Brain Research Bulktin, Vol. 20, pp. 825-829. 0 Pergamon Press plc, 1988. Printed in the U.S.A. 0361-9230/88 $3.00 + .oO

Thermal, Osmotic and Chemical Modulation of Neural Activity in

the Paraventricular Nucleus: In Vitro Studies

H. YAMASHITA, K. INENAGA AND R. E. J. DYBALL*

Department of Physiology, University of Occupational and Environmental Health School of Medicine, Kitakyushu, 807 Japan

and *Department of Anatomy, University of Cambridge, CB2 3DY UK

YAMASHITA, H., K. INENAGA AND R. E. J. DYBALL. Thermal, osmotic and chemical modulation of neural activity in the paraventricular nucleus: In vitro studies. BRAIN RES BULL ZO(6) 825-829, 1988.-To investigate the functions of the pamventricular nucleus (PVN) which plays an important role as an integration site for the nemoendocrine and autonomic nervous systems, the tiring activity of PVN neurons was recorded from hypothalamic slice preparations during thermal, osmotic and chemical stimulation. Neurons responded to environmental factors such as temperature and osmolarity and both warm-responsive and cold-responsive neurons were observed in the PVN. Some PVN neurons were also osmore- sponsive and unlike neurons in the supraoptic nucleus, most osmoresponsive PVN neurons decreased their fling rate during hyperosmotic stimulation. One of the classical transmitters, noradrenaline, exerted excitatory effects on PVN neurons through q- and P-receptors and inhibitory responses through a,-receptors. Atrial natriuretic polypeptide exerted inhibitory effects on putative parvocellular PVN neurons but it had no effect on putative magnocellular PVN neurons. An endogenous sugar derivative, 2-deoxytetronic acid, thought to be an endogenous satiety factor, elicited inhibitory effects, supporting the possibility that the PVN also may be related to feeding behaviour. Arginine-vasopressin and oxytocin which are synthesised in the magnocellular neurosecretory cells excited PVN neurons, suggesting that the PVN may have short circuits modulating neural activity within the nucleus itself. We conclude that neurons in the PVN may receive multiple information and act as one of the important integrative sites in the brain.

Paraventricular nucleus Thermosensitivity Osmosensitivity Noradrenaline Vasopressin Oxytocin Angiotensin II Atrial natriuretic polypeptide 2-Deoxytetronic acid

THE magnocellular neurons in the paraventricular nucleus (PVN) produce arginine-vasopressin (AVP) and oxytocin (OXT) which regulate the body fluid balance and control in the milk ejection reflex, respectively. Recent histological [14] and electrophysiological [6] evidence shows that the PVN has strong connection with parts of the brainstem which regulate the cardiovascular system. The PVN neurons send axons to the brainstem and vice versa. These studies demonstrated that the PVN may play an important role in the regulation of the autonomic nervous system as well as in the neurohypophysial system and suggested that the PVN may integrate the activity of these two systems.

In the PVN, there exist nerve terminals which contain neuropeptides as well as the classical neurotransmitters such as noradrenaline, acetylcholine, dopamine and histamine [14]. These substances, when released from the terminals, could modulate neural activity of PVN neurons so it is of interest to know how they act on the individual neurons of the PVN.

The neurons in the central nervous system (CNS) may be

sensitive to changes in the temperature and osmolarity of the microenvironment around them. Supraoptic (SO) neurons can be influenced by a change in osmolarity l7] and neurons in the preoptic area can be affected by temperature [l]. In a recent paper [3] we demonstrated that some PVN neurons are thermosensitive, that is, the PVN has warm- and cold- sensitive neurons. Here we report, additionally, that PVN neurons are osmosensitive.

There is also evidence that the PVN may have an impor- tant function in the regulation of behaviour [12,15]. A novel endogenous sugar acid, 2-deoxytetronic acid (ZDTA), has marked effects on feeding behaviour [ll] and we recently showed that it can act directly on PVN neurons [4] so that the PVN may be associated intimately with feeding be- haviour.

