EFFECTS OF REPEATED'INFUSIONS 0F 'HYPERTONIC SALINE SOLUTIONS ON THE ELECTRICAL ACTIVITY OF HYPOTHALAMIC NEURAL UNITS OF GOATS Thesis for the Degree of Ph‘ D. MICHIGAN STATE UNIVERSITY LARRY WILLIAM THORNTON 1969 IHI’S'? LIBRARY Michigan State University This is to certify that the thesis entitled EFFECTS OF REPEATED INFUSIONS OF HYPERTONIC SALINE SOLUTIONS ON THE ELECTRICAL ACTIVITY OF HYPOTHALAMIC NEURAL UNI S 0F GOATS presente g LARRY WILLIAM THORNTON has been accepted towards fulfillment of the requirements for Ph. D. degree in Psychology é/jg/M/Vk/ J M Major professor fl Date 2'2 fifil,”/1/Q/l//6¢ 0-169 v \ 3 1, BINDING BY ' ' NOAH & SflII ABSTRACT EFFECTS OF REPEATED INFUSIONS OF HYPERTONIC SALINE SOLUTIONS ON THE ELECTRICAL ACTIVITY OF HYPOTHALAMIC NEURAL UNITS OF COATS BY Larry William Thornton Areas of the hypothalamus have been shown to participate in parts of the motor sequence leading to water intake, and to underlie motivational components of thirst leading to the acquisition of an instrumental response sequence. Under some circumstances, the internal condition of an animal is in an adequate state to elicit drinking behavior, but the animal does not have immediate access to water. Maintained high blood NaCl concentration is related to a state of prolonged water need. This study investigated some of the characteris- tics of the internal mechanism that is sensitive to prolonged water need. In order to learn more about the mechanism, with- in the hypothalamus, that signals the need fcr water, electri- cal activity of single cells was recorded and analyzed. To determine the long term effects of high levels of blood NaCl concentrations on the electrical activity of hypo- thalamic osmosensitive neural units of goats, spike discharge activity of hypothalamic units was recorded after each of three infusions of 50cc cf 16% NaCl solutions. Eight neural units showed a decrease in spike discharge frequency, while two units showed an increase in spike discharge frequency fol- lowing an increase in blood NaCl concentration. All osmosen— sitive units showed a response to an increase in blood NaCl Larry William Thornton concentration which persisted after an increment in blood salinity. Five of the neural units showed a graded response of long duration to increments in blood NaCl concentration. Eight of the hypothalamic neural units showed a response to water loaded into the stomach of the goats. If the cells sampled in this study participate in the con- trol of drinking behavior, then the results of this experiment indicate that the hypothalamic activity signaling the need for water does not adapt over prolonged periods of thirst. Also, the activity of some of the hypothalamic cells may signal dif— ferent degrees of thirst. Finally, the activity of some of the hypothalamic cells alter their activity when water need is reduced. EFFECTS OF REPEATED INFUSIONS OF HYPERTONIC SALINE SOLUTIONS ON THE ELECTRICAL.ACTIVITY OF HYPOTHALAMIC NEURAL UNITS OF COATS By Larry William Thornton A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Psychology 1969 Gum/X 7-,-7a ACKNOWLEDGEMENTS I express deep appreciation to: Jeffrey Licht, Earl Walker, and Mark Wiltrakis to whom I'm indepted for their assistance throughout the research project; to Peter Rates, Edwin Rubel, and John Wright whose assistance was invaluable throughout a large part of the experiment; to C. Robert Almli, C. Thomas Bennett, G. I. Hattcn, Russel Newell, and Andrew Barton who rendered assistance when extra help was necessary; and the Department of Psychology and Laboratory of Comparative Neurology who provided support to carry out the research. I express thanks to: Dr. Glenn 1. Hattcn who served as chairman of the thesis committee and guided.my graduate pro- gram; tc Dr. Lawrence O'Kelly, Dr. Ralph Pax, and Dr. Robert Raisler who served on the thesis committee. I express special thanks to: Dr. John I. Johnson who provided support and time to make it possible for me to reify the idea into the deed; and to Sandy Thornton who baked cakes for the midnight watch, extracted brains, did the histology, and typed the many versions of the thesis. I dedicate this thesis to the Goat God. 11 TABLE OF CONTENTS LIST OF TABLES....................................... iv SLIST OF FIGURES...................................... v LIST 0FTAPPENDICES................................... vii Introduction......................................... 1 Method............................................... 6 SUbJectsooeoooecocoooooooeooooeoooooeoeeooo Apparatus.................................. Procedure.................................. Mta Analysis"............................ oro~Jo\ ResultSOO...0.00.00.00.00.00.0.0000...OOOOOOOOOOOOOOO 13 DiscussionOO0.00.00...OOOOOOOOOOOOOOOOOOOO0.0.0.0.... 52 Duration of the response................... 5 Direction of the response.................. 5 GradationOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO... 56 Water loaded into the stomach.............. 57 maintained act1V1tyOOOOOOOO.OOOOOOOOOOOOOOO 58 Future Research...OOOOOOOOOOIOOOOOOOOO0.... 59 summaryOOOOOOOOOOOOOO0.00000000IOOOOOOOOOOO 63 ReferenceSOOCOCOOOOOOOOO.OOOOOOOOOOOOOOOOOOOOOOOCOOOO 64 AppendiceS0.0000000000COOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 66 111 Table l. 2. 3. LIST OF TABLES Medians and semi-interquartile ranges of the pre-infusion electrical activity of ten hypo- thalamic naural unitsooooeoeoooeeeeooooooeoeoeoo Duration of saline effects following each 1nm810neoeooooooooooooooooeeooeeooooeoeoooeoeoo Summary of anatomical location and characteris- tics cf unit discharge frequency following re- peated increases in blood salinity and water loaded. into the Stomachoooooeeeooooeeeoeoooooooe iv Page 16 24 31 LIST OF FIGURES Figure l. 2. 9. 10. ll. hperimental deSIEnOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOQ Overall effects of hypertonic saline infusions and stomach water loading on the spike discharge frequencies of ten hypothalamic neural units...... The total effect of three infusions of hyper- tonic saline solution and stomach water load on the discharge frequency of each neural unit.... The duration of the effect of each infusion of hypertonic saline solution on the spike discharge frequencies of osmosensitive hypothalamic units... The cumulative effects of repeated infusions of hypertonic saline infusions on the spike dis- charge frequencies of osmosensitive hypothalamic unitSOOOOOOOOOOO00....0.000.000.0000...OOOOOCOCOOO A coronal section of the diencephalon of a goat brain showing a representation of the electrode track Of unit # leoooooocooooeoeoooooeoeoooeooeooo A coronal section of the diencephalon of a goat brain showing a representation of the electrode trBCk or unit # Zoooooooeoooeoooooocoeoooeeoeooeoe A coronal section of the diencephalon of a goat brain showing a representation of the electrode traCR Of Imit # Boooooooeoooooooooeooooocoo-cocoon A coronal section of the diencephalon of a goat brain showing a representation of the electrode track or unit # “OOOOOOOOOOOOOOO0.0.00.00.00.00... A coronal section of the diencephalon of a goat brain showing a representation of the electrode track or unit # 5.000000000000000...ooeooooooooooo A coronal section of the diencephalon of a goat brain showing a representation of the electrode track or unit # 6....00..0......OOOOOOOCOOOOOOOOOO V Page 11 15 19 22 2? 3h 36 38 40 42 44 Figure 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. A coronal section of the diencephalon of a goat brain showing a representation of the electrode track Of unit # 7...eoeeoeeeooeeoooooooeeoeeooeeooe A coronal section of the diencephalon of a goat brain showing a representation of the electrode track or unit # 80.00.000.000...OOOOOOOOOOOOOOOOOOO A coronal section of the diencephalon of a goat brain showing a representation of the electrode track or unit # 9 and # 10.00.000.000...ooooooeeeeo Histograms representing the effect infusions of hypertonic saline and load on the discharge frequency of Histograms representing the effect infusions of hypertonic saline and load on the discharge frequency of Histograms representing the effect infusions of hypertonic saline and load on the discharge frequency of Histograms representing the effect infusions of hypertonic saline and load on the discharge frequency of Histograms representing the effect infusions of hypertonic saline and load on the discharge frequency of Histograms representing the effect infusions of hypertonic saline and load on the discharge frequency of Histograms representing the effect infusions of hypertonic saline and load on the discharge frequency of Histograms representing the effect infusions of hypertonic saline and load on the discharge frequency of Histograms representing the effect infusions of hypertonic saline and load on the discharge frequency of Histograms representing the effect infusions of hypertonic saline and load on the discharge frequency of vi of repeated stomach water neural unit #1.. of repeated stomach water neural unit #2.. of repeated stomach water neural unit #3.. of repeated stomach water neural unit #4.. of repeated stomach water neural unit #5.. of repeated stomach water neural unit #6.. of repeated stomach water neural unit #7.. of repeated stomach water neural unit #8.. of repeated stomach water neural unit #9.. of repeated stomach water neural unit #10. Page 46 48 50 72 74 76 78 80 82 84 86 88 9O LIST OF.APPENDECES Appendix A: Apparatus and.materials..................... 67 Appendix B: Frequency histograms of different records of electrical activity of 68011 neural unlt............................ 69 Appendix C: 33" data.................................... 91 vii Introduction Verney (19“?) and Andersson (1952) showed that areas in the anterior hypothalamus participate in the regulation of intake and output of water. Since their studies, many dif- ferent experimental approaches have been used to locate and study the effects of hypertonic solutions on the water regup latory system in different animals. B. Andersson observed drinking behavior after infusing small amounts of NaCl solutions into the brain, electrically stimulating cells in different parts of the hypothalamus, or lesicning parts of the hypothalamus. Andersson et a1. (1951, 1952, 1953) has demonstrated that drinking behavior is elicit- ed in goats when NaCl solutions are placed in direct contact with cells around the paraventricular nucleus of the hypothal- amus. IAlso, Andersson (1955) has shown that drinking behavior can be elicited in goats when areas between the fornix and the mammillothalamic tract of the hypothalamus are electrically stimulated. Lesions of hypothalamic areas between the fornix and mammillothalamic tract were followed by partial or com- plete adipsia in goats and dogs (Andersson, 1957). Goats ac- quire a ”new” motor sequence (climbing stairs) to get access to water while being electrically stimulated in the hypothal- amus (Andersson, 1956). These studies by Andersson show that there is a neural substrate in the hypothalamus of the goat underlying parts of the motor sequence leading to water in- take, and underlying motivational components of thirst lead- ing to the acquisition of an instrumental response sequence. 