Intracellular study of nucleus parabrachialis and nucleus tractus solitarii interconnections

Brain Research, 492 (1989) 281-292

281

Elsevier BRE 14645

Intracellular study of nucleus parabrachialis and nucleus tractus solitarii interconnections Antonio R. Granata and S.T. Kitai Department of Anatomy and Neurobiology, College of Medicine, The University of Tennessee, Memphis, Memphis, TN 38163 (U.S.A.) (Accepted 27 December 1988)

Key words: Nucleus parabrachialis; Nucleus tractus solitarii; Intracellular recording; Intracellular labeling; Antidromic activation

Responses of the nucleus parabrachialis (PBN) neurons to electrical stimulation of the nucleus tractus solitarii (NTS) were investigated by intracellular recording technique in anesthetized rats. Excitatory postsynaptic potentials (EPSPs) were evoked by ipsilateral NTS stimulation in 38 PBN neurons. They were considered monosynaptic because their latencies did not change with either variations in stimulus intensity or high-frequency repetitive stimulation. The latencies of EPSPs ranged from 1.2 to 6.9 ms. PBN neurons were also antidromically activated by NTS stimulation, giving a mean axonal conduction velocity of 4.6 m/s. Some of these neurons also responded with monosynaptic EPSPs to NTS stimulation• Direct stimulation of these neurons by depolarizing current pulses elicited repetitive firing with frequencies up to 350 Hz. The morphological analysis of 5 PBN neurons labeled with horseradish peroxidase (HRP) indicates that the soma were fusiform in shape, and the size varied from 163 to 783/~m2. All neurons had 3-5 spiny primary dendrites which extended in a predominantly mediolateral direction. Axons arose from a proximal dendritic trunk, close to the soma. The results indicated that PBN is reciprocally connected with the NTS which elicits an excitatory effect on PBN neurons.

INTRODUCTION The nucleus parabrachialis (PBN) consists of a group of small to medium-size neurons surrounding the superior cerebellar peduncle in the dorsolateral pons 2. It has b e e n considered to be involved in a variety of visceral, behavioural and n e u r o e n d o c r i n e functions such as the taste 29"31, respiration 4'11"5°, cardiovascular control ~°'28, secretion of adrenocorticotropin ( A C T H ) 51, sleep 39 and attack grooming, and threat behaviors 3. The P B N is also considered a relay for visceral afferent inputs coming from the nucleus tractus solitarii (NTS) to the forebrain. Taste-sensitive neurons in the rostral part of the NTS 53 project ipsilaterally to the c a u d o m e d i a l P B N 21'31 which in turn sends projections to the thalamic taste area 33. Electrophysiological studies have d e m o n s t r a t e d that neurons in the rostral subdivisions of the NTS

responsive to taste and natural stimulation of the oral cavity were identified as s o l i t a r i o p a r a b r a c h i a l neurons 32. H o w e v e r , no a t t e m p t was m a d e to d e t e r m i n e the synaptic actions of NTS p r o j e c t i o n s upon PBN neurons. On the o t h e r hand, the neurons in the m e d i o caudal p o r t i o n of the NTS receiving p r i m a r y afterents from b a r o - and c a r d i o p u l m o n a r y m e c h a n o r e ceptors, as well as from o t h e r general visceral receptors 9'11 project heavily to the lateral subdivision of P B N and K o l l i k e r - F u s e ( K F ) nuclei 1'21' 23,30,35. In turn, these neurons p r o j e c t to the forebrain including the insular, the infralimbic and the frontal cortex, as well as the h y p o t h a l a m u s , the a m y g d a l a and the thalamus 14. Electrophysiological experiments d e m o n s t r a t e d that neurons in he lateral PBN and KF, were activated o r t h o d r o m i c a l l y by stimulation of carotid sinus and aortic d e p r e s s o r nerves 6.

