225

Journal of Physiology (1990), 431, pp. 225-241 With 9 figures Printed in Great Britain

THE OSMORECEPTOR COMPLEX IN THE RAT: EVIDENCE FOR INTERACTIONS BETWEEN THE SUPRAOPTIC AND OTHER DIENCEPHALIC NUCLEI

BY K. HONDA, H. NEGORO, R. E. J. DYBALL*, T. HIGUCHI AND S. TAKANO From the Department of Physiology, Fukui Medical School, Matsuoka, Fukui, 910-11, Japan and the *Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY (Received 28 February 1990) SUMMARY

1. Experiments were undertaken to provide evidence for the existence of a circuit of neuronal interconnections between the supraoptic nucleus (SON), the ventral anteroventral third ventricular region (including the organum vasculosum of the lamina terminalis; ventral AV3V) and the median preoptic nucleus (MnPO), and to determine the importance of these connections in the osmotic control of the neuronal activity of the SON. Extracellular recordings were made in the urethaneanaesthetized male rat from neurones in one of these three sites, while the other two sites were electrically stimulated. 2. During recording from the SON, electrical stimulus pulses applied either to the ventral AV3V or to the MnPO were followed by orthodromic excitation (OD+) or initial short-duration inhibition followed by long-duration excitation (OD - +) of most SON neurones (44/48). The latency of OD+ or OD+ component of OD - + response produced by electrical stimulation of the MnPO was significantly (paired t test, P < 0-01) shorter than that by the stimulation of the ventral AV3V. None of the neurones we recorded in the SON was activated antidromically by stimulation of either the ventral AV3V or the MnPO. Pressure injection of lidocaine (10 %, 50 nl) into the MnPO reversibly depressed the OD + effect after stimulation of the ventral AV3V in all the SON neurones tested (11/11), while injection of lidocaine into the ventral AV3V did not affect the OD + effect after stimulation of the MnPO in most neurones (7/9). Both types of observation are consistent with the presence of an excitatory input to SON through the MnPO. 3. Pressure injection of lidocaine into both the ventral AV3V and the MnPO reversibly blocked the activation of SON neurones following an i.P. injection of 1V5 MNaCl (1 ml) (ventral AV3V 11/11; MnPO, 10/10 cells tested). Injection of lidocaine at both sites, however, did not prevent activation of SON neurones by hypovolaemia (2 ml of blood was withdrawn through a cannula in the right atrium: ventral AV3V, 4/5; MnPO, 4/4 cells tested). The integrity of connections in the ventral AV3V and MnPO thus appeared to be essential for osmotic activation of the SON. 4. Of the 119 ventral AV3V neurones which were tested for their response to MS 8310 8

PHY 431

K. HONDA AND OTHERS 226 electrical stimulation of the SON, forty-nine neurones showed orthodromic excitation (OD + ; n = 33) or initial inhibition followed by excitation (OD - + ; n = 16). Thirty of the forty-nine OD+ or OD - + neurones also showed antidromic excitation (AD) after electrical stimulation of the MnPO. These AD cells provide a pathway by which excitation of the SON might activate the MnPO. 5. All the ventral AV3V neurones which were OD + or OD - + from the SON and also AD from the MnPO were excited (7/7) by hypertonic saline applied directly to them (0-2 M-NaCl applied by pressure ejection through the recording electrode), while none of the neurones in the hippocampus, thalamus and medial septum were excited by the same osmotic stimulation. Four out of seven ventral AV3V neurones of this type also increased their firing rate following an i.P. injection of 1-5 M-NaCl. It is thus likely that such neurones are important for osmoregulation. 6. Of the sixty-three MnPO neurones which were tested for their response to electrical stimulation of the ventral AV3V, thirty-one neurones showed either OD + (n = 20) or OD - + (n = 11) responses. Twenty-five of these neurones could also be antidromically activated by electrical stimulation of the SON. Most MnPO neurones of this type were excited by direction application of hypertonic saline (8/9) and by i.r. injection of 1P5 M-NaCl (8/11). Such MnPO neurones might augment the osmoresponsiveness of SON neurones. 7. Excitatory connections thus exist from SON to ventral AV3V, from ventral AV3V to MnPO and from MnPO to SON. Some neurones in each of these connections were activated by osmotic stimuli which are known to activate SON cells and disruption of the circuit prevented osmotic activation of SON cells. The circuit thus appears to constitute an osmoreceptor complex essential for the osmotic release of neurohypophysial hormones, the whole complex being more sensitive than any of its components alone. INTRODUCTION

The region of the hypothalamus just anterior and ventral to the third ventricle (AV3V) is known to play an important role in the osmotic control of neurohypophysial hormone release (Johnson, 1985a) and of the neuronal activity of the hypothalamic magnocellular neurosecretory neurones (Chaudhry, Dyball, Honda & Wright, 1989; Leng, Blackburn, Dyball & Russell, 1989; Honda, Negoro, Higuchi & Tadokoro, 1989). A prominent structure within the ventral part of the AV3V region (ventral AV3V) is the organum vasculosum of the lamina terminalis (OVLT), which is thought to lack a blood-brain barrier and is thus a likely osmoreceptor site (Ramsay, Thrasher & Keil, 1983; Thrasher & Keil, 1987). Electrolytic damage to this structure impairs the osmotically induced release of vasopressin (Thrasher, Keil & Ramsay, 1982; Sladek & Johnson, 1983; Thrasher & Keil, 1987) and oxytocin (Blackburn, Leng & Russell, 1987; Negoro, Higuchi, Tadokoro & Honda, 1988). Similar lesions reduce the osmosensitivity of the neurones of the paraventricular nucleus (PVN) (Honda et al. 1989) and supraoptic nucleus (SON) (Chaudhry et al. 1989). Local injection of hypertonic solution into the AV3V region was found to excite PVN magnocellular neurones (Honda, Negoro, Higuchi & Tadokoro, 1987). Neuroanatomical studies with retrograde tracers indicate the existence of direct projection from the OVLT to the PVN (Tribollet & Dreifuss, 1981; Johnson, 1985b)

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and to the SON (Tribollet, Armstrong, Dubois-Dauphin & Dreifuss, 1985). Interpreted together the two lines of evidence suggest that osmosensitive elements in the ventral AV3V provide a simple and direct osmotic excitatory drive to the magnocellular neurosecretory neurones.