The aim of this paper was to make a synthesis of our previous studies and to include some new data to give a more comprehensive insight into how the PVN acts to integrate the hypothalamo-neurohypophysial system with other parts of the autonomic nervous system.

825

YAMASHITA, 1NENAGA AND DYBALL

10 min

\* . . . . * \ I,. .

01, * . , , , ) . , 1.e.y

l a- 33 37 43

Temperature ( ‘C )

a 10 min

33 37 43 Temperature ( ‘C 1

FIG. 1. Typical warm-sensitive and cold-sensitive neurons from the rat PVN. (A) upper trace: slice temperature: lower trace: a rateme- ter record. (B) relationship between tiring rate and temperature was approximated by two successive straight lines (a and b) which inter- sected at 38°C. The Q,,) value and Troen of this neuron calculated from line were 14.2 and 0.52 spikes/set, “c, respectively. (C) upper trace: slice temperature; lower trace: a ratemeter record. (D) rela- tionship between firing rate and temperature of slice, from which Trarlf= -0.22 spikes/set “C (from Inenaga et at. [3] with permission from Brain Res).

METHOD

Brain tissue was taken either from Wistar rats (150-350 g) or from albino mice (25-35 g) which were stunned with a blow on the neck and rapidly decapitated. The brain was quickly removed and, after cooling in bathing medium at 4°C for approximately 1 min, was sliced coronally or parasagit- tally using a vibratome (350-500 pm). Some slices were further trimmed to obtain a small island of tissue containing only the PVN. Pieces of tissue were incubated in bathing medium at room temperature bubbled with 0, (95%) and CO, (5%) for at Ieast 1 hr before they were transferred to the recording chamber. Stable recordings were obtained for up to 18 hr thereafter.

The bathing medium had the following composition: NaCl 124 mM; KC1 5 mM; KH,PO, 1.24 mM; MgSO, 1.3 mM; CaCl, 2.1 mM (or 0.75 mM); NaHCOis 20 mM; glucose 10 mM. It was bubbled continuously with 95% O2 and 5% CO,, maintained at 35°C by a water jacket, and gravity fed from the storage chamber to the recording chamber at about 2 mlimin. The recording chamber (approximate volume 1 ml) was made of clear acrylic with a sylgard (Dow-Coming 184 silicon elastomer) mat glued to the bottom, and was illumi- nated from below. Excess medium was removed by suction, and the tissue was held in place on the sylgard mat by a nylon net and platinum weights. When the composition of the bath- ing medium was changed, the modified medium was supplied through a T valve from a second storage chamber. The tem- perature of the tissue was changed when required by altering the temperature of the perfusate while maintaining the perfusion rate constant. The temperature was monitored with a small copper-constantan thermocouple placed on the slice.

ExtraceIlular recordings were made with glass electrodes

275 mOsm t kg 1

B ,. 342 mannitol

FIG. 2. Osmotic response of a PVN neuron in the mouse. The os- molality of the normal medium was 295 mOsm/kg. Mannitol (B) and NaCl (C) were added to the normal medium; osmolality was reduced by decreasing NaCl concentration. This neuron responded both to changes in mannitol and NaCI concentration so it may be truly os- mosensitive and not just Na-sensitive.

filled with 0.5 M sodium acetate containing 2% Pontamine sky-blue. They were introduced into the PVN region under visual control and recording sites marked by pass- ing a current of 4 PA for 1-5 min (tip negative) down the electrode. The spot was located subsequently in 40 pm fro- zen sections of the tissue fixed in 4% paraformaldehyde. Recorded spikes were displayed using conventional tech- niques on a Tektronix 5000 series oscilloscope and a paper chart recorder together with a tape recorder and oscilloscope camera.

