Physiology & Behavior, Vol. 15, pp. 713-722. Pergamon Press and Brain Research Publ., 1975. Printed in the U.S.A.

Lateral Preoptic and Lateral Hypothalamic Units: In Search of the Osmoreceptors for T h i r s t I CHARLES S. WEISS 2 AND C. ROBERT ALMLI Ohio University, Athens OH 45701 (Received 14 April 1975) WEISS, C. S. and C. R. ALMLI. Lateral preoptic and lateral hypothalamic units: in search o f the osmoreceptors for thirst. PHYSIOL. BEHAV. 15(6) 713-722, 1975. - Single unit activity was recorded from a variety of brain structures after subcutaneous injection of 16 percent NaCl. Units of the lateral preoptic area (LPO), lateral hypothalamic area (LH) and tractus striohypothalamicus changed activity patterns prior to the behaviorally determined onset of drinking (5.73 min) and had estimated osmolality thresholds less than those associated with the behavioral onset of drinking (1.91 percent). Five distinct activity patterns were recorded for these osmotically sensitive cells. Units from other brain areas either showed no response or responded with activity changes not related to the onset of drinking. The role of LPO and LH cells as osmoreceptors for drinking behavior is discussed. Osmoreceptors Lateral preoptic area Singleunit recording Salt induced drinking Drinking thresholds Thirst

Lateral hypothalamic area

Drinking behavior

and the lateral preoptic area (LPO; [ 5,12] ). Damage to the LH produces temporary adipsia and a stereotyped recovery syndrome. For animals with LH damage, drinking is attenuated or abolished (as a function of lesion locus and extent) following cellular stimulation [5,36] and extracellular stimulation [5,36]. Complete and permanent adipsia has been shown to occur after damage to the posterior aspect of the LH [5, 29, 30]. Blass [11] noted that extensive damage to the frontalpole-area caused either a reduction in drinking or total lack of drinking in response to cellular stimuli. A more precise study, undertaken by Blass and Epstein [12], sought to identify the specific forebrain locus related to cellular dehydration and the drinking response. The authors showed that damage to the LPO resulted in a reduction of water intake to cellular dehydration but no change to hypovolemia. On the basis of this evidence and local chemical stimulation data, Blass and Epstein then concluded that the LPO is the 'osmosensitive zone for thirst'; the work of Peck and Novin [32] using rabbits supports this interpretation. Recently the viability of this hypothesis (stating that the LPO is the osmosensitive zone for thirst) has been seriously questioned [5]. Almli and Weiss [5] have presented evidence which is not in complete agreement with the work of Blass and Epstein [12]. Almli and Weiss showed that LPO damage resulted in elevated drinking thresholds ( 6 - 7 percent increases in osmotic pressure compared to the normal 2 - 3 percent), elongated latencies to drink; and

THE control of drinking in mammals is related to various cellular and extracellular deficits in body water content. An increase in effective plasma osmotic pressure [21] ultimately produces dehydration of the cellular space and has been shown to result in drinking for a variety of mammalian species [1, 6, 14] including man [42]. When plasma osmolality becomes elevated by 2 - 3 percent over ad lib levels (292 mOsm/kg; [4, 6, 22]) drinking is initiated [6,22] and cessation of drinking is associated with a subsequent 4 - 5 percent decrease in plasma osmolality [24]. This drinking threshold [22] appears to be quite consistent for rats across a wide variety of thirst producing treatments such as: hypertonic NaC1 injection [6, 22, 25], hemorrhage [2], polyethylene glycol injection [3,41], and eating dry food [18,22]. Drinking resultant from 24 h r water deprivation [6] or a chemically induced state of water deprivation [7] also appears correlated with this 2 - 3 percent osmolality threshold. In addition, daily drinking under natural home-cage conditions also is linked to a 2 - 3 percent rise in plasma osmolality [4]. The evidence suggesting an increase in osmolality (or perhaps sodium ion concentration; [8]) as an adequate internal stimulus for drinking behaviors is quite compelling; however, information concerning the neural detector mechanism [e.g. osmoreceptor] responsible for metering these osmolality levels has been much less convincing. Drinking to cellular stimuli may be controlled by two neural systems: the lateral hypothalamic area (LH; [5,36] )