SF0

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Fig. 1. Schematic representation of the neuronal connections between the supraoptic nucleus (SON), the ventral part of the anteroventral third ventricle region (vAV3V) and the median proptic nucleus (MnPO), which appear (on the basis of the present study) to be involved in the osmotic control of neurohypophysial hormone release. PVN, paraventricular nucleus; SFO, subfornical organ; Pit, pituitary; FX, fornix; AC, anterior commissure; OCH, optic chiasma.

In an earlier investigation we failed to demonstrate the involvement of neurones projecting directly from the ventral AV3V to the SON in such a role, whereas paradoxically we found that neurones in the ventral AV3V which were excited by electrical stimulation of the SON were also excited by raised plasma osmotic pressure (Chaudhry et al. 1989). Such results suggested that the neuronal interactions between the ventral AV3V and the SON involved not only a simple excitatory drive from the ventral AV3V to the SON but also a projection in the reverse direction. Thus osmotic drive from the ventral AV3V to the SON, if it existed, probably involved a polysynaptic pathway. Hormone release studies (Mangiapane, Thrasher, Keil, Simpson & Ganong, 1983; Gardiner, Verbalis & Stricker, 1985; McKinley, Congiu, Miselis, Oldfield & Pennington, 1988) suggested that the region more dorsal to the AV3V region, especially the median preoptic nucleus (MnPO), was also involved in the osmotic control of neurohypophysial hormone release. Neuroanatomical studies indicated the existence of direct projections from the MnPO to the SON (Sawehenko & Swanson, 1983; Johnson, 1985 b; McKinley et al. 1988; Wilkin, Mitchell, Ganten & Johnson, 1989). With these observations in mind, we made the hypothesis that 8-2

K. HONDA AND OTHERS 228 osmotic drive from the ventral AV3V to the SON was mediated through the MnPO and that such input might also be driven by the SON neurones themselves by their projection to the ventral AV3V (Chaudhry et al. 1989) (Fig. 1). The present experiments were undertaken to provide evidence for interconnections between the SON, ventral AV3V and MnPO and for the involvement of the interconnections in the osmotic control of neuronal activity in the SON. We also provide evidence that neurones in these regions, which have the interconnections we describe are themselves osmosensitive. A preliminary account of some of the observation has already been reported (Honda, Negoro, Takano, Higuchi & Dyball, 1989). METHODS

Male Wistar rats (400-500 g BW) were anaesthetized with urethane (1-3 g/kg, I.P.) and the trachea cannulated. Extracellular recordings were obtained using glass micropipettes (tip diameter, 1 /sm; impedance, 20-30 MCI), from neurones in one of the following three sites: SON, ventral AV3V, MnPO, while side-by-side bipolar stimulating electrodes (tip diameter and distance between the two tips, 0-1 mm and 50 ,um respectively) were inserted into the other two sites. In some experiments a glass micropipette (tip diameter; 7,um) filled with 10% lidocaine was attached to the stimulating electrode with cyanoacrylate adhesive (tip separation, 60 ltm) and introduced into the ventral AV3V and/or MnPO to anaesthetize these regions locally by pressure injection of the anaesthetic. The volume of the solution ejected by pressure was calibrated before inserting the micropipette into the brain; the tip of the pipette was immersed in oil and the diameter of a droplet of ejected solution was measured under a microscope. The pressure was fixed at 40 lbf/in2 and the ejection volume was controlled by changing the duration of the application period; application of pressure (40 lbf/in2) for 2-5 s ejected approximately 50 nl of solution. In a preliminary experiment diffusion of injected lidocaine from the MnPO to the ventral AV3V was assessed by recording neurones in the ventral AV3V which were antidromically activated by electrical stimulation of the SON. It was found that the threshold and latency of the antidromic spike was not affected by lidocaine injection with a volume of up to 100 nl (Fig. 2) that a volume of 50 nl was used in later experiments. For recording from the SON an additional stimulating electrode was placed on the neural stalk to identify SON neurosecretory neurones antidromically and a cannula was inserted into the right atrium through the jugular vein so that blood could be withdrawn for hypovolaemic stimulation (2 ml). A dorsal approach was used to insert recording or stimulating electrodes into the MnPO, and a ventral approach to insert the electrodes into the neural stalk, SON and ventral AV3V (see Chaudhry et al. 1989). All the stimulating electrodes except those placed on the neuronal stalk, which was held in place with a microelectrode holder, were introduced stereotaxically and secured in place with acrylic resin and self tapping screws applied to the skull before starting the recording. Extracellular recordings were made from single neurones in each region using conventional techniques. Spike trains were recorded on magnetic tape for subsequent analysis. Peristimulus time histograms and averaged spike waveforms were prepared using a signal processor (Signal Processor 7TO7A, SAN-EI Instrument, Tokyo, Japan). Ratemeter histograms were prepared using a penrecorder. Neurones encountered in each region were tested for their responses to electrical stimulus pulses applied to each of the other two regions (biphasic square-wave pulses, 0 7 mA, total duration 1ms). When orthodromic effects were evaluated, a more than 50 % change of firing rate for a period of more than 10 ms after the stimulus was regarded as a positive effect. The mean prestimulus firing rate was calculated from a 100 or 200 ms period before the stimulus. Some cells were further tested for their response to direct application of 0-2M-NaCl solution by pressure ejection through the recording electrode or to an intraperitoneal injection of 1 ml of 1-5M-NaCl. For the direct application of hypertonic saline, divided, theta glass micropipettes were used. The open end of one side was sealed with wax. A small hole was made in the wall of this side and 0 5Msodium acetate containing 2 % Pontamine Sky Blue 6B (Tokyo Kasei Kogyo, Japan) was introduced through the hole. The other side was filled with the hypertonic saline (0-2M) which was connected to the microelectrode amplifier (DPZ- 16A, Dia-Medical System, Tokyo, Japan) by a silver wire and acted as the electrolyte for recording. The entire end of the pipette was then so