RESULTS

ThrrmosPnsitivity of PVN Neurons

The thermosensitivity of 65 spontaneously active PVN neurons was tested [3 1. We found warm- and cold-sensitive neurons in the PVN and classified as warm-sensitive those neurons which had both a Qlo>2 and a Tvtrrrr>O. 1 spikesisec “C and as cold-sensitive those with a Tcuen< -0.1 spikesisec “C. An example of a warm-sensitive neuron is shown in Fig. 1A and B. The firing rate of the neuron increased as the temperature of the slice increased and decreased as it de- creased. Figure 1C and D shows an example of a cold-sensi- tive neuron. The firing rate of the neuron decreased as tem- perature of the tissue increased and there was a linear rela- tionship between the firing rate and temperature. Of 65 PVN neurons tested, 23 neurons were judged to be warm- sensitive. The mean Tvoefl of the warm-sensitive neurons was 0.58 spikes/set “C and the mean Q10 value was 10.9 spikes/ see “C. Nine PVN neurons were judged to be cold-sensi- tive. The Tcoeff of the cold-sensitive neurons was -0.47 spikeslsec @C. Of the PVN neurons tested 49% showed either warm- or cold-sensitivity.

Osmosensitivity of PVN Nrurons

The osmosensitivity of 89 spontaneously active PVN neurons was tested either by changing the NaCl concentra- tion or by adding mannitol to the normal perfusion medium. Six (7%) neurons were excited, 3(3%) showed excitation fol-

MODULATION OF PARAVENTRICULAR NEURAL ACTIVITIES 827

Sl 50

:

252 -L

NAT5W 0%

NA lo-5M NATM

C*iivsm.~

k~pr~renol Clonidyno 10% M 5110-6 M

5min

FIG. 3. Effects of noradrenergic and related drugs on a mouse PVN neuron. The activity of this neuron was increased by low concentra- tions of NA ( 10m6 and 3 x 10m6 M) but depressed by high concentra- tions (more than 8 x 10m6 M). Methoxamine (q-agonist) and isopro- terenol (@-agonist) slightly increased and clonidine (a,-agonist) strongly decreased the activity of the neuron. The effect of clonidine was prolonged but the spontaneous firing rate recovered to pre- treatment level 1 hr after its removal (from Inenaga et al. [2] with permission from Brain Res).

lowed by inhibition and 34 (38%) were inhibited when the osmolality of the medium was increased by 30 to 50 mOsm/kg above normal. The other 46 PVN neurons were unre- sponsive to osmotic stimulation. Figure 2 shows an example of a PVN neuron which was inhibited by increasing NaCl concentration or by adding mannitol to the normal medium. Decreasing the NaCl concentration increased the firing rate of the same neuron. Osmoresponsive PVN neurons were scattered throughout the whole PVN region and not grouped in specific areas of the PVN. By contrast similar osmotic stimulation elicited excitatory response in the SON [16].

Effects of Noradrenaline on PVN Neurons in the Periventricular Parts of the PVN

Thirty-seven neurons were recorded from the periven- tricular region of the PVN which contains mainly parvocellu- lar neurons and only a very few magnocellular neurons [14]. The dose-response relationship between firing rate and noradrenaline (NA) concentration was investigated in 4 PVN neurons. Representative responses of a PVN neuron to ap- plication of NA are shown in Fig. 3. The activity of this neuron was increased by low concentrations of NA (10m6 and 3 x 10e6 M) but depressed by high concentrations (more than 8 x 10mfi M). These results imply that individual periventricu- lar neurons can show two types of response to NA depend- ing on the concentration applied. When the effect of NA was determined at a concentration of 10e5 M, 13 (35%) PVN neurons were excited and 22 (5%) neurons were inhibited. We also investigated which subtypes of NA receptors were present on the PVN neurons. Six of 7 neurons were excited and one neuron inhibited by the q-agonists, methoxamine or phenylephrine. All of 4 neurons tested were excited by the @-agonist isoproterenol. The cu,-agonist clonidine strongly depressed the spontaneous activity of all 12 neurons tested and exerted an inhibitory effect over a long period. These results suggest that PVN neurons are excited through aI- and Preceptors and are inhibited through cy,-receptors.