1This research was partially funded by grants from NIMH (22482-01) and NICHHD (08504-01) to C. Robert Almli. Portions of this paper were presented at the meeting of the Eastern Psychological Association, April, 1975, New York. 2Reprint requests should be sent to Charles S. Weiss, Department of Psychology, College of the Holy Cross, Worcester MA 01610. 713

714 when the tissue destroyed was confined solely (in the anterior-posterior plane) to the LPO, then the total volume of water consumed to cellular or extracellular stimulation did not differ from the intakes of normal rats. Lesions which destroyed the LPO but also extended posteriorly to include some tissue of the anterior LH resulted in reduced water intake in addition to increased thresholds and elongated latencies. It should be noted that minor damage confined to this anterior LH tissue was ineffective in altering drinking behavior. Large LH lesions totally abolished drinking behavior as did simultaneous damage to LPO and LH (no animals survived past 21 days postlesion). The fact that LPO tissue damage affected only the latency and threshold components of drinking behavior led the authors to conclude that the LPO may be responsible for appetitive (latency-onset) aspects of drinking behavior. Also, since LH destruction reduced or abolished water intake (as a function of lesion size and anterior-posterior locus), it was postulated that the LH may be responsible for the consummatory (intake) aspects of water regulation. These results were interpreted as suggesting both the LPO and LH may contain thirst receptor cells which are stimulated by internal physiological stimuli and subsequently affect drinking behavior. Many investigators have used the electrophysiological technique to evaluate neural responsiveness to iontophoretic or intravenous administration of chemical stimuli. Results from these many studies indicate that a wide variety of brain structures show altered firing patterns (a sensitivity) in response to osmotic stimuli. These areas include: supraoptic nucleus [9,38], lateral hypothalamus [23, 31, 39, 40], preoptic area [26,28], anterior hypothalamus [ 261, periventricular nucleus [ 17], massa intermedia [17], medulla [15], and septum [13]. The direction of responding to the osmotic challenge may be facilitory, inhibitory or null, and single neurons from several neural areas can each respond to various types of stimuli (e.g. hyper-, iso- or hypotonic solutions, angiotensin; [ 10, 39, 40]). The apparent global responsiveness of the brain to osmotic stimulation has led Sudsten and Sawyer [35] to postulate that the whole brain may be osmosensitive. Given the evidence from the preceding studies, it is impossible to determine which of these areas are merely osmosensitive and which areas contain osmoreceptors involved in thirst and the drinking response. If a neural region is to be considered a detector mechanism for monitoring cellular stimuli, it should possess certain properties. First, ablation of the area should result in an abolition of drinking behavior and total lack of response to osmotic stimulation or at least in altered appetitive (latency-threshold) and/or consummatory (volume of intake) thirst components. Second, osmosensitive cells should be located there. Both the LPO and LH meet these criteria. Finally, a distinction between an osmosensitive cell and an osmoreceptive cell can only be determined with neuro-behavioral techniques. The onset of drinking following subcutaneous injection of hypertonic NaC1 occurs in 4 - 7 rain and is associated with a 2 - 3 percent increase in plasma osmolality (e.g. [5, 6, 22]). If cells in suspected detector sites (i.e. LPO and LH) function as osmoreceptors for drinking behavior, one might predict that neural activity should change before the behavioral act of drinking occurs ( 4 - 7 min) and units should respond to osmolality increases less than those associated with drinking ( 2 - 3 percent). Therefore the purpose of the present investigation is to

WEISS AND ALMLI evaluate the time-course of responses for single LH and LPO neurons. If neural activity patterns correspond temporally to the behavioral correlates of dipsogenic stimulation then the LH and/or LPO most probably contain units for detecting changes in osmolality (i.e. the osmoreceptors). METHOD Animals Fifty-seven 9 0 - 1 1 0 day old male rats (Holtzman Co., Madison, Wisc.) were housed individually or in pairs in stainless steel cages with Purina laboratory chow and tap water freely available. Colony temperature was maintained at 25 - 2 7 C and incandescent illumination was continuously provided. o