~ ~ ~>1

OSMORECEPTIVE CIRCUITS

229

connected to a pressure source (Pico-Pump, PV-820, WPI Instrument) by a nondistensible polyethylene tube. These techniques allowed the application of hypertonic solution directly to the recorded neurone by pressure without diffusion of dye from the other barrel and also allowed the deposition of dye into the recording site by passing a direct cathodal current of 5 ,ZA through a C

A Jr

*

Control

*

1 min after

1 min after

*

B

*,

Control

5 min after

Control

15 min after

1 min after

E LO

10 ms

Fig. 2. Traces showing antidromic activation of a ventral AV3V neurone following electrical stimulation of the SON (stimulus at arrows) with threshold current intensity (07 mA) before and after pressure injection of lidocaine into the MnPO (each trace indicates averaged waveform of five sweeps). The antidromic spike latency and threshold were not affected by the injection of 50 nl (A) and 100 nl (B), but they were obviously and reversibly affected by injection of 200 nl (C). The results indicate that lidocaine injections with a volume of up to 100 nl did not diffuse from the MnPO to the ventral AV3V in amounts sufficient to influence cell excitability in the ventral AV3V. silver wire placed in the dye barrel for 5 min. When direct application of hypertonic solution was not used, recordings were made with conventional single barrelled electrodes filled with sodium acetate and Pontamine Sky Blue. A response to direct application of hypertonic solution was regarded as positive if the firing rate (spikes/10 s) during the application period (ten periods counted) was significantly different (P < 005, Mann-Whitney U test) from that immediately before the application. When the responses of neurones to i.P. injection of hypertonic saline were tested, the firing rate was monitored for ten periods every 5 min as described above. A neurone was regarded as having been excited or inhibited by the injection if the firing rate 20 min after the hypertonic injection was significantly different (P < 0 05, Mann-Whitney U test) from that before the injection.

K. HONDA AND OTHERS

230

At the end of each experiment, a direct current of 0-7 mA was passed through each stimulating electrode for 5 s to make an electrolytic lesion at both stimulus sites in addition to the marks made at the recording sites. After perfusion fixation with 10% formaldehyde the brains were cut coronally in 50 ,um slices using a freezing microtome. The stimulating and the recording sites were A