Excitatory Effects of Vasopressin (AVP) and Oxytocin (OXT) on PVN Neurons

The effects of AVP and/or OXT were tested on 97 spon- taneously firing PVN neurons [S]. Following application of AVP at lo-’ M to a bath, 64 (66%) neurons showed excita- tory responses and 3 (3%) inhibitory responses. Application of OXT at 1O-7 M excited 39 (57%) of 68 neurons and inhib- ited 2 (3%) neurons. Individual PVN neurons responded to both application of AVP and OXT, but the magnitude and threshold of the responses varied from cell to cell. Neurons responsive to AVP and OXT were dispersed throughout the whole region of the PVN, suggesting that the neurons were parvocellular [5,141.

After blocking synaptic transmission with a medium con- taining a low [Ca2+] and a high lMg2+], all tested neurons (AVP, n=15; OXT, n=14) which had responded to applica- tions of AVP or OXT in normal medium still showed similar responses to the peptides, although the effect was less marked in half the cells.

We investigated which type of receptor was involved in the responses of PVN neurons to AVP, using AVP analogues. The AVP analogues available in this experiment were d(CH&DAVP (a V,-receptor antagonist), d(CH&d- TyrVAVP (a V,N,-receptor antagonist) and dDAVP (a V,- receptor agonist); Fig. 4 shows the effects of the V,-receptor antagonist. Of 12 PVN neurons tested, d(CHJ,DAVP (n=lO) and d(CH&D-TyrVAVP completely or partly blocked AVP-induced responses. The V,N,-receptor antagonist partly suppressed the effects of AVP on the PVN neurons. On the other hand, none of the 6 PVN neurons showing an excitatory response to AVP were affected by application of dDAVP lo-’ and lO-‘I M. It is thus probable that for AVP, the receptor of the PVN neurons was of the V,-

type. Effects of Atria1 Natriuretic Polypeptide (ANP) and Angiotensin ZZ (AZZ) on PVN Neurons

Effects of ANP and AI1 on the PVN neurons have already been investigated [lo]. Of the 40 neurons tested with each peptide at 10e7 M (which included 10 neurons antidromically activated by stimulation of the hypothalamo-hypophysial tract), 7 showed inhibitory responses to ANP and 14 were excited by AI1 (Fig. 5). However, one was excited by ANP and 2 were inhibited by AII. Thus, ANP had mainly inhibi- tory effects on PVN neurons while AI1 exerted excitatory effects. Perhaps surprisingly, no neurons showed both in- hibitory responses to ANP and excitatory responses to AI1 [lo]. We investigated whether the recorded PVN neurons were themselves influenced by ANP or not, using low [Ca2+] and high [Mgz+] solutions. Five PVN neurons (probably par- vocellular) which had shown inhibitory responses to ANP in the control perfusion medium were also inhibited by ANP in medium with low [Ca*+] and high mg*+]. On the other hand, two neurons antidromically activated by electrical stimula- tion dorsolateral to the fornix (presumably magnocellular neurons), which had been inhibited by ANP in control per- fusion medium, were not responsive to ANP in the medium with low [Ca”] and high lMg*+]. The results indicate that some PVN neurons (probably parvocellular cells) but not magnocellular PVN cells are directly sensitive to ANP.

Inhibitory Effects of 2-Deoxytetronic Acid (2-DTA), a Putative Endogenous Satiety Factor, on PVN Neurons

The endogenous sugar acid, 2-DTA, was investigated for

828 YAMASHITA, INENAGA AND DYBALL

AVP lO-8 M AVP 10-B M

VI antagonist lo-7 M

I t I AVP 10-8 M

1

AVP 10-B M AVP 10-B M

5 min “’

FIG. 4. Ratemeter records showing the effects of V~-an~gonist on a rat PVN neuron. Application of AVP increased the firing rate; the response was repeatable but application of a VI-antagonist (d(CHJ,DAVP) completely suppressed the ex- citation induced by AVP.