Correspondence: A.R. Granata, The University of Tennessee, Memphis, College of Medicine, Department of Anatomy and Neurobiology, 875 Monroe, Memphis, TN 38163, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

282 The function of the descending PBN projections to the caudal NTS 14'43 has been postulated to be involved in the modulation of the responses of NTS neurons to baroreceptor nerve inputs 13. For instance, electrical stimulation of PBN produced an attenuation of the baroreflex response accompanied by an increase in arterial pressure and heart rate 2s. Therefore, the study of reciprocal connections between the caudal NTS and the lateral subdivision of PBN and KF can be considered important in relation to a regulation of cardiovascular and other autonomic functions. Studies concerning the postsynaptic actions on PBN neurons elicited by caudal NTS inputs are very scarce, although putative transmitters which could mediate these responses have been postulated 26'27. Therefore, we also consider it important to study synaptic actions of caudal NTS inputs on the PBN neurons using intracellular recording techniques. Some recorded neurons were intracellularly labeled with horseradish peroxidase (HRP) to identify the sites of the recorded neurons and to study their morphology. MATERIALS AND METHODS Thirty-two male Sprague-Dawley rats (weighing 250-350 g) were used in these experiments. Rats were anesthetized with urethane (1.5-1.8 g/kg, i.p.) plus ketamine (30 mg/kg, i.p.). Supplemental doses of ketamine were administered (every 2 h) throughout the experiment. The trachea was cannulated. The animal was placed in a stereotaxic apparatus and suspended by clamps on the spinal process of T 2 and on the proximal section of the tail to obtain stable recording conditions. Some animals were paralyzed with tubocurarine chloride (0.12 mg/kg) and artificially ventilated (Harvard respirator pump) on 100% O3 which prevents the hypoxia produced by anesthesia. An occipitoparietal craniotomy exposed the cortical and the cerebello-pontomedullary surface, and the dura and arachnoid were removed. Then, the posterior vermis was gently retracted allowing access to the recording and stimulating areas including the caudal portion of the IVth ventricle. Bipolar stainless steel electrodes were fabricated from 00 insect pins, insulated with Epoxylite to within 0.2-0.5 mm from the tip and separated by a distance of 0.3 mm.

The electrodes were stereotaxically placed in the NTS by a micromanipulator. An example of stimulus electrode placement in the NTS is shown in Fig. 1. Electrical stimulation consisted of rectangular current pulses of 0.5 ms duration, 50-150/*A delivered at 0.5-1 Hz. The cortical surface was covered with a mixture of warm and liquid paraffin to reduce brain pulsations and to prevent the cortical surface from drying. lntracellular recording microelectrodes were made from borosilicate (2.0 mm o.d.) filament glass capillary tubing, beveled and filled with a solution of 4% H R P (Sigma Type IV) in 0.05 M Tris buffer (pH 7.6) and 0.5 M KCl (or potassium methylsulfate). The resistances of the electrodes measured in saline solution varied from 50 to 120 MD. Intracellular potentials were recorded in a conventional manner, and records were photographed by a kymograph camera (Grass Instrument Co.) from the oscilloscope screen. The data were also stored on a magnetic tape (Vetter model B) for off-line analysis. Only neurons with a resting membrane potential of 45 mV or more were intracellularly labeled with H R P by passing 2-5 nA depolarizing rectangular pulses of 150 ms duration at 3.3 Hz for 3-7 min. At the end of the experiment, an extra dose of urethane was administered (1.5 g/kg, i.p.) and the animal was perfused through the heart with saline solution followed by a mixture of 1% paraformatdehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The brain was removed and postfixed overnight in fresh fixative at 4 °C, then cut into 50 #m serial sagittal or transverse sections by a Vibratome. Sections were transferred into a cold 0.05 M phosphate buffer, and after that, processed for H R P histochemistry with 0.05% 3,3"-diaminobenzidine tetrahydrochloride and 0.03% H202. The histological sections containing intracellularly labeled neurons were postfixed in 2% osmiumtetroxide in a phosphate buffer, mounted onto gel-coated slides and air-dried before being dehydrated and coverslipped. Other sections were mounted and stained with Cresyl violet to determine the location of the stimulating electrodes or lesions. The morphological features of the intraceltularly labeled PBN neurons were examined with a light microscope: Five labeled neurons were serially reconstructed using a drawing tube and a 100× oil