B

~~~~~~~~~~16

16

E

200 ms

Ln

X16

200 ms

16

80 200 ms

200 ms

Fig. 3. Representative peristimulus time histograms (100 sweeps; stimulus at arrows) showing orthodromic excitation (A) and initial inhibition followed by excitation (B) of SON neurones in response to electrical stimulation of the ventral AV3V (upper traces) and the MnPO (lower traces).

histologically examined. Recordings were only included in the analysis if the stimulating and recording electrodes inserted into the MnPO were confirmed to be situated in the dorsal part of the nucleus (less than 350 /4am dorsal to the anterior commissure and 250 ,um from the mid-line). Those inserted into the ventral AV3V were confirmed to be situated in the region just dorsal (within 600 ,um) to the optic tract at the rostral end of the third ventricle and within 250 ,um of the mid-line. Those inserted into the SON were also confirmed to be situated within the nucleus. RESULTS

Recording from the SON Forty-eight neurosecretory neurones (twenty-three phasic, twenty-three continuous and two slow firing neurones) were recorded from the SON and they fell into four categories according to their responses to stimulus pulses (single or double pulses with 3 ms interval) applied to the ventral AV3V: orthodromic excitation (OD+, n = 15, latency and duration were 37-0±8-0 ms (mean± S.E.M.) and 327-0 + 290 ms), initial short-duration inhibition followed by long-duration excitation (OD - +, n = 30, latency and duration of OD + effect were 72-0 + 4 0 ms and 364-0 + 170 ms), simple inhibition (OD-, n = 2, latency and duration were 23-0 and 40 0 ms), and no effect (n = 1). These forty-eight neurones were also tested for their responses to stimulation of the MnPO and fell into four categories: OD+ (n = 27, latency and duration were 25-0+3-0 ms and 361V0+26-0 ms), OD- + (n = 19,

OSMORECEPTIVE CIRCUITS 231 latency and duration of OD + effect were 69-0 + 9 0 ms and 348-0 + 360 ms), OD (n = 1, latency and duration were 15-0 ms and 90 0 ms), and no effect (n = 1). Thus most (44/48) of the neurones tested showed an OD+ or OD - + response to stimulation of both sites. The latency of the OD+ or the OD+ component of the A

B

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200 ms UMn 16 E'-

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200 ms

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200 ms Fig. 4. Peristimulus time histograms (100 sweeps; stimulus at arrows) showing responses of a SON neurone to electrical stimulation of the ventral AV3V before (upper trace) and after (middle trace, 0-5 min; lower trace, 15-20 min) the pressure injection of lidocaine (50 nl) into the MnPO (A) and those showing responses of another SON neurone to electrical stimulation of the MnPO before (upper trace) and after (lower trace; 0-5 min) the injection of lidocaine into the ventral AV3V (B). Note that injection of lidocaine into the MnPO (A) reversibly depressed the orthodromic excitation following ventral AV3V stimulation, while injection into the ventral AV3V (B) did not affect the excitatory response following MnPO stimulation.

OD - + responses produced by stimulation of the MnPO was significantly (paired t test, P < 0-01) shorter than that following stimulation of the ventral AV3V. The threshold current intensity which evoked a clear OD+ or OD - + effect was 0-2 ±0-1 mA (n = 6 at each site). Representative responses are shown in Fig. 3. None of the SON neurones tested was activated antidromically (AD) by stimulus pulses applied to these regions. To test the possibility that the effect of stimulating the ventral AV3V on SON neurones was mediated through the MnPO, the local anaesthetic, lidocaine (10% solution, 50 nl) was injected by pressure into the MnPO through a glass micropipette

K. HONDA AND OTHERS

232

attached to the stimulating electrode in the MnPO. Such injections reversibly depressed the OD+ or OD+ component of OD - + responses to ventral AV3V stimulation in all the SON neurones tested (11/11) (Fig. 4A). On the other hand injection of lidocaine into the ventral AV3V did not affect the OD + or OD - + effect Lidocaine vAV3V

1.5 M-NaCI

Lidocaine MnPO

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10 min

Lidocaine vAV3V

1.5 M-NaCI c

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Fig. 5. Upper trace: ratemeter record showing inhibition of the osmotic activation of an SON neurone (i.P. injection of 1-5 M-NaCl, 1 ml) by pressure injection of lidocaine (50 nl) into the ventral AV3V or the MnPO. The increase in firing rate following i.P. injection of hypertonic saline was completely blocked after lidocaine injection into each of the two regions and the effects were reversible. Lower trace: ratemeter record showing an SON neurone which retained its capacity to respond to hypovolaemic stimulation (2 ml of blood was withdrawn through a cannula in the right atrium) during blockade of osmotic activation. Open and filled bars indicate the time at which blood was removed and replaced, respectively. Excitation of both neurones immediately after I.P. injection of hypertonic saline was probably caused by transient changes in blood pressure (Brimble & Dyball, 1977).

of MnPO stimulation in most (7/9) of the neurones tested (Fig. 4B). The OD+ responses of the remaining two neurones were depressed by the injection, although one of these two neurones showed a peristimulus time histogram with two peaks before the injection and only the second peak was depressed after the injection. To assess the functional importance of the pathway we proposed for the osmotic activation of SON neurones, either the ventral AV3V or the MnPO region was acutely and locally anaesthetized in rats given an i.P. injection of 1-5 M-NaCl (1 ml). Such anaesthesia reversibly inhibited the increase in firing rate of all the SON neurones tested (ventral AV3V, 11/11; MnPO, 10/10 cells tested; Fig. 5). The inhibition lasted for 14 1+ 3-6 min (n = 11) following the injection of lidocaine into the ventral AV3V and 16-5 + 3-1 min (n = 10) following injection into the MnPO. By contrast most of the SON neurones tested still responded to hypovolaemia during local anesthesia-induced inhibition of their osmotic activation (ventral AV3V injection, 4/5; MnPO injection, 4/4 cells tested; Fig. 5).

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To confirm the earlier findings that SON neurones have intrinsic osmosensitivity (Leng, 1980; Mason, 1980; Abe & Ogata, 1982), the responses of SON neurones to direct application of hypertonic saline (0-2 M) were examined and compared with those of hippocampal, thalamic and medial septal neurones. Most SON neurones 64 1 E 0

