: a review of biochemical mapping studies. Prog. Neurobiol. 23, 151-189. 28. Pellegrino L. J., Pellegrino A. S. and Cushman A. J. (1979) A Stereotaxic Atlas of the Rat Brain, 2nd edn. Plenum Press, New York. 29. Pelletier G., Leclerc R., Saavedra J. M., Brownstein M. J., Vaudry H., Ferland L. and Labrie F. (1980) Distribution of p-lipotropin, adrenocorticotropin and melanocyte-stimulating hormone in the rat brain. I. Origin of melanocytestimulating hormone in the rat brain. II. Origin of the extrahypothalamic fibers. Brain Res. 192, 43%440. 30. Raff H., Tzankoff S. P. and Fitzgerald R. S. (1982) Chemoreceptor involvement in cortisol responses to hypoxia in ventilated dogs. J. appl. Physiol. 52, 1092-1096. 31. Raff H., Shinsako L., Keil C. and Calhnan M. F. (1983) Vasopressin, ACTH and corticosteroid during hypercapnia and graded hypoxia in dogs. Am. J. Physiol. 244, E453-458. 32. Sanghera M. K., Harris M. C. and Morgan R. M. (1978) Effects of microiontophoretic and intravenous application of gonadotrophic hormones on the discharge of medial-basal hypothalamic neurones in rats. Brain Res. 140, 63-74. 33. Schwanzel-Fukuda M., Morrell J. I. and Pfaff D. W. (1984) Localisation of forebrain neurones which project directly to the medulla and spinal cord of the rat by retrograde tracing with wheat germ agglutinin. J. camp. Neurol. 226, l-20. 34. Share L. and Levy M. N. (1966) Effect of carotid chemoreceptor Am. J. Physiol. 210, 157-161.
stimulation on plasma antidiuretic hormone titre.
35. Strahlendorf H. F., Strahlendorf J. C. and Barnes C. D. (1981) Stimulation of hypothalamic arcuate inhibits
locus
916
D. BANKSand M. C. HARRIS
coeruleus unit activity: evidence for endorphin mediation. In The Role of Peprides and Amino Acids as Neurotransmitters. A.R. Liss, New York. 36. Versteeg D. H. G. (1980) Interactions on peptides related to ACTH, MSH and jI-LPH with neurotransmitters in the brain. Pharmac. Ther. 11, 535-557. 37. Watson S. J., Richard C. W., Ciaranello R. D. and Barchas J. D. (1980) Interaction of opiate peptide and noradrenalin systems: light microscopic studies. Peprides 1, 2330. (Accepted
30 July 1987)