283

Fig. 1. Photomicrograph of a coronal section of the medulla showing the location of stimulating electrodes in the NTS. Arrow indicates the electrode tips. NTS, nucleus tractus solitarii; XII, hypoglossal nucleus.

immersion objective. The soma area was measured by a planimeter method. RESULTS Electrophysiological characteristics of P B N neurons A total of 73 neurons was recorded intraceUularly from the PBN. Of these, 38 were activated orthodromically and 5 antidromically by caudal NTS stimulation. The amplitude of action potentials in these neurons was 40-75 mV and the duration 1.3-2.0 ms. In general, these neurons were spontaneously active; some neurons fired in a bursting manner with 2-5 spikes per burst. The firing rate varied between 5 and 100 Hz. In addition, the firing characteristics of PBN neurons were investigated by the injection of depolarizing current through the recording electrode which produced a high-frequency repetitive discharge (Fig. 2A,B,D,E). The relationship between the magnitude of current injected and the frequency of discharge was practically

linear up to 350 Hz of firing (Fig. 2C). Antidromic response in P B N neurons after stimulation of NTS A total of 5 neurons was antidromically activated by NTS stimulation. Fig. 3A represents a schematic diagram of recording and stimulation sites. Antidromic spikes were all or none, with a constant latency following NTS stimulation (Fig. 3B) at threshold of stimulation. No underlying synaptic potentials were observed when the action potential failed. Latencies of antidromic spikes varied from 0.9 to 2.0 ms (mean 1.3 ms; S.D. 0.4 ms). Considering the PBN-NTS conduction distance of 5-7 mm, the calculated conduction velocities of these PBN projection neurons ranged from 3.0 to 6.6 m/s. Three of these 5 antidromically activated neurons also responded with monosynaptic EPSPs (Fig. 3C). At lower intensities of stimulation (<50 hA) only excitatory postsynaptic potentials (EPSPs) with constant onset latency were evoked. At higher intensi-

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Fig. 4. A: changes in EPSP amplitudes with constant depolarizing (top trace) and hyperpolarizing (bottom two traces) current applied through the recording electrode. Second trace is control (cont) EPSP. Intensities of current are indicated on right hand side of each figure. Voltage calibration applies to all 4 traces. B: EPSPs and a spike evoked with different stimulus intensities. The onset latency of EPSPs did not change in spite of changes in stimulus intensity. At higher intensity of stimulation, a spike was triggered from the EPSP. C: EPSPs followed by an inhibitory postsynaptic potential (IPSP). Injection of constant hyperpolarizing current (-1 nA) increased the amplitude and duration of EPSPs and decreased the amplitude of IPSPs (bottom trace). D: a double stimulation at 10 ms interstimulus interval still evokes EPSPs without change in onset latencies.

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NTS stimulation: a depolarizing potential from which a spike potential was generated. H y p e r p o larizing current applied through the recording electrode (- 0.5; -0.9 nA) increased the amplitude of the depolarizing potential (Fig. 4A); while depolarizing current (+0.3 nA) produced the opposite effect (Fig. 4A). Therefore, these depolarizing potentials were defined as EPSPs. These EPSPs were considered monosynaptic because: (1) the onset latency was maintained constant in spite of variations in stimulus intensity (Fig. 4B) and (2) these EPSPs could follow relatively high frequency stimulation (e.g. 100 Hz) with constant latency (Fig. 4D). In only one neuron, the monosynaptic EPSP evoked by NTS stimulation was followed by a hyperpolarization potential (Fig. 4C). The amplitude of this hyperpolarizing potential decreased when the neuron was hyperpolarized by passing negative current (-1 nA) through the recording electrode, indicating this potential was an inhibitory postsynaptic potential (IPSP) (Fig. 4C, lower trace). During this period of hyperpolarization, the amplitude of the EPSP evoked by NTS stimulation increased, and its peak was prolonged in time indicating that the influence of the IPSP normally seen at resting m e m b r a n e potential is no longer present when the m e m b r a n e potential reaches values close to the IPSP equilibrium potential. The latency of the monosynaptic EPSPs varied from 1.2 to 6.9 ms (Fig. 5D). However, from the frequency histogram it is possible to differentiate two clearly separated groups: one with latencies shorter than 5 ms, with a mean value of 3.7 ms (S.D. 0.4 ms), and a second group with latencies between 5.1 and 7.0 ms, with a mean value of 6.1 ms (S.D.