1 2 Andersson's research is designed to study the effects of electrical and osmotic stimulation of the hypothalamus on drinking behavior when the goat is given.immediate access to water. Under some circumstances, the internal condition of an animal is in an adequate state to elicit drinking behavior, but the animal does not have immediate access to water. Studies in the literature suggest that changes in blood NaCl concentration continue to affect drinking behavior of animals when access to water is delayed after the adequate stimulus that usually results in drinking behavior. When rats are not given access to water for six hours after either a lcc injec- tion of 16$ NaCl or a .87} NaCl solution, they approach water more rapidly and drink more water after the 16$ injection than after the .875 injection (Thornton, 1966). Fitzsimons (1963) demonstrated that there is no difference in the change in body weight after drinking between rats who had immediate access to water after a hypertonic injection and rats who had access to water 24 hours after a hypertonic injection. Both studies suggest that there are internal mechanisms that are sensitive to water need over periods of delayed access to water. The study of electrical activity of hypothalamic cells in the neuralsubstrate underlying drinking behavior (as defin- ed by the results cf Andersson's research) is an indicator of the characteristics of the internal mechanisms that are sensi- tive to prolonged water need. Other studies are designed to locate and study electri- cally active neural units in the anterior hypothalamus while infusing hypertonic solutions into the carotid artery (Cross 3 and Green, 1959; Joynt, 1964). These studies sampled the electrical activity of osmosensitive neural units over short periods of time (14 seconds to 60 seconds). The present ex- periment was designed to sample the electrical activity of neural units over longer periods of time and to repeat the infusions several times. The experiment represents an ini- tial effort to furnish information on the effects of changes in blood salinity on the electrical activity of neural units in the goat anterior hypothalamus and to assess possible changes in the electrical response of neural units over ex- tended periods of time after repeated infusions of hypertonic saline solutions. Specifically, this study is centered around the charac— teristics of direction, gradation, and duration of the elec- trical response of osmosensitive neural units before, during, and for an extended period of time after changes in blood salinity. Direction refers to an increase and/or decrease of the electrical response (spikes/unit time) after an infusion of hypertonic saline solution. The gradation of electrical responses to increases in blood salinity was proposed by Von Euler (1953) as a crite- rion for an osmosensitive nerual unit located in the anterior hypothalamus. Gradation is a term referring to the effect on the electrical responses after repeated infusions of hyperton- ic saline solutions. If the electrical response of an osmo- sensitive unit is graded to increments in blcod NaCl concen- trations, then each successive infusion of saline solution should affect the electrical response of the neural unit u, in steps. Each step should be in the same direction and of greater magnitude than the previous step. Cross and Green (1959) and Joynt (1964) report that the electrical responses of osmosensitive neural units in the anterior hypothalamus of rabbits and cats show the characteristic of gradation af- ter repeated infusions of hypertonic saline solutions. Transitoriness is a term referring to the duration of the observed electrical response of the osmosensitive neural unit after an infusion of hypertonic saline solution. If the re- sponse changes during the infusion and then returns to pre- infusion level, then the electrical response is characterized as transitory. A modification of this transitory characteris- tic is a residual effect. A rasidual effect is an electrical response that is less than the initial transitory effect but greater than the pre-infusion level and persists after the in- crement in blood NaCl concentration. Cross and Green (1959) and Joynt (1964) indicate that increases of blood salinity have transitory effects on the electrical responses of osmo- sensitive neural units in the anterior hypothalamus. Their results might be due to the small amounts of NaCl solutions that they infused into the carotid artery during each stimu- lation. In this experiment, more NaCl was infused during each stimulation, and each infusion was repeated three times for each osmosensitive neural unit under observation. If a cer- tain increment in blood NaCl concentration is necessary before the electrical response is nontransitory, then pushing the 5 neural unit to its limits should increase the probability of a longer lasting electrical response to increases in blood NaCl concentration. Threshold is defined as the amount of infused NaCl solu— tion needed to cause a lasting electrical response. Vbn Euler (1953) proposed evidence of threshold as a criterion for an osmosensitive neural unit in the anterior hypothalamus. The purpose of this experiment is to determine if goat osmosensitive neural units in the anterior hypothalamus have a graded electrical response after repeated increases in blood salinity, and, if the electrical response to an increase in blood salinity is transitory when activity is sampled over a long period of thme. Goats were used in the experiment to extend Andersson's (1952) findings that osmosensitive areas are located in the hypothalamus of goats. Method Subjects Information for this study was obtained from five male domestic goats (952;; hircus, Toggenburg strain). The goats were obtained from a goat herd maintained by the Endocrine Research Unit at Michigan State University. They weighed (40.7, 36.2, 33.0, 33.6, and 32.6 kg.). Their age was be- tween one and two years. The goats were food deprived the day before surgery. The animals were initially anesthetized intraperitoneal- 1y with dial-urethane (157 mg of urethane and 49 mg of dially- barbituric acid per kilogram of body weight» One-half hour prior to the dial-urethane administration, the animals were injected with lcc (50 mg) of promazine hydrochlorine (Sparinégg Throughout an experiment, additional dial-ure- thane anesthesia was limited to one-fourth of the original dosage every eight hours. Sodium pentobarbital (20 mg/kg) was used when anesthesia was required during the eight hour interval between dial-urethane injections. The animals were tracheotomized and maintained on artificial respiration throughout the experiment. After the head was cemented into a holder, the left neo-cortex was exposed for access. The left carotic artery was cannulated with IntramedicQEIpolyeth- ylene #90 tubing which was filled with heperinized physiolo- gical saline and connected to a constant infusion pump. The animals' rectal temperature and heart rate were monitored throughout the experiment. The animals' temperatures varied between 33-36°C during the experiments. 6 W Glass insulated tungsten microelectrodes were used for recording. The recording electrodes were modifications of the Hubel (1957) tungsten.microelectrode. The electrodes were uninsulated for 30 to 100 microns from the tip. The shaft diameter was 40 to 80 microns at a point one millime- ter from the tip of the electrode. The electrode signals were amplified by an A. C. preamplifier and the vertical am- plifier of the oscilloscope. During the experbment the sig- nals were recorded on.magnetic tape. After the completion of the experiment, the signals were converted to standard pulses and put into a small computer which gave the number of spikes per 10 seconds. Procedure Electrodes were driven.manually by means of a micromani- pulator through the opening in the skull. When the electrode was presumed to arrive at a site in the hypothalamus, a unit was selected whose spike activity seemed to be relatively stable. Baseline activity was recorded and infusions began. To determine if the relationship between increasing blood tonicity and neural unit activity was graded, 5000 of 16$ sodium chloride solution was infused at a rate of Scc/ minute into the carotid artery three times in sequence, with 50 minutes between each infusion. A.magnetic tape record was taken of the electrical activity before, during, and immedi- ately after the infusion, and 20 minutes after the completion of the infusion. The independent variable was the amount of saline infused while recording from the neural unit. The 8 dependent variable was the total number of spikes per 10 seconds during and after the infusion of the saline. The same procedure was used to determine if the neural unit re- sponse to the infusion of saline was transitory. The over- all procedure for each unit consisted of three infusions of 50cc of 16$ saline solution followed by water loaded into the stomach after the series of three infusions. Magnetic tape records were taken of the neural unit electrical acti- vity before, during, after each infusion, and after 3 liters of water were placed in the stomach. Data Analysis Eight records of electrical activity were recorded on tape for each hypothalamic neural unit. Each record was for a certain length of time, according to the schedule below. There was a period of tune between each record when nothing was recorded. Refer to Figure 1 for a diagram of the follow- ing schedule. The periods of recording and non-recording for each neural unit were as follows: 1. IA 15 minute period of recording followed by a 5 minute period of non—recording. Record 1 is referred to as the pro-infusion period. 2. A 10 minute period of recording, during which 50cc of 16} NaCl solution was infused during the first 10 minutes, followed by a 15 minute period of non-recording. Record 2 is referred to as the first infusion period. 3. A 10 minute period of recording followed by a 5 minute period of non-recording. Record 3 is referred to as the post-first-infusion period. 