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200 ms

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Fig. 6. Trace (top left) showing antidromic activation of a ventral AV3V neurone after electrical stimulation of the MnPO (stimulus at arrows). Two stimulus pulses were applied just after the occurrence of spontaneous spike. No antidromic spike appeared after the first pulse (collision) but a spike appeared after the second one with constant latency (averaged waveform of five sweeps). The trace (top right) is a peristimulus time histogram (100 sweeps) obtained from the same neurone showing initial inhibition followed by excitation (OD - +) after application of stimulus pulse to the SON (stimulus at arrow). This neurone was asumed to receive synaptic input from the SON and also to project to the MnPO. The lower trace is a ratemeter record showing excitation of the same neurone by direct application of hypertonic saline (application at horizontal bars at pressure indicated). Since the spontaneous activity of this neurone was low, an electrical stimulus was applied to the MnPO every 10 s to monitor the antidromic spike.

(21/23) were found to be excited by such stimulation; two were unresponsive. By contrast none of the tested neurones in the hippocampus, thalamus or medial septum were excited by the same stimulation (10/11). The remaining one (thalamic neurone) was inhibited. Recording from the ventral A V3V In an earlier investigation we found that ventral AV3V neurones excited by i.P. injection of hypertonic saline showed OD+ or OD - + response after electrical stimulation of the SON (Chaudhry et al. 1989). We therefore set out to test the

K. HONDA AND OTHERS

234

possibility that such AV3V neurones might project to the MnPO and consequently affect the neuronal activity of the SON. One hundred and nineteen neurones were recorded from the ventral AV3V and they fell into six main categories according to their responses to stimulus pulses applied to the SON; AD (n= 17, latency and

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0

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ms ~~~~~~~~~~~~100

1.5 M-NaCI

10 min

Fig. 7. Traces (top) showing the same type of responses of a ventral AV3V neurone to electrical stimulation of the MnPO (left, AD) and the SON (right, GD -+) as those indicated in Fig. 6. The lower trace is a ratemeter record showing excitation of this neurone by I.P. injection of hypertonic saline (injection at arrow).

threshold were 12.0 +1-0 ms and 1-3+_0-2 mA), OD+ (n = 33, latency and duration were 61P0+9-O ms and 122@0+27@0 ins), OD- + (n = 16, latency and duration of OD± effect were 121PO+4S*0 ms and 236*O+ 124'0 ins), OD- (n = 20, latency and duration were 33 0_ +110 ms and 182*O0± 69*0 ins), no effect (n = 32) and complex cycle of excitation and inhibition (complex, n = 1). These 119 neurones were also tested for their responses to stimulation of the MnPO and fell into six categories: AD (n = 72, latency and threshold were 8-0+ 1S0 ms and 1-2+0S1 mA), OD + (n = 30, +110 ins), OD -+ (n = 11, latency latency and duration were 33 0 + 4 0 ms and 59O _ and duration of OD + effect were 103*0 +20*0 ms and 212*0 +42*0 ins), OD -(n = 3, latency and duration were 10*0+10 ms and 103*0+ 18*0 ins), no effect (n = 2), complex (n = 1). Of the forty-nine neurones which were OD + or OD-+ from the SON, thirty were AD from the MnPO. These neurones were assumed to receive either excitatory or inhibitory synaptic inputs from the SON and to project to the MnPO. We tested the intrinsic osmosensitivity of some of these neurones; 0-2 M-NaCl was applied to the recorded cell by pressure through the recording electrode. All the neurones which were OD + (nt = 6) or OD -+ (n = 1) from the SON and also AD from the MnPO were excited by direct application of hypertonic saline (Fig. 6). For comparison the responses of neurones of this type to the I.P. injection of 1a5M-NaCl were also tested. Four out of seven neurones which were OD + (two out of three) or

OSMORECEPTIVE CIRCUITS

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OD - + (two out of four) from the SON and also AD from the MnPO were excited by the injection (Fig. 7). The remaining three were unresponsive. Recording from the MnPO Since the MnPO is known to project to the SON (Sawchenko & Swanson, 1983; Johnson, 1985b; McKinley et al. 1988; Wilkin et al. 1989), we tested the possibility 16 n

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Fig. 8. Traces showing antidromic activation of a MnPO neurone following electrical stimulus pulses applied to the SON (top left, stimulus at arrows); see legend of Fig. 6 for detailed description, and a peristimulus time histogram (100 sweeps) obtained from the same neurone showing initial inhibition followed by excitation (OD - +) after electrical stimulus pulse applied to the ventral AV3V (top right, stimulus at arrow). This neurone was assumed to receive synaptic input from the ventral AV3V and also to project to the SON. The lower trace is a ratemeter recording obtained from the same neurone showing excitatory response to direct application of hypertonic saline (application at horizontal bars at pressure indicated).

that the neurones in this region also receive synaptic input from the ventral AV3V and transmit osmotic drive to the SON. Sixty-three neurones were recorded from the MnPO and they fell into five categories according to their responses to electrical stimulus pulses applied to the ventral AV3V: AD (n = 13, latency and threshold were 9-0 + 2-0 ms and 1-0 + 0-2 mA), OD + (n = 20, latency and duration were 36-0+7-0 ms and 53-0+ 12-0 ms), OD- + (n = 11, latency and duration of OD+ effect were 75-0 + 8-0 ms and 1 13-0 + 21-0 ms), OD - (n = 2, latency and duration were 18-0 ms and 186-0 ms), no effect (n = 17). These sixty-three neurones were also tested for their responses to SON stimulation and fell into five categories according

K. HONDA AND OTHE'RS

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to their response to stimulation at the second site: AD (n = 39, latency and threshold were 110+ 1 0 ms and 1 4+0 1 mA). OD+ (n = 8, latency and duration were 470+ 160 ms and 1040±350 ms), OD- + (n = 1, latency and duration of OD+ effect were 910 ms and 810 ms), OD- (n = 3, latency and duration were 32 cn E 5

ms

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Fig. 9. Traces (top) showing the same tvpe of responses of a MInPO neurone to electrical stimulus pulses applied to the SON (left. AD) and the Xventral AX3V' (right, OD- +) as those indicated in Fig. 8. The lower trace is a ratemeter recording showing excitation of this neurone after i.P. injection of hypertonic saline (injection at arrow).