FIG. 5. Ratemeter records showing the effects of ANP on rat PVN neurons. A shows inhibitory responses of a neuron to application of ANP. Inhibition was seen both in a control perfusing medium (left) and in a low [Ca2+] and high IMp2+] medium (right). B shows re- sponses of an antidromically activated PVN neuron to ANP in a control perfusing medium (left) and in a low [Ca’+] and high [Mg2+] medium (right). Note the disappearance of the responses to ANP in a low [Cat+] and high wg”+] medium (from Okuya and Yamashita IIOI).

its effects on the activity of PVN neurons [4]. Of 53 neurons tested, 23 (43%) were inhibited and 2 were excited. The other 28 (53%) did not respond. The inhibition in response to 2-DTA was dose-related. Since some PVN neurons were sensitive to changes in plasma osmotic pressure (as de- scribed above), we tested the effects of osmotic pressure change by increasing the sodium chloride concentration while recording from PVN neurons which were inhibited by 2-DTA. Addition of sodium chloride to the medium did not elicit any consistent response from the neurons, while addi- tion of the same concentration of 2-DTA produced very clear inhibitory responses. These results suggest that 2-DTA acts as a substrate related to the suppression of feeding behaviour in the PVN.

DISCUSSION

Histological [14] and electrophysiological [6] studies have demonst~ted that the PVN has connection with many brain regions, receives input through pathways involving classical nemotransmitters and peptides and controls both the hypo-

thalamo-neurohypophysial system and the autonomic nerv- ous system. It is thus reasonable to suppose that the PVN may be an integrative site for these two systems. However, very little is known about how the PVN might exert such actions.

Our results show that PVN neurons are influenced by chemical substances, wide spread in the brain, such as NA,

ANP, AII, AVP, OXT and 2-DTA. The PVN neuron re- sponse to NA was dependent on its concen~ation and it is probable that (Ye- and Preceptor activation elicits an increase in firing rate and &receptor activation elicits a decrease in firing rate. However, in some PVN neurons both excitatory and inhibitory responses were induced by NA with only a In-fold difference in NA concentration (Fig. 3). Such small changes in neurotransmitter concentration in the synaptic cleft might follow physiological changes in the membrane potential of the presynaptic terminals. Thus some of the changes seen in the activity of PVN neurons may be due to changes in the tone of input pathways involving NA.

Local refease of neurosecretory hormones within the PVN and the SON (by an unknown mechanism) may affect the activity of PVN and/or SON neurons @]. ~o~hoIo~ca~ investigation has demonstrated that AVP- and OXT- containing neurons have branches within the PVN and the SON [13]. Our results, obtained with electrophysiological techniques, clearly show that AVP and OXT in fact can ac- tivate PVN neurons. Thus there may be intranuclear con- nections in the PVN between AVP- and OXT-containing neurons and the other neurons. Such short circuits may con- tribute to the integration of neuroendocrine and autonomic functions.

Our results also show that some, at least, of the neurons in the PVN are thermosensiti~e and osmosensitive. Both factors strongly influence autonomic and neuroendocrine functions. Contrary to our expectation, however, PVN neurons showed inhibitory response to hyperosmotic stimu- lation. The osmosensitive neurons were diffusely distributed in the PVN. With the same protocol, cells in the SON showed more excitatory responses [16]. The two sets of ob- servations could be reconciled if the neurons which are inhibited by hyperosmotic stimulation are parvocellular and control the neuroendocrine and autonomic parts of the nerv-

MODULATION OF PARAVENTRICULAR NEURAL ACTIVITIES 829

ous system directly or indirectly. It is also possible that the tracerebroventricular injection 191. osmoresponsiveness of cells in the PVN may be related to The present study thus supports the suggestion that the drinking behaviour. Indirect support for such a suggestion PVN has multiple functions and that by responding to both may be given by evidence that the PVN is influenced by chemical and physical stimulation plays a role in the control ANP, a peptide which has an antidipsogenic action after in- of both neuroendocrine and autonomic mechanisms.