9 4. A 10 minute period of recording, during which 50cc of 16$ NaCl was infused, followed by a 15 minute period of non-recording. Record 4 is referred to as the second infusion period. 5. A 10 minute period of recording followed by a 5 minute period of non-recording. Record 5 is referred to as the post-second-infusion period. 6. A 10 minute period of recording, during which 5000 of 16$ NaCl was infused, followed by a 15 minute period of non-recording. Record 6 is referred to as the third infusion period. 7. A 10 minute period of recording followed by the period of time needed to load 3 liters of water into the stomach of the goat. Record 7 is referred to as the post-third-infusion period. 8. A,10 minute period of recording. Record 8 is referred to as the post-water-load period. The electrical activity from each of the tape records of the periods listed above was put into a Computer of Aver- age Transients. The computer was programmed to count the number of unit discharges occuring in successive ten second intervals. The computer only counted unit spike discharges of a predetermined amplitude. The amplitude was determined so that only the spike activity under study was counted. The computer output was printed on paper by a tale-typewriter. Periods of recorded unit electrical activity that lasted for 15 minutes had 90 ten second intervals. Periods of recorded electrical activity that lasted for 10 minutes had 60 ten 10 Figure 1 shows the experimental design for each hypotha- lamic neural unit. Infusion periods and non-infusion periods are super-imposed on the time (minutes) of the experiment on the neural unit. I, II and III refer to the periods of infu- sion of 500c of 16$ NaCl solution into the carotid artery. 0, 20, 30, etc. refer to the successive minutes of the experi- ment. The period of time in which 3 liters of water was load- ed into the stomach is represented at the right of the figure. Periods of recording the electrical activity of the neural unit is represented below the periods of infusion and non- infusion. The numeral 1 refers to the tape record before starting the series of infusions. The numerals 2, 4 and 6 refer to the tape records during the infusions. The numerals 3, 5 and 7 refer to the tape records after the infusions. The numeral 8 refers to the tape record after the completion of the water load. l 1... 000 Ltd Men-III. m: or 09 he M 9. 3 he or On ca .3 O a 43.5% L [ I { Ofn‘ogohOV.“ mro DAN... 02.3. M. 0.: Joe—Lm.rImCa o... le-Oohhfl m: d d 0 U 0.; tCOZ C [L OE." meavtwm 04.00.. T (390th #335 g 01.3qu 12 second intervals. The unit of observation in analyzing elec- trical activity is a ten second interval. The dependent variable used in analyzing the electrical activity of the neural unit was the mean number of spike dis- charges per second during each ten second interval. The aver- age discharge frequency was found by dividing the number of spike discharges occuring in a ten second interval by 10. Results The overall effects of repeated hypertonic saline infu- sions and stomach water loading en the diScharge frequencies of ten hypothalamic neural units are shown in Figure 2. Figures 15 through 24 in Appendix B show the effects of re- peated infusions and loading water into the stomach on the variability of spike discharge frequency of each unit. The figures are histograms showing the distribution of unit dis- charge frequency over each of the recorded samples of unit activity before, during, and after each infusion. They show the proportion of ten second intervals of the recorded sample of unit activity that had a certain discharge frequency speci- fied in mean number of spikes per second. The descriptive statistic used to compare the different treatment periods was the median discharge frequency. The average number of spike discharges per second during each ten second interval of a 10 or 15 minute treatment was found. The median discharge frequency refers to the particular mean spikes/second above which and below which fifty percent of the ten second intervals' discharge frequencies fell. Table 1 lists the median discharge frequencies and semi- interquartile ranges of the pre-infusion electrical activity of ten hypothalamic units. Experiments were done on more than one unit in two of the goats; consequently the goats had a history of infusions and stomach loading prior to the obser- vation of activity of some units. Five units were observed from goats who had previous infusions and stomach loading of water. 13 14 Figure 2 shows the overall effects of hypertonic saline infusions and stomach water loading on the spike discharge frequencies of ten hypothalamic neural units. Each panel represents a different neural unit. The median average spike frequency is represented on the vertical side of each panel. The different periods of recording are represented under the bottom panel. Each point within the panel represents the me- dian average discharge frequency of a unit during each suc- cessive period of recording, i.e., pro-infusion, first infu- sion, post-first-infusion, second infusion, post-second-infu- sion, third infusion, post-third-infusion, post-water-load. Units are grouped so that similar behaviors are next to each other. 15 Figure 2 1.4 M 3.4 as m mu" .b 4' .b l 4. I I I were...“ I" Put 6... 5" l}— 2.. 1‘ l‘ unint- (1‘- IS. -...--/..,---... .4- 01" d . g 6 O 0 73.91..» vrocusvuyu. €510£ 6 Q 7.. .813 L 3m t 0 .2.th Fan. ‘03,..th 7:. 1h ‘01 SVSH 9d C 0‘1“.th 1.9m COVJGCH Ltd :2...er H at... hme‘g pears. 4 term 29‘ .0 5L 16 Table 1 Medians and Semi-interquartile ranges of the pre-infusion electrical activity of ten hypothalamic neural units. Unit Previous infusions __ Median Semi-inter- number and stomach loading (x spikes/sec.) quartile into the goat. (iflggfies/sec.) 1 No .49 .18 2 No 10.28 ~40 3 No .93 .20 u No n.45 ~26 5 Yes 4.19 -“3 6 Yes .52 .18 7 No 1.90 ~32 8 Yes .29 .16 9 Yes 9.20 1.26 10 Yes 21.18 2-51 17 The oemosensitivity of a neural unit was determined by comparing the median spike discharge frequency of the unit before the first infusion with the median spike discharge frequency after the third infusion. If the discharge fre- quency of a neural unit after the final increase in blood salinity was different from the pro-infusion unit discharge frequency, then the neural unit was considered an osmosensi- tive neural unit. Refer to Figure 3. Ten units were sampled All units were affected by the combined salt load of the three infusions. 18 Figure 3 shows the total effect of three infusions of hy— pertonic saline solution and the water load on the discharge frequency of each neural unit. The histograms show the median average firing frequency as a function of the treatment time that the electrical activity was sampled. Ere refers to the electrical activity of the unit before beginning the series of three infusions. ‘ngt refers to a ten minute record of activity after the completion of the three infusions. W§£e£_ refers to a ten minute record of activity after the water load. The median refers to the particular E spikes/second above which and below which fifty percent of the ten second intervals' average discharge frequencies fell. For example, if the median firing frequency of unit 1 before infusions was .4 spikes/sec- ond, then fifty percent of the ten second intervals during the time of recorded activity had an average discharge frequency equal to or less than .4 spikes/second. 19 FIGURE 3 unit 1 unIt 2 unn 3 10 1.0 1.5 5 '5 .5 ‘ I | I l I I unit 4 unit 5 and 6 5 15‘ .5 4 3 2 .2 unit 7 unit 8 unit 9 6'01 1o 2 1.0 5 1 l l . ‘ ' o 0 0 } unlt 1O 0 i‘ ll. .2 c 31 U 0 E pre post water peruod 20 The direction of the effect of increased blood NaCl con- centration on the discharge frequency was determined bysee- ing if the change in unit discharge frequency after the final increase in blood salinity was above or below the pre-infusion discharge frequency. Two units increased their discharge fre- quency, while eight units decreased their discharge frequency after the final increase in blood salinity. To determine the duration of the effect of each infusion, the median average discharge frequencies before, during, and after each infusion were compared. Refer to Figure 4. Only effects which were in the same direction as the discharge fre- quency after the third infusion were considered to be due to increased salinity. If the change in discharge frequency dur- ing infusion was in the direction opposite to the total effect of the salt load on the unit (as indicated by the difference between pro-infusion discharge frequency and post-third-infu- sion discharge frequency) then the question of duration of saline effects could not be determined. If the change in dis- charge frequency during the infusion was in the same direction as the total effect of the salt load on the unit, but the dis- charge frequency of the unit returned to or above the pre- infusion discharge frequency after the termination of the in- fusion, the effect of the saline was considered transitory. If both the records of unit activity during infusion and after the infusion were in the same direction as the effect of the total salt load on the unit, the saline effect was considered to be non-transitory or of long duration. Table 2 lists the classification of each unit. 21 Figure 4 shows the duration of the effect of each infu- sion of hypertonic saline solution on the spike discharge fre- quencies of osmosensitive hypothalamic units. Each of the ten histograms represent the spike discharge activity of an osmo- sensitive hypothalamic unit. There are three triads of bars or medians in each histogram. Each triad represents the ef- fects of a hypothalamic saline infusion on the discharge ac- tivity of the osmosensitive unit. The three bars of a triad represent the median average discharge frequency (i spikes/ second) of the osmosensitive unit during different periods of recorded electrical activity. The first bar represents the median average discharge frequency during a period of time before the infusion. The second bar represents the median av- erage discharge frequency during the infusion. The third bar represents the median average discharged frequency during a period of time after the termination of the infusion. The median refers to the particular value (E spikes/second) above which and below which fifty percent of the ten second inter- vals fell during the period of recorded activity. For example, if the median average discharge frequency of unit 2 for the period of recorded electrical activity occuring before infu- sion 3 was 0.0 spikes/second, then fifty percent of the ten second intervals during the period of recorded activity had an average spike discharge frequency equal to or less than 0.0 spikes/second. The arrow indicates the threshold infu- sion which resulted in an effect of long duration. 