260+ 130 ms and 1090+270 ms), no effect (n = 12). Of the thirty-one neurones which were OD + or OD - + from the ventral AX3V. twenty-five were AD from the SON. These neurones were assumed to receive either excitatorv or inhibitory synaptic inputs from the ventral AN3V and also to project to the SON. We tested the intrinsic osmosensitivity of some of these neurones. Eight out of the nine neurones, which were OD+ (three out of four) or OD- + (five out of five) from the ventral AV3V and AD from the SON, were excited by the direct application of hypertonic solution (Fig. 8). The remaining one was unresponsive. We also tested eleven neurones which were OD + (n = 8) or OD - + (n = 3) from the ventral AV3V and AD from the SON for their responses to i.P. injection of hypertonic saline: eight were found to be excited (Fig. 9). The remaining three (OD+ from the SON) were unresponsive. DISCUSSION

Electrical stimulus pulses applied to the ventral AV3V' and to the MnPO produced orthodromic excitation or initial short duration inhibition followed by long duration excitation in most SON neurosecretory neurones tested. The results confirm the

OSMORECEPTIVE CIRCUITS 237 earlier findings (Leng et al. 1989) that the AV3V region provids a predominantly excitatory influence to the SON. Since injection of local anaesthetic into the MnPO blocked the effect, the results also suggest that the effect of stimulating the ventral AV3V on SON neurones was mediated by MnPO neurones although we cannot exclude the possibility that the injection of lidocaine acted not only on cell bodies but also on fibres of passage. Since, as discussed later, we found that many neurones in the MnPO receive synaptic inputs from the ventral AV3V and also project to the SON, it seems very likely that the effect of lidocaine injection into the MnPO was due to anaesthesia of the cell bodies of neurones in the MnPO. If the pathway through the MnPO is specifically involved in the osmotic control of activity in the SON and transmits osmotic information to the SON, blockade of the pathway would be expected to inhibit the osmotic activation of SON neurones. By contrast, SON neurones might be expected to respond to non-osmotic stimuli even during blockade of the pathway. The suggestion is supported by the blockade by lidocaine injected into the MnPO or ventral AV3V of the excitation of SON neurones in rats given an i.P. injection of 1P5 M-NaCl solution. Local anaesthesia of each of these regions completely blocked osmotic activation of SON neurones but they were still capable of responding to hypovolaemic stimulation (Fig. 5). These results, however, do not exclude the possibility that osmotic activation of SON neurones is brought about by a small membrane depolarization of the magnocellular neurosecretory neurones themselves in combination with excitation by tonic inputs from the AV3V region which are themselves not necessarily osmosensitive (Leng, Mason & Dyer, 1982). Although an in vitro study by Sayer, Hubbard & Sirett (1984) indicated that some OVLT neurones are osmosensitive, we know of no evidence that neurones in the ventral AV3V projecting directly to the magnocellular neurosecretory neurones are osmosensitive. By contrast an earlier attempt to demonstrate the osmosensitivity of ventral AV3V neurones projecting directly to the SON was unsuccessful; instead we found that neurones which were OD+ or OD - + from the SON were excited by raised plasma osmotic pressure (Chaudhry et al. 1989). In the present series of experiments, therefore, we made recordings from the ventral AV3V to test the hypothesis that osmoresponsive neurones in the ventral AV3V region may affect the neuronal activity of the SON by synapsing in the MnPO. Electrical stimulus pulse applied to the SON produced both orthodromic and antidromic excitation of ventral AV3V neurones. The result was in good agreement with our previous findings that the neuronal interactions between the ventral AV3V and the SON involve not only projections from the ventral AV3V to the SON but also those in the reverse direction (Chaudhry et al. 1989). We cannot completely exclude the possibility that the effects of stimulating the SON on ventral AV3V neurones may result from stimulus spread outside the nucleus. However, we found in the previous experiments that stimulus pulses applied to the neural stalk instead of the SON produced qualitatively similar orthodromic effects on ventral AV3V neurones (Chaudhry et al. 1989). Since in the present experiments we used stimulus conditions similar to those used earlier, the effects of stimulating the SON which we reported here are most probably mainly caused by the activation of SON neurones themselves. If this was the case, axon collaterals or processes from SON neurones may mediate

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the orthodromic effects on ventral AV3V neurones. In fact, Leng et al. (1989) found that a small proportion of SON neurones responded antidromically to electrical stimulation of the AV3V region. However, it is also likely that the orthodromic effect is mainly produced by polysynaptic -connections, since we encountered no SON neurones which responded antidromically to electrical stimulation of the ventral AV3V. Electrical stimulation of the MnPO also affected ventral AV3V neurones. The results suggest the existence of reciprocal neuronal interconnections between the two regions. Of these interconnections, a direct projection from the ventral AV3V to the MnPO appeared to be of major importance since 61 % of ventral AV3V neurones responded antidromically to electrical stimulation of the MnPO. Such a strong connection was also suggested by the neuroanatomical study of Saper & Levisohn (1983). The results of the present experiments further indicated that 61 % of the neurones in the ventral AV3V which received excitatory synaptic inputs (OD+ or OD - ±) from the SON projected to the MnPO. All the neurones of this type were osmosensitive (excited by direct application of hypertonic saline) and many of them increased their firing rate when plasma osmotic pressure increased. Our results, therefore, suggest that osmotic drive produced by the ventral AV3V, part of which may also be driven by the SON, is transmitted to the MnPO. In the next series of experiments, recordings were made from neurones in the MnPO to test whether or not osmotic drive from the ventral AV3V was transmitted to the SON by the MnPO. Neurones in the MnPO responded in a number of