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Apparatus and Procedure Six rats were adapted for 3 days (1 hr/day) to drinking boxes [22] equipped with a stainless steel spout and water tube. On the forth day the rats received a 1 ml/350g body weight injection of 16 percent (w/v) NaC1 subcutaneously in the back region and were immediately placed into the drinking boxes. Latency to initiate drinking (3 sec of continuous licking at the spout) was timed with a stopwatch and occurred at (~nean -+ SE) 5.73 -+ 1.42 min with the range of latencies between 3.58 and 11.96 rain. Other rats were then anesthetized with dial-urethan (Ciba-Geigy, 0.9 ml/kg body weight; [16]) and surgically prepared for recording. Each animal had cardiac electrodes attached to the chest and was positioned in a stereotaxic instrument with the skull rendered horizontal (lambda and bregma on the horizontal plane). The scalp was incised and the dura mater was exposed (by a 2 mm trephine hole in the calvarium over the LPO or LH) and removed by microdissection. The exposed brain tissue was covered with mineral oil. Electrode placements were at the following coordinates (in mm) with anterior coordinate relative to the intra-aural plane, lateral coordinate relative to the sagittal suture and ventral position measured from the surface of the brain: LPO: A + 9 . 0 0 - 1 0 . 4 , L + - 1.0-2.0, V - 7 . 0 - 8 . 5 ; LH: A + 5.5-7.5, L -+ 1.2-2.2, V - 8 . 0 - 9 . 2 5 . Only one unit was monitored per rat. Body temperature was maintained at 37 ° + 0.5°C by heating tape and electronic feedback circuit [33] with temperature input recorded by rectal thermistor thermometer probe. Once the animal was surgically prepared, an epoxylite insulated stainless steel, tungsten or tungsten-carbide platinum-coated microelectrode (Haer & Co. or Transidyne General) with exposed tip measuring 5 - 2 0 microns (diameter of 1 - 1 0 microns) and impedance of 2 - 7 megohms (at 1000 Hz) was lowered by micromanipulator to the neural site. Isolation of a single neuron was accomplished by use of amplitude discriminator windows on a neuronal spike analyzer (Mentor N-750). The discriminated output of the spike analyzer (spike frequency and inter-spike interval) provided visual (oscilloscope) and audio (audiomonitor) on-line data feedback. Discriminated action potentials were recorded on magnetic tape for computer histogram plotting and analysis. Upon isolation of a single neuron, base-rate action potentials were monitored for 15 rain. During the first 10 min, several tests for the influence of external sensory stimuh (olfactory, auditory, visual, somatic) on the unit's activity were performed. First a cotton applicator was soaked in xylene and positioned approximately 3 cm from

OSMORECEPTORS F O R THIRST

715 RESULTS

the animal's nose and m o u t h for 15 sec. Second, 5 loud hand-claps were made near the rat's ears (although in many cases the animal's tympanic membranes were ruptured from the stereotaxic ear pins). Third, a moving beam of light from a flashlight was shone into the animal's eyes for 15 sec from a distance of approximately 40 cm. Finally, a dissecting pin was firmly probed into the hind foot and back region of the animal for 15 sec. After application of each external sensory stimulus, neuronal activity was noted for approximately 2 min. The data reported in the Results section of this study are based solely on ceils that did not show altered activities to these peripheral stimuli. After the sensory stimulation tests, animals had base-rate spikes recorded on magnetic tape for 5 min. Then, a subcutaneous injection o f 16 percent NaC1 (w/v in tap water; 1 ml/350g body weight) or a subcutaneous injection of physiological saline (1 ml/350g body weight; 0.87 percent NaC1 in demineralized water) was given through an acutely implanted cannula located in the back region, Injection of physiological saline was used to control for blood volume changes resultant from NaC1 treatment. Immediately at the completion of the recording session a 9 VDC electric current (Grass stimulator source) was passed through the electrode tip for 1 sec. This procedure created a microlesion at the electrode tip which was 5 0 - 1 5 0 microns in diameter. Then, the animals were intracardially perfused with 0.87 saline solution followed by 10 percent formal-saline. Brains were extracted and stored in 10 percent formal-saline for at least 24 hr prior to sectioning. Frozen serial sections ( 5 0 - 7 5 microns) made through the site of the microlesion were mounted and stained with cresyl violet. The stained sections were projected onto figures from the atlas of K6nig and Klippel [27] where the site of the microlesion was marked. Various neural structures (e.g. optic chiasm, anterior commissure, fornix, optic tracts) were used as guides for defining the precise locus of the microlesion.