22 mm. 'gem. FIGURE 4a the II: unit 2 unit 1 tptkol/goc. 1 av- 1 E "I'd inn flrst second third IOVG INFUSION unIt 4 1" unIt 3 mpleu median - 0plk00/00c. unit 5 + I first unlt 7 00cond infusion 23 FIGURE 4b thIrd unit 6 unit 8 A unlt 9 unit 10 24 Table 2 Duration of saline effects due to each infusion. (The number under each category indicates the first, second, or third infusion.) Unit # Transitory Non-transitory Indeterminate WQVQU‘dFWNH H 0 1,2 3 1,2 1,293 1.2.3 1,3 2 1,2,3 1,2,3 1,2 1.3 1,2,3 25 The present experiment was designed to determine the effects of increased blood NaCl concentration for an extended period of time after the infusion. Infusion threshold is de- fined as the number of infusions of 5000 of 16$ NaCl necessary to get a change in spike discharge frequency that persists af- ter the termination of an infusion. The infusion which is followed by a change in spike discharge frequency after the termination of the infusion and which is in the same direction as the total salt load effect is called the threshold infusion. Refer to Figure 4. The infusion threshold was infusion one, for units # 2,3,5,6,7,8,9 and 10. The infusion threshold was infusion three, for units # l and 4. Gradation of saline effects was determined by comparing the pro-infusion, post-first-infusion, post-second-infusion, and post-third-infusion discharge frequencies. In order to be considered graded, the spike discharge frequency of the unit, after each infusion, must be different from the saline effect after the previous infusion and in the same direction as the total salt load effect. The question of gradation is only considered after the threshold infusion that was follow- ed by effects of long duration. Refer to Figure 5. The ef- fects of repeated saline infusions are considered graded for five units - units 2,5,6,7 and 10. Unit 2,6 and 10, reached their limit of change before the completion of the series of infusions, but one infusion demonstrated gradation of saline effects. Unit 3 reached its limit of change after the first infusion. Blood salinity did not reach threshold until infu- sion 3 for units 1 and 4. The effect of repeated infusions was irregular for units 8 and 9. 26 Figure 5 shows the cumulative effects of repeated infu- sions of hypertonic saline infusions on the spike discharge frequencies of osmosensitive hypothalamic units. Each histo- gram of the ten histograms represents a hypothalamic osmosen- sitive unit. Each histogram has four bars or median. The four bars represent the cumulative effects of repeated hyper- tonic saline infusions on the spike discharge frequency of the osmosensitive unit gfggg_the termination of the infusion. The four bars represent the median average spike discharge frequency (E'spikes/second) of the osmosensitive unit during different periods of electrical activity. The first bar re- presents the median average discharge frequency during a period of time before beginning the series of infusions. The second, third, and fourth bar represent the median average discharge frequency of the period of electrical activity af- ter the termination of the first, second, and third infusion, respectively. The median average discharge frequency refers to the particular value (E'spikes/second) above which and be- low which fifty percent of the ten second intervals fell dur- ing the period of recorded activity. mod Ion 09|k00/..¢_ .1 I 1-0‘I unit 1 unit 3 P 1 period 2 3 27 FIGURE 53 unit 2 12—. unit 4 modisn spikes/g.c_ unII 5 4-1 p 28 FIGURE 123 period unit 7 unit 9 unit 6 unit 8 unit 1° 24- 29 Water loaded into the stomach affected eight of the ten units. Refer to Figure 3. Fer seven of the eight affected units, the spike discharge frequency changed in the direc- tion opposite to that of the salt load. An attempt was made to locate the bottom of the electrode track for each neural unit. A tracing was made of the brain section showing the lower limit of each electrode track. Fig- ures 6 through 14 show the tracings of the hypothalamic regions in which the bottom of the electrode puncture for each of the ten units was located. The approximate locations of the units were classified according to four regions of the hypothalamus: (l) Pre-optic region, (2) Supra-optic region, (3) Tuberal re- gion, and (4) Mammillary region. The regions are defined in an anterior-posterior direction, as follows: (1) Pro-optic region is anterior to the optic chiasma; (2) Supra-optic region is above the optic chiasma; (3) Tuberal region is from the optic chiasma to the mammillary body; and, (4) the Mammillary region is around the mammillary body. There are no lateral limits in the definition of the regions. Four units were located in the pre-optic region. Two units were located in the tuberal region. One unit was located in the mammillary region. Table 3 summarizes the approximate location of each neural unit and the effects of changes in blood salinity on unit spike discharge activity. The characteristics of the units in the supraoptic region were similar in that they showed a decreased response of long duration to the first increment of blood NaCl con— centration, showed graded responses to repeated increments of 30 blood NaCl concentration, and showed a response to a stomach load of 3 liters of water in a direction cpposite to the re- sponse to increased blood NaCl concentration. 31 Table 3 Summary of anatomical location and characteristics of unit discharge frequency following repeated in- creases in blood salinity and water loaded into the stomach. Unit Location Direction Threshold Gradation Direction # of stomach water load effect 1 Mammillary region increase third ------------ 2 Supraoptic region decrease first yes increase 3 Tuberal region decrease first limited increase 4 Tuberal region decrease third ------ increase 5 Supraoptic region decrease first yes increase 6 Preoptic region decrease first yes ------ 7 Supraoptic region decrease first yes increase 8 Preoptic region increase first irregular increase 9 Preoptic region decrease first irregular increase 10 Preoptic region decrease first yes increase 32 The following section is made up of figures (Figures 6- 14) and ten photomicrographs. Each figure represents a coro- nal section of the hypothalamus of a goat brain. The figure is a tracing of the section showing the approximate location of the microelectrode tip during the recording of the neural unit electrical activity. The arrow in each figure indicates the representation of the electrode track and unit number. The magnification for tracing each section was X 14. Follow- ing each figure is the photomicrograph of the brain section showing the electrode track. The magnification is X 15. .AHA AHL .APO A. Sept. A. Sept. L CA CF C int. C Mm Ch. 0 FMT FR EX NRA NHDM NHVM NPV NSO Ped. Cer. PM RE RET SCH T0 33 Key to abbreviations used in tracings of brain sections (Richard, 196?). area hypothalamia anterior area hypothalamia lateralis area preoptica area septalis area septalis lateralis commissura anterior campi foreli capsula interna corpus mammillare mediale chiasma opticum fasiculus mammillothalamicus fasiculus retroflexus fornix nucleus hypothalamicus anterior nucleus hypothalamicus dorsalis medialis nucleus hypothalamicus ventralis medialis nucleus paraventricularis hypothalami nucleus supraopticus pedunculus cerebri pedunculus mammillaris nucleus reuiens nucleus reticularis nucleus suprachiasmaticus tractus opticus 34 Figure. 6 =Inm 35 C. InT =Imm Figure. .7 37 39 40 r‘ 41 42 Figure. \0 C INT “II #5 N H A 43 . '("ITTV’YTFT'I I . .3. 3"" figure- ““II [.2 I MIIIIIndcr A See‘- L A Sept I“ 45 «2. 46 Fiqurg I1 CA unit 07 A PO ‘ c 0 CHO- 47 48 Figure I3 :Imm I+9 5O Fiswre. I ‘1 =Imm 51 c. J; ...V..:,. . , we”... 04“.. I... .. I... 1.1.". 1 .r ,x fowl“... I)... (In; It. , O. .. HA 2 . . .. . J”. I A. I? J; . 'I . 1'}. .l .. I . , l ,., a 1 1+0 .~., .. .I. r 4.... fr... V. awn»... (”viz/I 1.1.1.5 , at. I . . Discussion The problem under investigation in this study was the long-term effects of increments in blood NaCl concentration on the discharge patterns of osmosensitive hypothalamic neural units. The results indicate the following answers to the questions presented in the introduction: 1. Do the responses of osmosensitive hypothalamic neural units persist after a change in blood NaCl concentrations? The change in discharge frequen- cies of all ten osmosensitive units to increments in blood NaCl levels persisted for twenty to thirty minutes after the change in blood NaCl levels. 2. What is the direction of change of responses of long duration? Eight of the osmosensitive neural units showed a decrease in discharge frequency af- ter the increase in blood NaCl concentration, while two osmosensitive neural units showed an increase in discharge frequency. 3. Are the responses of long duration graded with re- peated increments in blood NaCl concentrations? All of the osmosensitive neural units did not show a graded response to increments in blood NaCl con- centration. Five of the ten units showed the char- acteristic of gradation of response to changes in stimulus intensity. The remainder of this section will be a more detailed discussion of the response characteristics of duration, direc- tion, and gradation, of an osmosensitive hypothalamic neural 52 53 unit. Further, findings about the effects of water loaded into the goat's stomach on the responses of osmosensitive hypothalamic neural units will be discussed. The findings from the results of the locations of the units will be dis- cussed. Finally, hypotheses suggested by the results and future areas of research will be discussed. Duration of the response The findings of other research looking at the effects of changes in extracellular NaCl concentration levels on the water regulatory system suggest that the activity of parts of the neural substrate underlying the control of water consumption and.ADH release should persist for long periods of time. It has been demonstrated that the rate of release of .ADH does not accomodate in dogs when high blood NaCl levels are maintained for 40 minutes (Verney, 1947). Fitzsimons (1963) interprets results of an experiment as showing that receptors for thirst do not adapt. He demonstrated that when water was withheld for 24 hours after injection of NaCl solu- tion into nephrectomized rats, there was no difference in the change in weight between.anhmals who