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0.6 ms). Both groups were c o m p a r e d using Student's t-test, P < 0.001. Considering a conduction distance of approximately 5-7 m m from the NTS to the PBN in the rat, and a synaptic delay time of 0.6 ms, the calculated conduction velocities of these monosynaptic projections ranged from 1.5 to 4.0 m/s and from 0.9 to 1.2 m/s for the fast- and slow-conduction fibers, respectively. The duration time of these EPSPs varied according to the latency: the mean

TABLE I Somatic and dendritic characteristics o f intracellularly labeled neurons with H RP, located in the P B N

All neurons were orthodromically stimulated. VL-PBN, ventrolateral parabrachial nucleus; mVL-PBN, medial border of ventrolateral parabrachial nucleus. Neuron

Soma size (~m )

Soma area (l~m2)

No. of primary dendrites

Dendritic field (l~m)

Soma location

1 2 3 4 5

7x 8x 11 x 10 x 6x

367 248 783 357 163

5 4 3 3 3

395 x 320 x 310 x 207 x 340 x

mVL-PBN VL-PBN VL-PBN VL-PBN VL-PBN

40 46 61 28 24

700 965 390 580 795

286

A

B

Fig. 6. Camera lucida drawing of two HRP,labeled neurons reconstructed from serial sections. The arrows in A and B indicate the axon of these neurons. The stars in the insets show the location of the labeled neurons in the medial border of ventrotateral PBN in A and ventrolateral PBN in B. PBL, lateral division of parabrachial nucleus; IC, inferior colliculus; V, motor trigeminat nucleus. Bar = 50/~m and applies to A and B.

value of the short-latency group was 6.5 ms (S,D. 0.6 ms), while that of the long-latency group was 16.1 ms (S.D. 8.1 ms). Similar variations occurred with EPSPs amplitudes; the mean values were 4.9 mV

(S.D. 2.8 mV) and 11.5 mV (S.D. 7.4 mV) for the short and long latency groups, respectively. In 3 neurons, it was possible to evoke an early monosynaptic EPSP followed by a late EPSP with

Fig. 7. Photomicrograph of the same neuron shown in Fig. 6A. The axon is indicated by an arrow in A and an arrow with !a' in B. The 's' in A points to somatic spines. Arrows in B point to somatic and dendritic spines. The inset in B shows a distal dendrite with dendritic spines (indicated by arrows) at higher magnification. Bar = 25/~m in A; 10 pm in main frame in B and 7 pm in inset in B.