different ways to electrical stimulation of the ventral AV3V and of the SON. The most commonly encountered response to stimulation of the ventral AV3V appeared to be orthodromic excitation or initial inhibition followed by excitation (49 %), and that to stimulation of the SON appeared to be antidromic activation (62 %). The finding of the latter response was in good agreement with an earlier electrophysiological study which indicated the existence of direct projection from the MnPO to the PVN (Tanaka, Saito & Kaba, 1987) and with neuroanatomical tract tracing studies (Misells, Shapiro & Hand, 1979; Sawchenko & Swanson, 1983; Johnson, 1985b; Tribollet et al. 1985; Wilkin et al. 1989). The results of the present experiments indicated in addition that many (64 %) of MnPO neurones projecting to the SON also received synaptic input (OD + or OD - +) from the ventral AV3V. Most of MnPO neurones of this type increased their firing rate when plasma osmotic pressure was raised. The results clearly suggest that osmotic drive from the ventral AV3V is transmitted to the SON via the MnPO neurones which project to the SON. Such a relay may provide the basis for the amplification of the signal at the MnPO, since many MnPO neurones which have the appropriate neuronal connections were themselves osmosensitive. In an attempt to draw all these observations together we now propose that both the neuronal interconnections between the SON, the ventral AV3V and the MnPO, and the intrinsic osmosensitivity of neurones in these regions contribute to the osmotic control of SON neuronal activity. When plasma osmotic pressure rises, neurones in each of these regions may increase their firing rate to some extent because of their own intrinsic osmosensitivity. Neurones in the SON, ventral AV3V and MnPO may, in addition, drive the next neurones in the chain that is in the

OSMORECEPTIVE CIRCUITS 239 ventral AV3V, MnPO and SON, respectively (Fig. 1). The intrinsic osmosensitivity of neurones in each region and the neuronal interconnections may thus co-operate to increase the firing rate of neurones in each region. We observed an initial inhibition followed by excitation (OD - +) in a considerable proportion of neurones in each region. The nature of this type of response is unclear at present. One of the possible explanations of the OD - + response is that two pathways were stimulated, although we have no direct evidence for this. The direct application of hypertonic saline to ventral AV3V and MnPO neurones excited greater numbers of neurones than the i.P. injection of hypertonic saline. A possible explanation for these observations may be that the change in the extracellular osmolarity was more profound when hypertonic saline was applied locally to the recorded cell compared with the situation when i.P. injection was used. It is unlikely that the direct osmotic stimulation we used in the present study was nonspecific, since none of neurones in other parts of the central nervous system which are not thought to be related to osmoregulation were excited by the same stimulus. Further, Leng (1980) reported that the excitatory effect of local applications of hypertonic saline, at a concentration which was more than 5-times higher than that used in the present experiments, was comparatively ineffective in exciting nonneurosecretory neurones close to the SON when compared with its effects on SON neurosecretory neurones. Whichever stimulus (direct application or i.P. injection) more nearly mimics physiological conditions, the argument remains the same since both stimuli excite ventral AV3V neurones which project to MnPO and MnPO neurones which project to SON. The results of the present experiments do not exclude the involvement of other neuronal interconnections in the osmotic control of magnocellular neurosecretory neurones such as connections between the subfornical organ (SFO) and the SON (Sgro, Ferguson & Renaud, 1984) or the PVN (Ferguson, Day & Renaud, 1984). The SFO is a circumventricular organ, the electrolytic destruction of which also attenuates the osmotically induced release of vasopressin (Mangiapane, Thrasher, Keil, Simpson & Ganong, 1984) and of oxytocin (Leng et al. 1989). Local anaesthesia of the SFO attenuates the osmotic activation of some PVN vasopressin neurones (Tanaka, Saito & Yagyu, 1989). Gutman, Ciriello & Mogenson (1988) indicated that some neurones in the SFO projecting either to the SON or the PVN were excited by intracarotid injection of hypertonic saline. On the other hand an in vitro study (Buranarugsa & Hubbard, 1979) indicated that neurones in the SFO were not osmosensitive and SFO neurones which were activated antidromically by electrical stimulation of the SON were unresponsive to the i.P. injection of hypertonic saline (Dyball & Leng, 1989), so that the osmosensitivity of SFO neurones and their role in the osmotic control of magnocellular neuronal activity is still unresolved. We conclude that the neuronal interactions between the ventral AV3V and the SON which regulate osmotically induced vasopressin and oxytocin secretion are more complex than a simple excitatory drive from the ventral AV3V to the SON. There is also a projection in the reverse direction, which extends back to the SON via the MnPO. Both these neuronal connections and intrinsic osmosensitivity of the neurones involved are probably important for the osmotic control of neuronal activity in the SON.

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A part of this work was supported by grants-in-aid for scientific research (60770141, 61770118) from the Ministry of Education, Science and Culture of Japan and the Medical Research Council. We also wish to thank Miss R. Tsurube for her assistance in the preparation of this manuscript. REFERENCES ABE, H. & OGATA, N. (1982). Ionic mechanism for the osmotically-induced depolarization in neurones of the guinea-pig supraoptic nucleus in vitro. Journal of Physiology 327, 157-171. BLACKBURN, R. E., LENG, G. & RUSSELL, J. A. (1987). Control of magnocellular oxytocin neurones by the region anterior and ventral to the third ventricle (AV3V region) in rats. Journal of Endocrinology 114, 253-261. BRIMBLE, M. J. & DYBALL, R. E. J. (1977). Characterization of the responses of oxytocin- and vasopressin-secreting neurones in the supraoptic nucleus to osmotic stimulation. Journal of Physiology 271, 253-271. BURANARUGSA, P. & HUBBARD, J. I. (1979). The neuronal organization of the rat subfornical organ in vitro and a test of the osmo- and morphine-receptor hypotheses. Journal of Physiology 291, 101-116. CHAUDHRY, M. A., DYBALL, R. E. J., HONDA, K. & WRIGHT, N. C. (1989). The role of interconnection between supraoptic nucleus and anterior third ventricular region in osmoregulation in the rat. Journal of Physiology 410, 123-135. DYBALL, R. E. J. & LENG, G. (1989). Hypothalamic microcircuits involved in osmoregulations. Biomedical Research 10, suppl. 