drank after 24 hours and animals who drank.immediately after the injection. Both of these findings suggest that osmosensitive units in the hypo- thalamus may respond as long as blood concentration of NaCl is higher than normal. The results of the present experiment show, that in all osmosensitive neural units sampled, a re- sponse to an increment in blood NaCl concentration persists 54 for 20 to 30 minutes after the change in blood NaCl con- centration was present. This persistent response occurred if the change in blood NaCl concentration was high enough. Several studies have shown that the electrical re- sponses of different hypothalamic areas are transient after an increase in blood NaCl concentration (Brooks, Ushiyama, and Lange, 1962; Cross and Green, 1959; Koizumi, Ishikawa, and Brooks, 1964; Nakayma, 1955; Novin and Dzrham, 1969; Sawyer and Gernandt, 1956; Von Euler, 1953). A possible reason for the difference in the findings from this experi- ment and theirs is that the increase in blood NaCl concen- tration from the infusions in their experiments was not large enough to maintain the blood NaCl concentration above the threshold value. The results of this experiment indi- cate that the amount of NaCl solution infused into the blood can determine if a response to an increment in blood NaCl concentration persists after the termination of the infusion. While eight of the units showed a response to an increase in blood NaCl concentration after the first infusion of 5000 of 16$ NaCl solution, two of the units did not show a persistent response until after three infusions or 15000 of 16$ NaCl so- lution. Direction .All neural units whose electrical activity was sampled were affected by the increase in blood NaCl concentration. Eight of the units decreased their spike discharge frequency, while two units increased their spike discharge frequency. The units were located throughout the hypothalamus. One of 55 the units which increased its activity to increased blood NaCl concentration was located in the pre-optic area of the hypothalamus, while the other unit was located in the mammillary area of the hypothalamus. Many studies show that, in general, the units in the supraoptic nucleus increase their activity with an increase in the blood NaCl concentra- tion. (Brooks, Ushiyama, and Lange, 1962; Cross and Green, 1959; Joynt, 1964; Novin and Durham, 1969; Zeballos, Wang, Koizumi, and Brooks, 1967). In this study, none of the units were located in the supraoptic nucleus. Cross and Green (1959) report that units in the paraventricular nucleus decrease their activity. .Also Suda et al. (1963) report that the spontaneous activity of supraoptic units increased their acti- vity when an island of hypothalamus containing the supraoptic nucleus was isolated from other neural connections. They in- terpret their results as providing evidence that cells in the supraoptic nucleus are under inhibitory control from other neural structures. Since the supraoptic nucleus seems to be the final neural area involved in the release of ADH it seems reasonable that cells in the supraoptic nucleus show an in- crease in their activity with an increase in blood NaCl con- centration. Wayner and Kahan (1969) have reported that, in general, units in other parts of the hypothalamus, thalamus, and midbrain also show an increase in activity with an in- crease in blood NaCl concentration. The difference between the findings in this experiment and others gives support to a conclusion made by Cross (1964). "Lability of unit discharges in the hypothalamus tends 56 to erode confidence in the concreteness of the 'centres' demarcated by the methods of stimulation and ablation.” (p. 163.) The findings of the present study suggest that, when activity is sampled over a longer period of time than in pre- vious studies, more extra-supraoptic units, in general, de- crease their activity with an increase in blood NaCl concen- tration, than increase their activity. Gradation Novin and Durham (1969) and Von Euler (1953) have stated that in order for a unit to be considered an osmoreceptor it must show the property of responding to increments in the concentration of NaCl in the blood. The results from both of their studies demonstrate that the magnitude of slow poten- tials in the supra0ptic nucleus varies proportionally with blood NaCl concentration. Several studies have shown that the changes in spike discharge frequency is proportional to incre- ments in blood NaCl concentration, during and shortly after the injection of NaCl solutions into the blood. (Cross and Green 1959; Joynt, 1964). The results of this experiment show that in five of the ten osmosensitive units the change in discharge frequency was graded to increments in blood NaCl concentrations for 20 to 30 minutes after the termination of the infusion of NaCl solutions into the blood. There are osmosensitive units in the hypothalamus that continue to re- spond to different concentrations of NaCl in the blood after the initiation of blood NaCl concentration change. 57 Five osmosensitive units did not show the property of gradation of response with repeated increases in blood NaCl concentration. Two of the units did not show a persistent response to an increment in blood NaCl concentration until after the third or final infusion. One unit reached its limit of response change after the first infusion. Two units showed irregular responses to repeated increments in blood NaCl concentration. Not all of the osmosensitive units show- ed the property of gradation of response. Water loaded into the stogggh. Eight of the ten units showed a change in discharge fre- quency after loading water into the stomach of the goat. Seven of the eight units showed a response in the direction opposite to the response to increased blood NaCl concentra- tion. The response to the water load persisted through the ten minutes after the completion of loading water into the stomach. Other studies report different results. Generally, they report that osmosensitive units do not respond to water infused into the blood (Brooks, Ushiyama, and Lange, 1962; Cross and Green, 1959; Joynt, 1964). Some studies have reported that slow potentials show a change in the opposite direction if water is infused into the blood. (Nakayama, 1955; Novin and Durham, 1969). Some experiments demonstrate that water infused into the carotid artery has an effect on electrical activity of osmosen- sitive neural units while other experiments reported no effect from water infused into the carotid. The differences in re— sults of the different experiments might be due to the 58 salinity of the blood prior to infusing water into the carot- id artery. If the blood NaCl concentration remains above the threshold concentration to which a neural unit is sensitive, then the unit could respond to water diluting the blood. Many experiments infuse small amounts of NaCl solution into the blood before looking at the effects of water infusion on the unit activity. If the small infusions of NaCl solution are not large enough to keep the blood at a high NaCl concen- tration, then the units will not respond. In this study, all units continued to resPond to increased blood NaCl concentra- tions after the termination of the infusions. If the con- tinued response of the unit indicates that the stimulus is above threshold, then the probability is high that the unit will respond to decrement in blood NaCl concentration due to loading water into the stomach. The finding suggested by the results of this experiment indicates that the blood NaCl con- centration has to be above the threshold amount at which a unit responds to increased blood salinity before a response to a decrease in blood salinity can be demonstrated. Maintained activity There is also interest in the 'maintained activity' of units. The present study reports results of the maintained activity for 15 minutes prior to beginning the series of infu- sions. Five of the ten units sampled cannot be compared to the findings of other studies because there was a previous history of changing blood NaCl concentrations. The average discharge frequency of the remaining five units varied be- tween .49 spikes per second to 10.28 spikes per second. 59 The findings of this experiment are in agreement with others, which report unit maintained activities from less than 1 spike per second to 40 spikes per second (Cross and Silver, 1966). Future research The finding that all units, sampled in this experiment, eventually responded to increased blood concentration and the finding that the units were located in diverse areas of the hypothalamus suggest a hypothesis about sensitivity of hypo- thalamic neurons. It is possible that all neural units are sensitive to an increment in blood NaCl concentration, if the increment is large enough. The hypothesis suggests a re- search design in which the response of hypothalamic units in different areas are measured as a function of small incre- ments in blood NaCl concentration. The procedural difference of this design from previous studies is to increase blood NaCl concentration until the unit electrical activity is affected. It is predicted that the blood NaCl concentration to which the cells in the supraoptic nucleus would respond would be lower than other areas of the hypothalamus. It would be in- teresting to see the results of measuring differential sensi- tivities of hypothalamic units to increments in blood NaCl concentrations. It was found that the procedure of loading water into the stomach of the goat eventually affected the electrical activi- ty of osmosensitive units when the blood NaCl concentration was large. Further research is necessary to determine how the water in the stomach affected the unit electrical activity. Can the findings, that water loaded into the stomach was 60 related to a change in discharge frequency of an osmosensi— tive unit, be repeated by infusing water directly into the 'blood, A better research design is necessary to determine if the units are responding to the water loaded or to the ‘water absorbed or both. It appears from conflicting find- ings in the literature and the findings of this experiment that the blood NaCl concentration before infusing water into the blood is of critical importance. It is hypothesized that if the blood NaCl concentration is large enough that an osmo- sensitive unit will respond to a decrease in blood NaCl con- centration. If the blood NaCl concentration is not large enough, the electrical activity of an osmosensitive unit will not be sensitive to a decrease in blood NaCl concentration. The characteristics of the changes