REFERENCES

1.

2.

3.

4.

5.

6.

7.

8.

Hori, T.; Shinohara, K. Hypothalamic thermo-responsive neurones in the new-born rat. J. Physiol. 294:541-560; 1979. Inenaga, K.; Dyball, R. E. J.; Okuya, S.; Yamashita, H. Char- acterization of hypothalamic noradrenaline receptors in the su- praoptic nucleus and periventricular region of the-paraventricular nucleus of mice in vitro. Brain Res. 369:3447: 1986. Inenaga, K.; Osaka, T.; Yamashita, H. Thermosensitivity of neurons in the paraventricular nucleus of the rat slice prepara- tion. Brain Res. 424: 126-132; 1987. Inenaga, K.; Oomura, Y.; Shimizu, N.; Kasai, M.; Yamashita, H. Inhibitory effects of 2-deoxytetronic acid, a putative endog- enous satiety factor, on paraventricular neurons of rats, in vitro. Neurosci. Lett. 79:123-127; 1987. Inenaga, K.; Yarnashita, H. Excitation of neurones in the rat paraventricular nucleus in vitro by vasopressin and oxytocin. J. Physiol. 370: 165-180; 1986. Kannan, H.; Osaka, T.; Kasai, M.; Okuya, S.; Yamashita, H. Electrophysiological properties of neurons in the caudal ven- trolateral medulla projecting to the paraventricular nucleus of the hypothalamus in the rats. Brain Res. 376:342-350; 1986. Mason, W. T. Supraoptic neurons of rat hypothalamus are os- mosensitive. Nature 287:15C156; 1980. Mason, W. T.; Hatton, G. I.; Ho, Y. W.; Chapman, C.; Robin- son, I. C. F. Central release of oxytocin, vasopressin and neurophysin by magnocellular neurone depolarization: evi- dence in slices of guinea pig and rat hypothalamus. Neuroendo- crinology 42:31 l-322; 1986.

9. Nakamura, M.; Katsuura, G.; Nakao, K.; Imura, H. Antidipsogenic action of atria1 natriuretic polypeptide adminis- tered intracerebroventricularly in rats. Neurosci. Lett. 58: l-6; 1985.

10. Okuya, S.; Yamashita, H. Effects of atrial natriuretic polypep- tide on rat hypothalamic neurones in vitro. J. Physiol. 389:717- 728; 1987.

11. Plata-Salaman, C. R.; Oomura, Y.; Shimizu, N. Effect of en- dogenous sugar acid derivative acting as a feeding suppressant. Physiol. Behav. 28:35%373; 1986.

12. Shiraishi, T. Gastric related properties of rat paraventricular neurons. Brain Res. Bull. 18:315-323; 1987.

13. Sofroniew, M. V.; Glasmann, W. Golgi-like immunoperoxidase staining of hypothalamic magnocellular neurons that contain vasopressin, oxytocin or neurophysin in the rat. Neuroscience 6:619-643; 1981.

14. Swanson, L. W.; Sawchenko, P. E. Hypothalamic integration: organization of the paraventricular and supraoptic nuclei. Annu. Rev. Neurosci. 6:269-324; 1983.

15. Weiss, G. F.; Leibowitz, S. F. Efferent projections from the paraventricular nucleus mediating q-noradrenergic feeding. Brain Res. 347:225-238; 1985.

16. Yamashita, H.; Inenaga, K. Osmosensitive neurons in the su- praoptic and paraventricular nucleus of the rat hypothalamus. J. Physiol. Sot. Jpn. 44381; 1982.