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288 lower threshold and longer duration (Fig. 5A-C). In the lower trace of Fig. 5C, the intensity of stimulation was adjusted to produce an action potential from the late EPSP. However, when the intensity of stimulation was increased, the early EPSP produced an action potential (upper trace) and the ensuing spike afterhyperpolarization prevented the second EPSP from reaching the spike generation level. Neuronal characteristics of the HRP-labeled neurons in P B N PBN neurons were intracellularly labeled with HRP. Five neurons were well labeled and preserved, and a full reconstruction and analysis of somatodendritic (see Table I) and axonal characteristics were performed on these neurons which were located in the ventrolateral PBN. Neurons located in the KF demonstrated some sign of degeneration and were not included in Table I. Other HRP-labeled neurons with some sign of degeneration were used only to identify their location in PBN and KE All 5 labeled neurons responded with monosynaptic EPSPs following NTS stimulation. Four neurons were recovered from coronal sections and one from sagittal sections. All injected neurons were located in the PBN, mainly in the ventrolateral, dorsolateral and KF subdivisions. The neuronal soma were fusiform in shape and the soma surface size varied from 163 to 783/am 2 (Table I). Some neurons had a few spines on their soma but most of them were without spines. The number of primary dendrites varied from 3 to 5, and their diameters ranged from 0.6 to 4.1/am. The dendrites bifurcated near the soma (distance of bifurcation: 2-140/am); after that, some of them gave a secondary and tertiary bifurcation (distance: 2-290/am) before terminating. Terminal dendrites were thin (0.3-1 /aM). The dendritic appendages were sparse to moderate spines, varicosities and filiform processes. The orientation of the dendrites varied from a predominant mediolateral (Table I, cells 1, 2, 4, 5) to rostrocaudal (cell 3) direction. The axon of the labeled neurons could be traced in 3 neurons. The axon arose from a proximal dendritic trunk close to the soma. Axon collaterals could be traced in only one neuron. However, as we could not trace these axons for a long distance, a conclusive statement about axon collaterals in PBN

neurons could not be made. Figs. 6A and 7A,B show a neuron (Table I, cell 1) located in the medial border of the ventrolateral PBN. The neuron had fusiform soma with a few spines (see Fig. 7) measuring 7 x 40 #m. Five primary dendrites of varying diameters (1.0-2.4 pm) arose from the soma and were covered with sparse to moderate spines. They branched close to the soma (9-48/am) and extended in some cases for 114/am before branching again. Secondary dendrites were tortuous with sparse to moderate spines, and all dendrites terminated with fine processes. The dendritic field of this neuron sectioned frontally, extended more extensively in the dorsomedial-ventrolateral direction (700/am) than in the rostrocaudal direction (395/am). The axon arose from a proximal dendrite close to the soma and was directed medio. ventrally. No axon collaterals were observed. Fig. 6B illustrates an intracellularly labeled neuron (Table I, cell 2) located in the ventrolateral PBN. This neuron had smooth soma, an ovoid shape, and measured 8/am in the minor and 46/am in the major axis. Four primary dendrites of varying diameters (1.0-1.4 /am) arose from the soma and branched close to it (1.2-43/am). Primary dendrites presented sparse spines, while secondary and tertiary dendrites had sparse to moderate spines and fine filiform processes. Diameters of secondary dendrites varied from 0.4 to 1.4/am. The dendritic field of this neuron extended 965/am mediolaterally and 320/am rostrocaudally. The axon arose from a ventromedially oriented proximal dendrite and headed in the same direction. Axon collaterals were not observed. DISCUSSION The results of this study demonstrate that, in the rat, electrical stimulation of the caudal NTS at the level of the calamus scriptorius induces monosynaptic EPSPs in neurons located in the lateral subdivisions of PBN. The possibility that those EPSPs were produced by the stimulation of neurons located in the caudal portion of the NTS is supported by the following: (1) it was anatomically verified that the stimulating electrodes were located in the caudal NTS; (2) areas surrounding the NTS were probably not activated by current spread. This statement is