in electrical acti- vity of osmosensitive units that did not show a graded re- sponse to repeated increments of blood NaCl concentrations are interesting. One of the units reached its limit of re- sponse change after the threshold infusion. Other units also reached a limit of response change before the completion of the repeated increments in blood NaCl concentration. These findings suggest the possibility that within certain ranges of blood NaCl concentration, osmosensitive units show a graded response to increments in blood NaCl concentration. An example of this possible characteristic is shown by unit 10 in Figure '5b. If the blood NaCl concentration is larger than the upper limit of that range, the unit does not show an increased change with an increment in blood NaCl concentration. An example of this characteristic is shown by unit 3 in Figure 5.8 o: 61 Finally, two units showed an irregular response to re- peated increments in blood NaCl concentration which resulted in a clear response of the unit. These findings suggest the possibility that some osmosensitive units show an all-or- nothing response to an increment in blood NaCl concentration. Below a certain concentration of NaCl in the blood, the osmo- sensitive unit is not sensitive to changes in blood salinity. Above the same concentration of NaCl in the blood, the unit discharge frequency changes to a new level. Any further changes in blood salinity do not change the response of this type of osmosensitive unit. The unit discharges only in response to the blood salinity being below or above a certain concentration. This suggestion is similar to Corbit's (1969) proposal that parts of the neural regulatory system underlying drinking be- havior function in an all-or-none manner. This experiment has demonstrated that some neural units throughout the hypothalamus are sensitive to changes in blood NaCl concentrations for an extended period of time after the termination of the infusion of NaCl solutions into the blood. Some of the units showed a graded response to increments in blood NaCl concentration for these extended time periods. Also, there are osmosensitive units in the hypothalamus which respond to water loaded into the stomach by changing its discharge fre- quency in the direction opposite to the reSponse to increments in blood NaCl concentration. One of the purposes of this experiment was to study char- acteristics of internal mechanisms that underlie a prolonged need for water. Maintaining a high blood NaCl concentration 62 for a long period of time is related to a state of prolonged water need. Recording and analyzing the electrical activity of hypothalamic cells during periods of maintained high blood NaCl concentration is a way of studying characteristics of internal mechanisms that underlie a prolonged need for water. To conclude that an osmosensitive unit in the hypo- thalamus participates in drinking behavior is speculation, since there is no way at present to clearly identify a single cell in the hypothalamus as a neural cell directly participa- ting in the control of drinding behavior. If the cells sam- pled in this experiment participate in the control of drink- ing behavior, than the electrical activity that signals the need for water in the hypothalamus does not adapt over pro- longed periods of thirst. Also, for at least some of the hypothalamic cells, the electrical activity signals different degrees of thirst. Finally, the electrical activity of some of the hypothalamic cells signal the reduction of the need for water. l. 2. 3. 4. 5. 7. Summary All neural units whose electrical activity was sampled were affected by increased blood salinity. Eight of the units decreased their activity, while two increased their activity. The changes in spike discharge frequency of the ten neural units persisted for at least 20 to 30 minutes after the termination of the final change in blood salinity. Two of the ten units required a larger change in blood salinity than the other eight units before showing a change in discharge frequency of long duration. The responses of five of the ten neural units were graded to repeated increases in blood salinity. Four units reached their limit of response change before the completion of the series of changes in blood salinity. Eight of the ten units showed a change in discharge fre- quency after loading water into the stomach of the goat. Seven of the eight units showed a response in the opposite direction of the response to increased blood NaCl concen- tration. The units showing a response to increased blood salinity were located in different regions of the hypothalamus. 63 References Andersson, B. The effect and localization of electrical stim- ulation of certain parts of the brain stem in sheep and goats. Acta h siolo ia scandinavia, 1951, 23, 1-16. Andersson, B. Polydipsia coused by intrahypothalamic injec- tggns of hypertonic NaCl - solution. Egperientia, 1952, 157- l . Andersson, B. The effect of injections of hypertonic NaCl . solutions into different parts of the hypothalamus of goats. Acta physiologia scandinavia, 1953, 29, 188-201. Andersson, B. and McCann, S. M. Drinking, antidiuresis and milk ejection from electrical stimulation within the hypo- thalamus of the goat. Acta h siolo ia scandinavia, 1955, 35, 191-201. Andersson, B. and McCann, S. M. The effect of hypothalamic lesions on the water intake of the dog. Acta physiologia scandinavia, 1956, 35, 312-320. Andersson, B. and Wyrwicka, W. Elicitation of a drinking motor conditioned reaction by electrical stimulation of the hypothalamic ”drinking area” in the goat. Acta physiologia scandinavia, 1957, 41, 194. Brooks, C. McC., Ushiyama J. and Lange, G. Reactions of neurons in or near the supraoptic nuclei. American Journal of Ph siolo , 1962, 202, 487-490. Corbit, J. D. Osmotic thirst: Theoretical and experimental analysis. Journal of Com rative and Ph siolo ical Ps chclo , 1969’ 67’ 3-1 0 Cross, B. A. The hypothalamus in mammalian homeostasis. In Symposia of the society for experimental biology. Number XVIII, Homeostasis and feedback mechanisms, 19 , Cambridge: at the university press. Cross, 8. A. and Green, J. D. .Activity of single neurons in the hypothalamus: Effect of osmotic and other stimuli. Jour- nal of Ph siolo , 1959, 148, 554-569. Cross, B. A. and Silver, 1. A. Electrophysiological studies on hypothalamus, British Medical Bulletin, 1966, 22, 254-260. Fitzsimcns, J. T. Drinking thresholds and adaptation. Jour- nal of Ph siolo , London, 1963, 167, 344-354. Hubel, D. H. Tungsten microelectrodes for recording from single units. Science, 1957. 125. 549-550- 64 65 Joynt, R. J. Functional significance of osmosensitive units in the anterior hypothalamus. Journal of Neurolo , 1964, Koizumi, K., Ishikawa, T. and Brooks, C. McC. Control of activity of neurones in the supra optic nucleus. Journal of Neuro h siolo , 1964, 27, 878-892. Nakamma, T. Hypothalamic electrical activities produced by factors causing discharge of pituitary hormones. Japanese Journal of Ph siolo , 1955, 311-316. Novin, D. and Dirham, R. Unit and D-C potential studies of the supraoptic nucleur. Annals of the New York Academ of Scienceg, 157, Art. 2, 740-754. ~ Richard, P. .Atlas stereotaxi ue du cerveau do Brebis. Institut Nations de la Recherche Aqronomique, l 9, 967, rue de Grenelle - Paris 7e. Sawyer, C. H. and Gernandt, B. E. Effects of intracarotid and intraventricular injections of hypertonic solutions on electrical activity of the rabbit brain. American Journal of Physiology, 1956, 185, 209-216. Suda, 1., Koizumi, K. and Brooks, C. McC. Study of unitary activity in the supraoptic nucleus of the hy othalamus. Ja 030 Journal of Ph siolo , 1963, 13, 37 -385. Thornton, L. The influence of subcutaneously injected sodi- um chloride solutions on readiness to drink and amount of water consumed b a bino rats. Unpublis ed experiment from masters thesis, I966, Michigan State University. Verney, E. B. The antidiuretic hormone and the factors which determine its release. Proceedi s of the R0 al Societ , London, 3., 1947, 135, 25-106. Vbn Euler, C. Apreliminary note on slow hypothalamic ”osmo- potentials". Acta h siolo ia scandinavia, 1953, 29, 133-136. Wayner, M. J. and Kahan, S. A. Central pathways involved dur- ing the salt arousal of drinking. Annals of the New Ygrk Academy of Sciences, 1969, 157, 701-722. Wolf, A. V. Osmometric analysis of thirst in man and dog. American Journal of Ph siolo , 1950, 161, 75-86. Zeballos, G. A., Wang, M. B., Koizumi, K. and Brooks, C. McC. Studies on the anterior hypothalamus of the opossum. Neuro- endccrinology, 1967, 2, 88-98. APPENDICES 66 Appendix A Apparatus used for infusipg solutions 1. Compact infusion pump. Harvard Apparatus Co., Inc. 2. Sodium Heparin. Panheprin®, Abbott Laboratories. 3. Polyethylene tubing. Inside diameter = .034” Outside diameter = .050'. Intramedic ® PE 90. WW salts—Wm The amount of NaCl in the solutions infused into the blood were determined by equations developed by Wolf (1950). Given the body weight and blood volume, the equations predict the change in blood NaCl concentration following the infusion of a salt load into the blood. The equations predict that an infusion of 5000 of 16$ NaCl would result in a 2.9 to 3.1$ change in blood NaCl concentration, given the weights of the goats used in this experiment. Solutions were maintained at 34° centigrade in a water bath prior to infusion. Appgpatus and.materials used in manufacture of microelectrodes 1. Tungsten wire. 2. Pyrex glass tubing. 3. The wire was sharpened with current passing through potassium nitrate. 4. Sharpened wire was insulated with glass by means of a vertical pipette puller (Model 7000, David Kopf Instruments.) 5. Glass was etched away from tip of electrode with hydrofluoric acid. 67 68 Apmatus used for respiration 1. 2. Respirator, Bodine Electric Company, Type NSR-34 RR. Rate of 15 cycles/minute, and volume of 30000 per cycle. Apmatus used for recordipg electrical activity 1. 2. 3. 4. 5. Tungsten.microelectrode. Preamplifier. voltage gain = 11000. Frequency range = 80 hertz to 40,000 hertz. Tektronix, Type FM 122. Vertical amplifier of dual beam.oscillosccpe. Output voltage from vertical amplifier varied between 2 and 6 volts. Signals monitored with audio monitor. Grass Instrument Company, Model AM 5 Electrical activity recorded on Scotch recording tape (Low print, 1.5 Mil Acetate) by a Magneccrd tape re- corder, model 1028. Appgpgtus used to analyze recorded electrical activity 1. 2. 3. a. 5. 