To assess the effects of the experimental treatment on neuronal activity, several considerations were devised. First, stable baseline unit activity should be established. This was defined as a basal activity pattern in which none of the 30 basal bins (5 min, 30 ten-sec bins) exceeded +-50 percent of the mean for that baseline period. A time bin of posttreatment activity was then determined to be reliably different from pretreatment (basal) activity if: (1) a posttreatment bin was not contained within +50 percent of the mean for that basal activity period (when a stable baseline was achieved); or (2) a posttreatment bin exceeded the most extreme bin of the basal period (in the event that a stable baseline was not achieved). Therefore, for the remainder of this study, a reliable difference in neuronal (pre- versus posttreatment) activity is defined as posttreatment unit spike frequency that is not contained within +-50 percent of the basal period mean or extreme basal data points. Preversus posttreatment bins were determined equivalent when posttreatment points did not exceed +50 percent of the basal mean or extreme basal bins. Finally, posttreatment activities were catagorized into those patterns which displayed a maintained change to stimulation and those which exhibited intermittent change to the treatment. These two major pattern types can be further classified as those showing increased or decreased activity compared to the basal levels. Thus 4 types of patterns were found as well as those cells which never changed from basal levels. Figure 1 shows a cell exhibiting a maintained increase following the injection (indicated by an arrow). It can easily be seen from this figure that a large increase in spike frequency occurred after the injection. This cell is classified as a maintained increase because the increase in spike frequency was greater than +50 percent of basal mean levels (horizontal dotted lines) for the majority of the recording session. Although it is evident that the unit's activity was quite variable after the injection, spike

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FIG. 1. Example of a cell displaying a maintained increase in neural activity (Cell 33, LH) to 16 percent NaCI injection. Arrow indicates time of injection, dotted lines represent -+50 percent of the mean basal activity. For the computer histograms a maximum displayed frequency of 200 spikes/10 sec bin was used although in many cases spike activity greatly exceeded this value.

716

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FIG. 2. Example of a cell displaying a maintained decrease in neural activity (Cell 31, LH) to 16 percent NaC1 injection. Arrow indicates time of injection, dotted lines represent -+50 percent of the mean basal activity. 8

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FIG. 3. Example of a cell displaying an intermittent increase in neural activity (Cell 27, LPO) to 16 percent NaC1 injection. Arrow indicates time of injection, dotted lines represent -+50 percent of the mean basal activity.

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FIG. 4. Example of a cell displaying an intermittent decrease in neural activity (Cell 51, LH) to 16 percent NaC1 injection. Arrow indicates time of injection, dotted lines represent -+50 percent of the mean basal activity. level was consistantly greater than + 5 0 percent o f basal activity for 1 2 - 1 5 min. Figure 2 s h o w s a maintained decrease while Figures 3 and 4 represent an i n t e r m i t t e n t increase and intermitten.t decrease respectively. N o t e on Figure 4 that a stable baseline was n o t achieved and o n l y

decreases in activity were f o u n d after the injection. Figure 5 indicates a unit w h i c h did not change to stimulation. Analysis o f the microlesion loci for animals receiving 16 percent NaC1 s h o w e d that the electrode tips were c o n f i n e d to 12 neural sites according to the atlas o f K6nig and

OSMORECEPTORS FOR THIRST

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FIG. 5. Example of a cell displaying no change in neural activity (Cell 20, LH) to 16 percent NaCI injection. Arrow indicates time of injection, dotted lines represent -+50 percent of the mean basal activity. TABLE 1 RESPONSES

OF

LATERAL

HYPOTHALAMIC (1 ML/350

G BODY

UNITS

TO

WEIGHT,

16 PERCENT

Cell Number

Basal Spikes / 10 sec

Latency (min)

Osmolality Threshold (%)