289 supported by the fact that we employed lower intensities of stimulation than those which would produce tongue and throat movements by activation of neurons in the hypoglossal nucleus; (3) anatomical studies demonstrated that neurons in the caudal NTS project heavily to the ventrolateral PBN and KF21'23'26'27'35; (4) although the stimulus could have activated neurons in the rostral gustatory portion of the NTS, they mainly project to the medial part of PBN31; (5) neurons in areas very close to the NTS, as for instance, the dorsal motor nucleus of the vagus, the area postrema, and the nucleus gracilis, could have been included in the zone stimulated in some experiments. However, while vagal motoneurons project preferentially to the medial PBN 21, there has been controversy concerning the existence of the projection from the area postrema to the PBN complex8A8'21'23'27'4°. Moreover, neurons in the nucleus gracilis project to the inferior colliculus, but not to PBN 2~. The possibility that fibers of passage originating in the medullary and spinal dorsal horn may be stimulated is excluded, because, although these fibers terminate in the lateral PBN and KF, they ascend the length of the spinal trigeminal complex within the spinal trigeminal tract through its nuclei and the ventral reticular formation 34. In addition, a group of medullary neurons in the lateral tegmental field and parvocellular reticular formation send major projections to PBN and KF. However, these ascending fibers run a ventrolateral route in the subtrigeminal region, dorsomedial to the spinocerebellar tract, far away from the stimulation site 21. Different anatomical and electrophysiological studies also demonstrated that lateral PBN neurons receive projections from neurons in lamina I of the spinal and medullary dorsal horn, probably related to somatosensory information 7A4'16'25'52'54. However, these studies did not provide evidence that these fibers run close to the stimulation area. The possibility that some monosynaptic EPSPs induced by NTS stimulation could have been produced by the stimulation of recurrent collaterals to the PBN originating from descending projections terminating or passing through the NTS can not be excluded on the bases of the present results, and future investigations will be necessary. Corticosolitary projections particular to the caudal NTS are

remarkably smaller in number than NTS-PBN projections 21'38'49. However, they innervate both the NTS and PBN complex 37'41'42'47'49. The paraventricular nucleus of the hypothalamus, as well as the lateral hypothalamic area also innervate the NTS and PBN 27'44-46. The neurons recorded were located in the ventrolateral and KF subdivisions of PBN, where terminals of efferents from the caudal NTS are found 21'23'26'27'35. The location of these neurons was verified by intracellular labeling with HRP. The morphological features of the intracellularly labeled neurons were analyzed in 5 weU-preserved neurons, while those neurons demonstrating signals of degeneration were used to determine the recording site. The present results demonstrated that the intracellularly labeled neurons were medium in size and fusiform in shape; several primary dendrites originated from the soma with sparse to moderate spines and fine appendages were observed in some cases. Serial reconstruction of these PBN neurons demonstrated that they have an extensive dendritic field oriented more in the mediolateralis direction, making them suitable to synapse with different ascending and descending projections. The data concerning axonal characteristics were limited. However. the results demonstrated that the axons originated from the proximal dendrites and axonal branching was absent in the majority of these neurons. Only 6% of the PBN neurons responded with antidromic spikes following NTS stimulation. The scarcity of PBN neurons antidromically activated from the NTS is compatible with anatomical evidence that few neurons in the middle third of PBN and KF project to the caudal NTS 14. The calculated conduction velocities for the PBN-NTS descending projections reported in the present experiments in the rat, are similar to those found in the cat for the same pathway 13. Three neurons in PBN were antidromically activated, as well as monosynaptically excited by NTS stimulation. These results indicate that PBN is reciprocally connected with the caudal NTS. Felder and Mifflin ~3, found that stimulation of PBN projections to the caudal NTS evoked EPSPs (probably monosynaptic) followed by inhibitory responses into NTS neurons. In addition, these authors claim that baroreceptor nerve inputs are modulated by PBN-