6. Magneccrd tape recorder, Model 1028. Tektronix 502 A D131 Beam Oscilloscopes. Peak Detector. Baseline is adjustable from 0.5 to 10.0 volts. Win- dow is adjustable from 0.0 to 10.0 volts above the baseline. Frequency range is 100 hertz to 1500 hertz. Technical Measurement Corporation, Model 607. Computer of Average Transients. Mnemotron Corporation, CAT 400 B. Stimulator. Grass Instruments Company, Model S 4. Teletype Type-Punch-Read Unit. Technical Measurement Corporation, Model 535. Apmtus ES materials used for histology 1. 2. 3. Animal perfused with 10$ formalin solution. Block of hypothalamus from goat brain frozen and 50 u sections cut with a microtcme (American Optical Com- pany, Model 860,0 Sections mounted on glass slides and stained with cresylecht violet solution. Appendix B Frequency histograms of different records of electrical activity of each neural unit Appendix B The following section is made up of ten pages of figures (15 - 24). Each page of histograms represent the effects of the repeated infusions of hypertonic saline solutions and stomach water loading on the discharge frequency of a hypo- thalamic neural unit. Each page is made up of eight histograms. The histograms represent distributions of average discharge frequencies for periods of time before, during and for an ex- tended period of time after each infusion of hypertonic saline into the common carotid artery and for a period of time after loading water into the stomach. Each period of time represent- ed by the histogram is either 10 or 15 minutes. Each 10 or 15 minute period is divided into 60 or 90 ten second intervals. A discharge frequency for a particular ten second interval is expressed in.mean number of spikes per second (E'spikes/second). Each histogram presents the proportion of ten second intervals of a specified period of time represented by the histogram in which the neural unit has a certain average discharge frequency. Fbr example, a neural unit might have had an average discharge frequency of two spikes/second for 20$ of the ten second inter- vals during the first infusion of hypertonic saline into the carotid artery. 70 71 Figure 15 The histograms represent the effect of repeated infusions of hypertonic saline and stomach water load on the discharge frequency of neural unit #1. The neural unit activity was re- corded from goat #12. There were 0 series of three hypertonic saline infusions followed by water stomach loads prior to the observations of this unit. This unit was 1 of 1 units record- ed from the same electrode site. The electrode tip was esti- mated after histology to be in the mammillary region of the hypothalamus. Refer to Figure 6. Pre-infusion refers to the electrical activity of the neural unit #1 prior to beginning the series of infusions. The his- togram represents 15 minutes of tape recorded electrical acti- vity. The median average discharge frequency is .49 spikes/ second. The semi-interquartile range is 0.1 spikes/second. First infusion refers to the electrical activity of unit #1 during 10 minutes of infusion. The median average discharge frequency is 0 spikes/second. The semi-interquartile range is 0.05 spikes/second. ' Post-first-infusion refers to the electrical activity of unit #I occuring 20 to 30 minutes after the termination of the first infusion. The median average discharge frequency is 0.23 spikes/second. The semi-interquartile range is 0.16 spikes/second. 8 00nd infusion refers to the electrical activity of unit #1 Euring 10 minutes of infusion. The median average discharge frequency is .10 spikes/second. The semi-interquartile range is .10 spikes/second. Post-second-infusicn refers to the electrical activity of unit #1 occuring 20 to 30 minutes after the termination of the second infusion. The median discharge frequency is 0.20 spikes/second. The semi-interquartile range is 0.22 spikes/ second. Third infusion refers to the electrical activity of unit #1 Euring IO minutes of infusion. The median discharge fre- quency is 0.23 spikes/second. The semi-interquartile range is 0.16 spikes/second. Post-third-ingpsion refers to the electrical activity of unit occuring 20 to 30 minutes after the termination of the third infusion. The median average discharge frequency is 1.45 spikes/second. The semi-interquartile range of average discharge frequencies is 1.86 spikes/second. Pcst-water-load refers to the electrical activity of unit #1 occuring 10 to 20 minutes after stomach loading 300000 of water. The median average discharge frequency is 1.47 spikes/ second. The semi-interquartile range is 0.33 spikes/second. 72 in. Figure 'I ‘5- L . . “3% .9 n Pr: - m eusmn J . Post- water- load CL; PCVIOJ Per'od -2 c .6 ‘ + 3 g a J" 3‘ 4 3’ c . n‘.’ a 1 . 4. e ' 1:, ‘5, i Y j. ' £3 ‘BVJV niefub' ”133:; 7 soukeslsec. F|.f'$+ {n¥US|.0n POS‘!" ‘Firsfo m‘ruslon .‘Uh—W Period 3. Paruod "’ '1 .4. V ]r T v v f 7 *1 f f v A ° u ‘4 3 ' 1.1 5.9 ' ' 1 . . i - SECond {n+usl'on ’ PostfiIrf‘FUSIan . FT Perubd ' ‘ ’" Pen“; ' 1 J ‘ .44 q»— -1 3 J a." is F‘ . ' 3:, ’3, f t Th'."d {nfusc'on 8‘ Pos+ - +kv'r‘d-I'n‘gusl'cn ' . F' Period 9.....4 "q .4. I 1 v - fi‘ f ,f . _ f v Q- . m 5.3 ’ 13 53 8.3 u «u m as 73 Figure 16 The histograms represent the effect of repeated infu- sions of hypertonic saline and stomach water load on the dis- charge frequency of neural unit #2. The neural unit activity was recorded from goat #14. There were 0 series of three hy- pertonic saline infusions followed by water stomach loads pri- or to the observations of this unit. This unit was 1 of 1 units recorded from the same electrode site. The electrode tip was estimated after histology to be in the supra optic region of the hypothalamus. Refer to Figure 7. Pre-infusion refers to the electrical activity of the neural unit #2 prior to beginning the series of infusions. The his- togram represents 15 minutes of tape recorded electrical ac- tivity. The median average discharge frequency is 10.28 spikes/second. The semi-interquartile range is 0.40 spikes/ second. First infusion refers to the electrical activity of unit #2 during 10 minutes of infusion. The median average discharge frequency is 8.45 spikes/second. The semi-interquartile range is 0.41 spikes/second. Post-first-infusion refers to the electrical activity of unit #2 occuring 20 to 30 minutes after the termination of the first infusion. The median average discharge frequency is 0.62 spikes/second. The semi-interquartile range is 0.23 spikes/ second. Second infusion refers to the electrical activity of unit #2 during 10 minutes of infusion. The median average discharge frequency is 0.22 spikes/second. The semi-interquartile range is 0.18 spikes/second. Post-second-infusion refers to the electrical activity of unit #2 occuring 20 to 30 minutes after the termination of the sec- ond infusion. The median discharge frequency is 0.0 spikes/ second. The semi-interquartile range is 0.08 spikes/second. Third infusion refers to the electrical activity of unit #2 during 10 minutes of infusion. The median discharge frequency is 0.0 spikes/second. The semi-interquartile range is 0.0 spikes/second. Post-third-infusion refers to the electrical activity of unit #2 occuring 20 to 30 minutes after the termination of the third infusion. The median average discharge frequency is 0.0 spikes/second. The semi-interquartile range of average discharge frequencies is 0.0 spikes/second. Post-water-load refers to the electrical activity of unit #2 occuring 10 to 20 minutes after stomach loading 3000cc of water. The median average discharge frequency is 1.42 spikes/ second. The semi-interquartile range is 0.19 spikes/second. cond mfcrwl! 91’ ' “Frog: ton . ct 74 Flogur-e‘ lb to" Pre- infusion Pos‘f- wth— load SW Period .81 Period .5‘ -— 1 .‘l‘ jT .. 'J v T ‘ writ. ; r i A— ? spikes/sec. 1! Hrs" infusion 0‘ n P051” ‘Fc'rs‘l’nh‘fusion .8 . Period ' Period ‘[ 1 .N 3:” f 1 L .311 "0 __ c I ‘L 1 W Second infusion ‘ Posf- second-infusion .8 Pern'od .8 Period 4 3" ”cm L ‘ o i 0‘ { WT Th‘I’J .‘nfusion 81—- p0$+' +h;V.A‘ ‘I‘TYUS‘Oh '81 Pen-(cc! ' Percod H1 ,4” 4 o "i o ‘ .L 75 Figure 17 The histograms represent the effect of repeated infusions of hypertonic saline and stomach water load on the discharge frequency of neural unit #3. The neural unit activity was re- corded from goat #15. There were 0 series of three hyper- tonic saline infusions followed by water stomach loads prior to the observations of this unit. This unit was 1 of 1 units recroded from the same electrode site. The electrode tip was estimated after histology to be in the tuberal region of the hypothalamus. Refer to Figure 8. Pro-infusion refers to the electrical activity of the neural unit #3 prior to beginning the series of infusions. The his- togram represents 15 minutes of tape recorded electrical ac- tivity. The median average discharge frequency is 0.93 spikes/ second. The semi-interquartile range is 0.20 spikes/second. First infusion refers to the electrical activity of unit #3 during 15 minutes of infusion. The median average discharge frequency is 0.88 spikes/second. The semi-interquartile range is 0.16 spikes/second. Post-first-infggion refers to the electrical activity of unit 3 occuring 20 to 30 minutes after the termination of the first infusion. The median average discharge frequency is 0 spikes/second. The semi-interquartile range is 0 spikes/sec- on . Second infusion refers to the electrical activity of unit #3 during minutes of infusion. The median average discharge frequency is 0 spikes/second. The semi-interquartile range is 0 spikes/second. Post-second-infusion refers to the electrical activity of unit #3 occuring 25 to 30 minutes after the termination of the second infusion. The median discharge frequency is 0.0 spikes/second. The semi-interquartile range is 0.0 spikes/ second. Third infusion refers to the electrical activity of unit #3 during 15 minutes of infusion. The median discharge fre- quency is 0.0 spikes/second. The semi-interquartile range is 0.0 spikes/second. Post-third-ingusion refers to the electrical activity of unit 3 occuring 20 to 30 minutes after the tenmination of the third infusion. The median average discharge frequency is 0.0 spikes/second. Pgst-watgf-logg refers to the electrical activity of unit #3 occuring 0 to 20 minutes after stomach loading 300000 of water. The median average discharge frequency is 0.77 spikes/ second. The semi-interquartile range is 0.18 spikes/second. 76 .3: .1: H" l-Sf Y spikes/sec. Fi rs+ in‘Fusion -8 Period WI I l I I—I o .3; .13 u: I-SS a S Etcnd 40‘?“ 5“” ' Pern'od I't—v—r I O .3! .1! Third ln‘FuS'Ion .8 Perl-0A .9 . 9 Figure I7 ‘.I43 . 4‘43 pre- m-Fusuon Pos'f— wafer“ loaJ 0 g . . Cl; .8 Pert'od ,3 Pcr'od .2 5'» *C 3' 8.