Pattern and Direction

5

38.0

10.00

2.96

I+

20 28 30 31 33 35

24.8 15.4 38.1 26.9 11.2 8.7

1.67 4.67 1.16 1.50 7.16

0.91 1.65 0.79 0.87 2.26

0 M+ M-M-M+ I+

46 47 50 51

50.2 131.1 14.4 60.3

2.33 3.16 1.16 4.67

1.08 1.28 0.79 1.65

M-M-I+ I--

38.1 ± 10.5

3.75 -+ 0.93

1.42 ± 0.23

91%

_+SE

NACL

INJECTION

SC)*

Intermittent Change at: (min) 10.00 12.33 16.83 22.50

Duration of Intermittent Change (min) 0.16 1.33 1.83 2.83

7.16 16.33 18.50

6.83 0.33 1.00

1.16 4.67 8.50 13.00

1.66 0.16 0.66 8.00

Post/Basal Activity 1.57 1.81 2.16 2.33 2.87 0.38 0.07 9.20 3.68 9.19 3.86 0.04 0.08 2.40 0.27 0.17 0.12

*Means ± SE are presented for basal spikes, latency and osmolality threshold. (+) indicates increased frequency, (--) indicates decreased activity, and (0) indicates no change from basal levels. M = maintained activity, I = intermittent activity. Percentage indicates proportion of cells showing an activity change~ Klippel [ 2 7 ] . Responses o f units in these sites were evaluated on the basis of: ( 1 ) F r e q u e n c y of basal activity; (2) Direction o f activity change, if a change occurred; (3) Latency to the onset o f neural change; (4) Response patterns o f the activity change, i.e. i n t e r m i t t e n t or maintained responders; (5) Onset and duration of each intermittent activity period; (6) Percent change from basal activity for a given interval; (7) Percent of cells within a specific neural locus displaying an altered activity pattern. A tabular description o f unit responses to h y p e r t o n i c NaC1 injection is given on Tables 1, 2, and 3. Eleven cells were located in the lateral h y p o t h a l a m i c area (LH; see Table 1). Basal cell frequencies ranged from

8.7 to 131.1 spikes/10 sec (mean ± SE: 38.1 ± 10.5) with 10 of the 11 cells (91 p e r c e n t ) d i s p l a y i n g altered activity patterns following NaCl injection. F o r these l 0 sensitive cells, the mean latency of the change occurred in 3.75 -+ 0.93 min. (with changes occurring as early as 1.16 min. or as late as 10.00 min postinjection). When cells were categorized on the basis of response pattern, 4 cells were f o u n d o f the i n t e r m i t t e n t type while 6 displayed maintained changes. The histological locus (and histological key) o f these cells within the LH and the direction of change are given in Fig. 6. F o r this and the subsequent histological figure, the most anterior sections are found at the figure top-left and posterior aspects at the bottom-right. Open

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FIG. 6. Location and responses of neurons recorded caudal to the level of the anterior commissure to 16% NaC1 injection. The histological key to this figure and Fig. 7 is as follows: ac: anterior commissure, CC: corpus callosum, cn: caudate nucleus, CO: optic chiasm, D-LPO: dorsal to the lateral preoptic area, F: fornix, GP: globus paMdus, hI-FMP: lateral hypothalamic area-median forebrain bundle, IC: internal capsule, mt: mammilothalamic tract, ON: optic nerves, pol-FMP: lateral preoptic area-median forebrain bundle, pore: medial preoptic area, py: pyriform cortex, re: nucleus reuniens, RTI: radiatio thalami inferior, st: stria terminalis, TD: tractus diagonalis (Broca), TO: optic tract, TSTH: tractus striohypothalamicus, vmn: ventromedial nucleus (hypothalami), ZI: zona inserta. Blackened boxes indicate increased activity. Open circles indicate no change in activity. Blackened circles indicate decreased activity. circles on these figures indicate a lack of unit change to stimulation, blackened circles denote a decrease in responding and blackened boxes indicate cells which increased firing rate. Electrode tips were found at the most anterior LH aspect (A 6280 u) and e x t e n d e d to the posterior LH regions (A 4620 u). As shown in Fig. 6 and Table 1, spike activities of LH neurons p r e d o m i n a n t l y changed after the NaC1 injection. There appeared to be no locus-dependent specificity of responses (increases, decreases, no changes, maintained or i n t e r m i t t e n t ) within the LH boundaries. The lateral preoptic area (LPO) was invaded by 10 microelectrode tips. The recorded basal activity for these cells was 25.2 + 10.2 spikes/10 sec (range: 2 . 9 - 9 5 . 5 spikes/10 sec) and 9 (90 percent) of the units changed their activity pattern in response to osmotic stimulation (See Table 2). Five cells responded with increased activity, 4