3, 21-32. FERGUSON, A. V., DAY, T. A. & RENAUD, L. P. (1984). Subfornical organ efferents influence the excitability of neurohypophyseal and tuberoinfundibular paraventricular nucleus neurons in the rat. Neuroendocrinology 39, 423-428. GARDINER, T. W., VERBALIS, J. G. & STRICKER, E. M. (1985). Impaired secretion of vasopressin and oxytocin in rats after lesions of nucleus medianus. American Journal of Physiology 249, R681-688. GUTMAN, M. B., CIRIELLO, J. & MOGENSON, G. J. (1988). Effects of plasma angiotensin II and hypernatremia on subfornical organ neurons. American Journal of Physiology 254, R746-754. HONDA, K., NEGORO, H., HIGUCHI, T. & TADOKORO, Y. (1987). Activation of neurosecretory cells by osmotic stimulation of anteroventral third ventricle. American Journal of Physiology 252, R1039-1045. HONDA, K., NEGORO, H., HIGUCHI, T. & TADOKORO, Y. (1989). The role of the anteroventral 3rd ventricle area in the osmotic control of paraventricular neurosecretory cells. Experimental Brain Research 76, 497-502. HONDA, K., NEGORO, H., TAKANO, S., HIGUCHI, T. & DYBALL, R. E. J. (1989). The role of neuronal circuits between supraoptic nucleus, ventral anteroventral third ventricular region and median preoptic nucleus in osmoreception in the rat. 4th International Conference on the Neurohypophysis (abstract), 22. JOHNSON, A. K. (1985a). Role of periventricular tissue surrounding the anteroventral third ventricle (AV3V) in the regulation of body fluid homeostasis. In Vasopressin, ed. SCHRIER, R. W., pp. 319-331. Raven Press, New York. JOHNSON, A. K. (1985b). The periventricular anteroventral third ventricle (AV3V): its relationship with the subfornical organ and neural systems involved in maintaining body fluid homeostasis. Brain Research Bulletin 15, 595-601. LENG, G. (1980). Rat supraoptic neurones: the effects of locally applied hypertonic saline. Journal of Physiology 304, 405-414. LENG, G., BLACKBURN, R. E., DYBALL, R. E. J. & Russell, J. A. (1989). Role of anterior peri-third ventricular structures in the regulation of supraoptic neuronal activity and neurohypophysial hormone secretion in the rat. Journal of Neuroendocrinology 1, 35-46. LENG, G., MASON, W. T. & DYER, R. G. (1982). The supraoptic nucleus as an osmoreceptor. Neuroendocrinology 34, 75-82. MCKINLEY, M. J., CONGIU, M., MISELIS, R. R., OLDFIELD, B. J. & PENNINGTON, G. (1988). The lamina terminalis and osmotically stimulated vasopressin secretion. In Recent Progress in Posterior Pituitary Hormones, ed. YOSHIDA, S. & SHARE, L., pp. 117-124. Elsevier, Amsterdam.

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MANGIAPANE, M. L., THRASHER, T. N., KEIL, L. C., SIMPSON, J. B. & GANONG, W. F. (1983). Deficits in drinking and vasopressin secretion after lesions of the nucleus medianus. Neuroendocrinology 37, 73-77. MANGIAPANE, M. L., THRASHER, T. N., KEIL, L. C., SIMPSON, J. B. & GANONG, W. F. (1984). Role for subfornical organ in vasopressin release. Brain Research Bulletin 13, 43-47. MASON, W. T. (1980). Supraoptic neurones of rat hypothalamus are osmosensitive. Nature 287, 154-157. MISELLS, R. R., SHAPIRO, R. E. & HAND, P. J. (1979). Subfornical organ efferents in neuronal systems for control of body water. Science 205, 1022-1025. NEGORO, H., HIGUCHI, T., TADOKORO, Y. & HONDA, K. (1988). Osmoreceptor mechanism for oxytocin release in the rat. Japanese Journal of Physiology 38, 19-31. RAMSAY, D. J., THRASHER, T. N. & KEIL, L. C. (1983). The organum vasculosum laminae terminalis: a critical area of osmoreception. Progress in Brain Research 60, 91-98. SAPER, C. B. & LEVISOHN, D. (1983). Afferent connections of the median preoptic nucleus in the rat: anatomical evidence for cardiovascular integrative mechanism in the anteroventral third ventricular (AV3V) region. Brain Research 288, 21-31. SAWCHENKO, P. E. & SWANSON, L. W. (1983). The organization of forebrain afferents to the paraventricular and supraoptic nuclei of the rat. The Journal of Comparative Neurology 218, 121-144. SAYER, R. J., HUBBARD, J. I. & SIRETT, N. E. (1984). Rat organum vasculosum laminae terminalis in vitro: responses to transmitter. American Journal of Physiology 247, R374-379. SGRO, S., FERGUSON, A. V. & RENAUD, L. P. (1984). Subfornical organ-supraoptic nucleus connections: an electrophysiological study in the rat. Brain Research 303, 7-13. SLADEK, C. D. & JOHNSON, A. K. (1983). Effect of anteroventral third ventricle lesions on vasopressin release by organ-cultured hypothalamo-neurohypophyseal explants. Neuroendocrinology 37, 78-84. TANAKA, J., SAITO, H. & KABA, H. (1987). Subfornical organ and hypothalamic paraventricular nucleus connections with median preoptic nucleus neurons: an electrophysiological study in the rat. Experimental Brain Research 68, 579-585. TANAKA, J., SAITO, H. & YAGYU, K. (1989). Impaired responsiveness of paraventricular neurosecretory neurones to osmotic stimulation in rats after local anesthesia of the subfornical organ. Neuroscience Letters 98, 51-56. THRASHER, T. N. & KEIL, L. C. (1987). Regulation of drinking and vasopressin secretion: role of organum vasculosum laminae terminalis. American Journal of Physiology 253, R108-120. THRASHER, T. N., KEIL, L. C. & RAMSAY, D. J. (1982). Lesion of the organum vasculosum of the lamina terminalis (OVLT) attenuate osmotically-induced drinking and vasopressin secretion in the dog. Endocrinology 110, 1837-1839. TRIBOLLET, E., ARMSTRONG, W. E., DUBoIs-DoUPHIN, M. & DREIFUSS, J. J. (1985). Extrahypothalamic afferent inputs to the supraoptic nucleus area of the rat as determined by retrograde and anterograde tracing techniques. Neuroscience 15, 135-148. TRIBOLLET, E. & DREIFUSS, J. J. (1981). Localization of neurones projecting to the hypothalamic paraventricular nucleus area of the rat: a horseradish peroxidase study. Neuroscience 6, 1315-1328. WILKIN, L. D., MITCHELL, L. D., GANTEN, D. & JOHNSON, A. K. (1989). The supraoptic nucleus: afferents from areas involved in control of body fluid homeostasis. Neuroscience 28, 537-584.

The osmoreceptor complex in the rat: evidence for interactions between the supraoptic and other diencephalic nuclei.

1. Experiments were undertaken to provide evidence for the existence of a circuit of neuronal interconnections between the supraoptic nucleus (SON), t...
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