290 NTS projections. It is possible then to speculate that mutually excited PBN-NTS neurons forming a reverberating circuit would modulate the baroreceptor reflex response, as well as, other cardiovascular and autonomic afferent inputs to NTS. We could differentiate two types of NTS-PBN excitatory inputs: one type with short-latency, smallamplitude and short-duration EPSPs; and a second type with longer-latency, larger-amplitude and longer-duration EPSPs. It is important to note that the resting membrane potentials were similar in both groups of neurons. Even though a further analysis (e.g. antidromic responses evoked upon caudal NTS neurons by PBN stimulation) will be necessary to confirm the hypothesis that NTS excitatory inputs are mediated by fibers with different velocities of conduction, the fast-conducting smaller EPSPs amplitude could suggest that this projection represents a small proportion of the NTS-PBN pathway. However, other possibilities should be considered: the position of the stimulating electrode closer to the group of fibers which mediate the long-latency EPSPs, or whether the synaptic contact takes place in proximal or distal dendrites. In 3 neurons, a monosynaptic EPSP with short latency, small amplitude and short duration was followed by a late EPSP with larger amplitude and longer duration. The threshold to produce the late EPSP was always lower than that to produce the early EPSP. If an action potential was triggered from the early EPSP, the relatively large afterhyperpolarization that followed the early spike prevented the late EPSP from producing an action potential. By this neuronal mechanism, PBN neurons could possibly discriminate two different inputs conveyed by fast and slow NTS pathways. When early EPSPs failed to produce an action potential, an action potential could be triggered from the late EPSPs. However, when both the early and the late EPSPs are potentially effective in producing spike potentials, only the early EPSP can trigger an action potential. This observation can be related to the fact that in cat motoneurons the repetitive firing is regulated by afterhyperpolarizing conductance 5, and the discharge frequencies are related to the time course of afterhyperpolarizing potentials TM. Moreover, during the afterhyperpolarization that follows an antidromic spike potential, the monosynaptic

EPSPs evoked by large afferent fibers (Group Ia) are smaller than the control response 12. Also, in cat motoneurons an early (with higher threshold) and a late (with lower threshold) monosynaptic EPSP can be evoked by cortical stimulation. These EPSPs were the result of fast and slow pyramidal tract stimulation x7. Parabrachiai neurons could fire at high frequency when depolarizing current was applied through the recording electrode. The relationship between the intensity of current injected and the frequency of discharge would indicate that PBN neurons are very sensitive to excitatory inputs such as the ascending NTS projections demonstrated in this study. We have observed IPSP evoked by NTS stimulation in only one PBN neuron, and this was preceded by monosynaptic EPSP. It was very difficult to determine the IPSP onset, due the temporal overlapping with the preceding EPSP. However, it is very unlikely that this IPSP was monosynaptically mediated by NTS inputs. Nevertheless, the IPSP seems to curtail the EPSP regulating the duration of depolarization. The caudal NTS at the level of the obex is the site of termination of primary afferents from cardiovascular and pulmonary baro- and mechanoreceptors, as well as the location of neurons related to cardiovascular and respiratory activity9A1'19'24'36'48. Moreover, the PBN complex is known to be involved in the mediation of a potent pressor response with increase in both the cardiac output and the total peripheral resistance 28. It is reasonable then, to postulate that the excitation evoked on PBN neurons by NTS stimulation conveys cardiovascular and autonomic information. The excitatory effects of NTS inputs to PBN neurons could mediate the increase in arterial pressure and heart rate which is reported to be elicited by electrical stimulation of NTS, especially at higher frequencies aS. Therefore, the NTS-PBN excitatory pathway could be an alternative pathway by which the NTS can modulate the sympathetic tone and the circulation. A unique feature of the PBN neuron to discriminately fire from time-dependent inputs (e.g. between early and late EPSPs) could be a mechanism by which this nucleus can select appropriate inputs (e.g. baroreceptor vs chemoreceptor) from the NTS. Our findings also indicated that the NTS-PBN pathway is a

291

very secure pathway in that NTS induced EPSPs in

ACKNOWLEDGEMENTS

PBN n e u r o n s are fast-rising and that the PBN n e u r o n s could fire at high frequency. This security in

This work was supported by U S P H S

Grants

conduction may be important not only in the role the PBN n e u r o n s play in modulation of peripheral

NS-20702 and NS-23886 to S.T.K. and A m e r i c a n Heart Association G r a n t 861294 to A . R . G . The

sympathetic function, but also to faithfully convey the a u t o n o m i c and visceral afferent information from the lower brainstem to the higher centers.

authors

REFERENCES

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also

express

their

appreciation to

Dr.

Salmon Afsharpour and Mrs. Holly Stiles for their technical assistance.

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