decreased, and 1 cell remained unchanged from basal levels. The latency to change occurred between 1.33 and 6.50 min with the mean being 3.35 -+ 0,50 rain. The locations of units in the LPO (see Fig. 7) ranged from A 7890 u to A 7020 u with no evidence suggesting locus-dependent response specificity. However, also evident on this figure are m a n y open circles surrounding the LPO indicating a lack of change to stimulation for nearby tissues. Recordings from 4 of the 5 tractus striohypothalamicus (TSTH) cells (basal mean = 33.6 -+ 16.3; range = 5 . 8 - 9 3 . 1 spikes/10 sec) which were m o n i t o r e d responded with changes to the h y p e r t o n i c saline challenge. The mean latency to change occurred in 3.70 + 1.16 min (range: 1.00 to 8.50 rain) with 3 cells showing increased, one cell showing decreased and one cell with unchanged activity. Histological locations of T S T H units are also given in Fig. 7. Three units were located just dorsal to the LPO and

OSMORECEPTORS FOR THIRST

719 TABLE 2

RESPONSES OF LATERAL PREOPTIC UNITS TO 16 PERCENT NACL INJECTION (1 ML/3$0 G BODY WEIGHT, SC)*

Cell Number

Osmolality Threshold (%)

Pattern and Direction

6.50

2.10

M+

1.33

0.83

I+

Basal Spikes / 10 sec

Latency (min)

8

5.4

12

7.7

Intermittent Change at: (rain)

Duration of Intermittent Change (min)

Post/Basal Activity 5.75

1.33

3.33

2.63

11.67

1.67

2.09

1.50

0.67

14

8.0

1.50

0.87

I+

16

6.6

2.50

1.12

M--

23

2.9

3.50

1.36

I+

3.50

0.33

27

57.9

2.00

0.99

I+

2.00

0.16

2.35

3.50

0.16

2.35

4.50

0.16

1.97

6.33

0.67

2.64

2.37 0.30 22.24

44

8.1

45

5.2

3.67

1.40

M--

0.38

48 49

54.7 95.5

4.00 5.16

1.14 1.77

M-M--

0.15 0.17

g+_ SE

25.2 +- 10.2

3.35 -+ 0.57

1.33 +- 0.14

90%

0

*Means _+ SE are presented for basal spikes, latency and osmolality threshold. (+) indicates increased frequency, (--) indicates decreased activity, and (0) indicates no change from basal levels. M = maintained activity, I = intermittent activity. Percentage indicates proportion of cells showing an activity change. TABLE 3 RESPONSES OF TRACTUS STRIOHYPOTHALAMICUS UNITS TO 16 PERCENT NACL INJECTION (1 ML/350 G BODY WEIGHT, SC)*

Cell Number

Basal Spikes /10 sec

Latency (min)

Osmolality Threshold (%)

Pattern and Direction

1.00

0.75

I+

2

8.6

24

5.8

26

17.6

36

43.0

Intermittent Change at: (min)

Duration of Intermittent Change (min)

Post/Basal Activity

1.00

1.33

2.88

8.50

0.16

2.06

9.50

0.16

2.35

12.00

0.16

2.64

12.83

1.00

2.47

14.50

0.33

2.21

16.00 4.16

0.33 1.00

2.62 2.61

5.50 8.00

2.00 6.67

7.66 8.14

14.67 17.33

2.16 1.67

5.13 3.17

0 8.50

4.16

2.59

1.58

I+

I+

43

93.1

1.16

0.79

M--

g-+ SE

33.6 +- 16.3

3.70 +- 1.16

1.41 -+ 0.43

80%

0.19

*Means _+ SE are presented for basal spikes, latency and osmolality threshold. (+) indicates increased frequency, (--) indicates decreased activity, (0) indicates no change from basal levels. M = maintained activity, I = intermittent activity. Percentage indicates proportion of ceils showing an activity change.

720

WEISS AND ALMLI

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TD FIG. 7. Location and responses of neurons rostral to the level of the anterior commissure to 16 percent NaCI injections. Histological key given in Fig. 6. slightly ventral to the crossing of the anterior commissure (See Fig. 7). This brain area has not been named (according to K~Snig and Klippel [27] ) and will be referred to in this paper as dorsal to the lateral preoptic area (D-LPO). Mean basal activity was 9.7 -+ 2.0 spikes/10 sec (range = 7 . 4 - 1 3 . 1 ) and none of the 3 D-LPO cells responded to stimulation. Three cells of the caudate nucleus (See Fig. 7) had a mean basal activity of 12.7 -+ 4.7 spikes/10 sec (range: 4.2-20.5). One of the three cells (33 percent) decreased while the other 2 cells were unchanged from basal activity level. This sensitive cell showed a maintained activity change in 10.83 min. One of 2 cells in radiatio thalami inferior decreased (maintained change) to the osmotic stimulus (11.33 min). The other cell did not show a frequency change. One cell of the zona inserta responded with an intermittent increase at 9.83 min. Neither of 2 cells of the globus pallidus nor a cell in the tractus diagonalis (Broca) responded to hypertonic

NaC1 treatment. One cell located in the internal capsule showed an intermittent decrease which occurred at 6.50 min, while one cell of the anterior commissure displayed an intermittent increase in 6.83 min. Of the 2 cells of the pyriform cortex, one cell did not change frequency while the other cell showed an intermittent decrease in 9.67 min. Locations of these units can be found on Figs. 6 and 7. Figure 8 is a bar graph showing latency to drink and latency to neural change for brain areas where at least three units were recorded. Also indicated on this figure are the proportion of cells for each area which displayed a frequency change to 16 percent NaC1 injection. It can be easily seen from this graph that units of the LH, LPO and TSTH responded at or slightly prior to the behavioral latency to drink (5.73 min) while cells of the D-LPO and caudate nucleus showed a neural frequency not corresponding with behavior. Not only do cells of the LH, LPO and TSTH show responses within the appropriate time constraints but also a very high proportion of responsive cells

OSMORECEPTORS FOR THIRST

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z uJ

change from basal activities (mean + SE: 6.5 + 2.4 spikes/10 sec.; range = 1.6-12.2). The remaining 2 cells exhibited changes to the control injection but these maintained decreases occurred with long latency (12.83 and 15.67 min). Of the 5 LPO cells (basal mean: 8.1 -+ 3.8 spikes/10 sec; range = 0 . 5 - 2 0 . 7 ) only one cell responded to the control treatment. This cell showed a maintained increase in 2.33 min postinjection. The results of control treatment find 3 of 9 cells responding to the isotonic injection. Compared to the hypertonic saline condition, the proportion of sensitive LH and LPO cells is very small when the control treatment is administered. DISCUSSION

DRINK

HL'FMP POL-FMP TSTH

O-LPO CAUDATE

FIG. 8. Mean latency to drink, latency to spike frequency change and proportion of sensitive cells for neural loci where 3 or more cells were recorded to 16 percent NaCI injections. are found in these areas (91, 90 and 80 percent, respectively). By using data provided by Hatton and Thornton [25], which was confirmed in our laboratory, we calculated plasma osmolality increase at the onset of neural activity change, and then translated these values into estimated neural-osmolality thresholds. The equations for this procedure are: OsmolalitYest = 297.2 + 0.718 (X - 5.2) and Neural Threshold = Osm°lalitYest-292 x 100 292 where 297.2 is the mean osmolality, 0.718 is the slope of the regression line and 5.2 is the mean latency to drink. Tables 1, 2 and 3 give the estimated neural osmolality thresholds for cells of the various neural loci. It can be seen that the majority of cells from the LH, LPO and TSTH demonstrate altered activities to osmolality increases of less than 2 percent (9 of these 23 cells responded to osmolality thresholds of 1 percent or less). This finding lends support to the concept of Verney [37] who hypothesized that a 1 percent increase in plasma concentration may be sufficient to trigger central detector ceils. The data of this investigation reinforce such a hypothesis since mean neural osmolality thresholds for LH, LPO and TSTH neurons are 1.42, 1.33 and 1.41 percent respectively. As a control treatment, physiological saline (1 ml/350g) was injected subcutaneously in the back region and the unit recording procedure was conducted as previously described. Nine ceils' activities were recorded during the control injection treatment. Four cells were located in the LH with 5 cells found in the LPO. Two of 4 LH cells showed no

Neural elements located in the LH and LPO for the most part did not respond to the injection of physiological saline. One cell of the LPO and 2 in the LH showed changes that were maintained in nature. These changes occurred with a long latency (over 12 min; except for one LPO cell which changed in 2.33 min) and can most probably be attributed to differences between the concentration of the body fluids and the control injection. Reliable responding to isotonic saline has recently been reported by Blank and Wayner [ 10]. These authors concluded that neural activity changes resulting from intravenous isotonic NaC1 occur because the solution was not truly isotonic with the organism's fluids. In the present study, animals were selected for recording during various times of the day so that they may have just eaten, drunk or have been about to eat or drink prior to anesthesia [4]. If the plasma was above or below a level of isotonicity, an injection of 0.87 percent NaC1 would most probably cause some change in ongoing neuronal activity. Hypothalamic unit sensitivity to hypertonic saline injections agrees with the works of Blank and Wayner [10], Hatton and Almli [23], Oomura e t al. [31], Wayner e t a l . [39,40] and Cross and Green [17]. The critical information not available from these prior investigations is the time-course of neural and behavioral activities following hypertonic treatment. When a sensory system model is applied to the proposed detector for metering cellular stimuli for thirst, the following results should be expected: (1) Total destruction of the receptors should result in lack of responsiveness or at least deficient responding to cellular stimuli [5, 12, 3 6 ] ; ( 2 ) T h e receptors should be sensitive to cellular stimuli [ 10, 17, 23, 31, 39, 40] ; and (3) The receptor cells should show activity changes preceeding the onset of drinking and should have osmolality thresholds less than the behavioral drinking threshold [22]. Units of the lateral preoptic area, lateral hypothalamus and tractus striohypothalamicus showed significant (+ 50 percent of basal levels; see Tables 1, 2, and 3 and Fig. 8) activity changes in 3.35, 3.75 and 3.70 rain and had osmolality thresholds of 1.33, 1.42 and 1.41 percent, respectively. Since the behavioral onset of drinking occurred in 5.73 min and is associated with drinking thresholds of 1.91 percent, the unit responses occurred in the fashion required in statement 3 above. Using the criteria of the sensory system analysis, it appears that cells of the LPO and LH may be receptors functioning to monitor osmolality levels and perhaps mobilize the drinking response. Since information concerning criteria (1) and (2) are not presently available for the TSTH, any speculation as to the functions of the TSTH units is premature.

722

WEISS A N D A L M L I

The i m p o r t a n c e o f t h e variety of response p a t t e r n s a n d d i r e c t i o n of change is n o t o b v i o u s to t h e s e a u t h o r s . However, in a highly speculative sense, cells w h i c h r e s p o n d w i t h b r i e f bursts of activity ( i n t e r m i t t e n t cells) m a y f u n c t i o n to activate or p r i m e t h e o r g a n i s m for m o t o r outp u t ( d r i n k i n g ) w h e n s t i m u l a t i o n i n t e n s i t y reaches a threshold level. Units displaying a m a i n t a i n e d change to t h e s t i m u l u s m a y f u n c t i o n to p r o m o t e d r i n k i n g u n t i l sufficient w a t e r has b e e n a b s o r b e d t o reduce s y s t e m i c o s m o l a l i t y

levels. Also while the present s t u d y gives evidence t h a t o s m o r e c e p t o r s p r o b a b l y exist in b o t h the LPO and LH, it is possible t h a t t h e o s m o r e c e p t o r s m a y reside in o n l y one of these areas a n d i n f o r m a t i o n is t h e n rapidly t r a n s f e r r e d t o t h e o t h e r site via (e.g.) the m e d i a n f o r e b r a i n b u n d l e . The data f r o m t h e present investigation c a n n o t rule o u t s u c h a possibility. B o t h o f t h e above p r o b l e m s are c u r r e n t l y u n d e r investigation, a n d t h e results s h o u l d define the o s m o r e c e p tors for thirst.

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