“.4 .4 3 c o. owl +— 'IfiI I I I v I o .3: .17 Ms :5! Posf- ‘Fi rs+- {n'?u3\'or\ 3‘ Panto J .4‘ 0 f3: ] p.51. sewn-a Manon Per iod '1 I" J a .35” __ Pos‘f— “15rd - imcusa'on .8 Period 3’ 77 Figure 18 The histograms represent the effect of repeated infu- sions of hypertonic saline and stomach water load on the dis- charge frequency of neural unit #4. The neural unit activity was recorded from goat #16. There were 0 series of three hy- pertonic saline infusions followed by water stomach loads prior to the observations of this unit. This unit was 1 of 1 units recorded from the same electrode site. The electrode tip was estimated after histology to be in the tuberal region of the hypothalamus. Refer to Figure 9. Pre-infusion refers to the electrical activity of the neural unit #4 prior to beginning the series of infusions. The histogram represents 15 minutes of tape recorded electrical activity. The median average discharge frequency is 4.45 spikes/second. The semi-interquartile range is 0.26 spikes/ second. First infusion refers to the electrical activity of unit #4 during 10 minutes of infusion. The median average discharge frequency is 0.41 spikes/second. The semi-interquartile range is 1.22 spikes/second. Post-first-infusion refers to the electrical activity of unit #4 occuring 20 to 30 minutes after the termination of the first infusion. The median average discharge frequency is 5.40 spikes/second. The semi-interquartile range is 0.44 spikes/second. Second infusion refers to the electrical activity of unit #4 during 10 minutes of infusion. The median average discharge frequency is 3.10 spikes/second. The semi-interquartile range is 0.93 spikes/second. Post-second-infusion refers to the electrical activity of unit occuring 20 to 30 minutes after the termination of the sec- ond infusion. The median discharge frequency is 4.78 spikes/ second. The semi-interquartile range is 0.51 spikes/second. Third infusion refers to the electrical activity of unit #4 during 10 minutes of infusion. The median discharge fre- quency is 0.53 spikes/second. The semi-interquartile range is 0.31 spikes/second. Post-third-infusion refers to the electrical activity of unit #4 occuring 20 to 30 minutes after the termination of the third infusion. The median average discharge frequency is 0 spikes/second. The semi-interquartile range of average dis- charge frequencies is 0.12 spikes/second. Pcst-water-load refers to the electrical activity of unit #4 occuring 10 to 20 minutes after stomach loading 300000 of water. The median average discharge frequency is 2.45 spikes/ second. The semi-interquartile range is 0.79 spikes/second. o‘F or'fio n seco P p... +en nd in‘l’ervo's 78 w Figure '8 V Prc-{nfusu'on “ P051“ wo+er- load .8 ’ Pernod .8“ Period .4' F ‘ .4p ”4» .ll’ 4 .1 4 ' __ I; I: 5.1 o .q 1.: 1.3 1.: m x sPukes/sec. o ' a r .g. 9.3+ mus... .9] 9.5+-1c...+-;nm.. Period Period SJ “*4 '1 T 0 a u 3.5 to? Jr'E-r' 5.1 ' Second infufion Pos+~ second-MWSiON .84 Period .94 Damien! T 31 flat ‘ ‘ I' , __..._._I I_.i o J 14 3.! 1.: £1 £1 1.: 4 u 1.3 1.: 5.1 M ' Third {Infusion ‘ P9515 'Hu'rd - u'n‘tusion "7 Period .8' 04 a. 79 Figure 19 The histograms represent the effect of repeated infusions of hypertonic saline and stomach water load on the discharge frequency of neural unit #5. The neural unit activity was re- corded from goat #16. There were 1 series of three hyper- tonic saline infusions followed by water stomach loads prior ‘ to the observations of this unit. This unit was 1 of 1 units recorded from the same electrode site. The electrode tip was estimated after histology to be in the supraoptic region of the hypothalamus. Refer to Figure 10. Pro-infusion refers to the electrical activity of the neural unit #5 prior to beginning the series of infusions. The his- togram represents 15 minutes of tape recorded electrical ac- tivity. The median average discharge frequency is 4.19 spikes/ second. The semi-interquartile range is 0.48 spikes/second. First infusion refers to the electrical activity of unit #5 during 10 minutes of infusion. The median average discharge frequency is 0.34 spikes/second. The semi-interquartile range is 0.38 spikes/second. Post-first-infusion refers to the electrical activity of unit #5 occuring 20 to 30 minutes after the termination of the first infusion. The median average discharge frequency is 0.25 spikes/second. The semi-interquartile range is 0.10 spikes/second. Second infusion refers to the electrical activity of unit #5 during 10 minutes of infusion. The median average discharge frequency is 0.38 spikes/second. The semi-interquartile range is 0.25 spikes/second. Post-second—infusion refers to the electrical activity of unit occuring 20 to 30 minutes after the termination of the sec- ond infusion. The median discharge frequencz is 0.12 spikes/ second. The semi-interquartile range is 0.1 spikes/second. Third infusion refers to the electrical activity of unit #5 during 10 minutes of infusion. The median discharge frequency is 0 spikes/second. The semi-interquartile range is 0 spikes/ second. Post-third-infusion refers to the electrical activity of unit #5 occuring 25 to 30 minutes after the termination of the third infusion. The median average discharge frequency is 0 spikes/second. The semi-interquartile range of average dis- charge frequencies is 0 spikes/second. Post-water-load refers to the electrical activity of unit #5 occuring 10 to 20 minutes after stomach loading 300000 of water. The median average discharge frequency is 14.26 spikes/second. The semi-interquartile range is 0.54 spikes/ second. 80 Figure W L” g :3" . . Q6? Pre — m‘Fusuon :18 Pct-1.04 <5 5.4. 4— U '- to 0 .4 LU. o {.1 of w 4. | v T r v I 1.1 :11 $1 5.1 ; srikes/sec. a p.551. .‘nfusion ' Per-{ad J! oi 31 ' I'1 8 SECOna in'FHSIbn Peru'oa Third {n‘quIon '8 pefiod Pgd" Fl'rs‘r- En‘Susion .9 Period ." o ' 3' ET 903+ ‘ Wolfe“ ’ load Period «.1 11.1 "-1 NJ “-1 IB-‘l Pod - seConJ-infusim Parked Al Pos+ _ +hu'rd . [nfusfon .8 Pariod 81 Figure 20 The histograms represent the effect of repeated infusions of hypertonic saline and stomach water load on the discharge frequency of neural unit #6. The neural unit activity was re- corded from goat #16. There were 2 series of three hypertonic saline infusions followed by water stomach loads prior to the observations of this unit. This unit was 1 of 1 units record- ed from the same electrode site. The electrode tip was esti- mated after histology to be in the preoptic region of the hy- pothalamus. Refer to Figure 11. Pro-infusion refers to the electrical activity of the neural unit #3 prior to beginning the series of infusions. The histogram represents 15 minutes of tape recorded electrical activity. The median average discharge frequency is 0.52 spikes/second. The semi-interquartile range is 0.18 spikes/ second. First infusion refers to the electrical activity of unit #6 during 10 minutes of infusion. The median average discharge frequency is 0.29 spikes/second. The semi-interquartile range is 0.12 spikes/second. Post-first-infusion refers to the electrical activity of unit occuring 20 to 30 minutes after the termination of the first infusion. The median average discharge frequency is 0.15 spikes/second. The semi-interquartile range is 0.13 spikes/second. Second infusion refers to the electrical activity of unit #6 during 10 minutes of infusion. The median average discharge frequency is 0.10 spikes/second. The semi-interquartile range is 0.08 spikes/second. Post-second-infusion refers to the electrical activity of unit occuring 20 to 30 minutes after the termination of the sec- ond infusion. The median discharge frequencK is 0.0 spikes/ second. The semi-interquartile range is 0.0 spikes/second. Third infusion refers to the electrical activity of unit #6 during 10 minutes of infusion. The median discharge fre- quency is 0.0 spikes/second. The semi-interquartile range is 0.0 spikes/second. Post-third-infusion refers to the electrical activity of unit #6 occuring 20 to 30 minutes after the termination of the third infusion. The median average discharge frequency is 0.0 spikes/second. The semi-interquartile range of average discharge frequencies is 0.0 spikes/second. Post-water-load refers to the electrical activity of unit #6 occuring 10 to 20 minutes after stomach loading 30000 of water. The median average discharge frequency is 0.0 spikes/second. The semi-interquartile range is 0.06 spikes/second. 82 J Figure 20 c as} to Pre- infusion P051"- wafer-loud c .e "'04 3 Period C" .24 l. t: 8.: 4 A o “E; .1 ° 1 -; g;“:/I¢:° I I. WI. ' I} . ‘- Firs+ n'nfusc'on Po 31' - ‘Fir’f- ln‘usion -8 period .8 Pet-(0A .‘I 'l a—r-l ' '0 ‘ b l B a I i T $1 ‘ Second in‘usion 1 P0015 second- ‘mFuu'on .8 Period 9- Pen'oé ." '0 £7 '1 ' .4 ' .1 o it). '7 Th; rd infusion 1 905+ - ‘Hfira- fiv‘uflen '3 paid .8- Per-(04 U ,4. 83 Figure 21 The histograms represent the effect of repeated infu- sions of hypertonic saline and stomach water load on the dis- charge frequency of neural unit #7. The neural unit activi- ty was recorded from goat #18. There were 0 series of three hypertonic saline infusions followed by water stomach loads prior to the observations of this unit. This unit was 1 of 1 units recorded from the same electrode site. The electrode tip was estimated after histology to be in the supraoptic region of the hypothalamus. Refer to Figure 12. Pre-infusion refers to the electrical activity of the neural unit #7 prior to beginning the series of infusions. The histogram represents 15 minutes of tape recorded electrical activity. The median average discharge frequency is 1.90 spikes/second. The semi-interquartile range is 0.32 spikes/ second. First infusion refers to the electrical activity of unit #7 during 10 minutes of infusion. The median average discharge frequency is 0.20 spikes/second. The semi-interquartile range is 0.48 spikes/second. Post-first-infusion refers to the electrical activity of unit #7 occuring 20 to 30 minutes after the termination of the first infusion. The median average discharge frequency is 0.81 spikes/second. The semi-interquartile range is 0.16 spikes/second. Second infusion refers to the electrical activity of unit #7 during 10 minutes of infusion. The median average discharge frequency is 0.25 spikes/second. The semi-interquartile range is 0.25 spikes/second. Post-second-infusion refers to the electrical activity of unit #7 occuring 20 to 30 minutes after the termination of the second infusion. The median discharge frequency is 0.0 spikes/second. The semi-interquartile range is 0.0 spikes/ second. Third infusion refers to the electrical activity of unit #7 during 10 minutes of infusion. The median discharge fre- quency is 0.0 spikes/second. The semi-interquartile range is 0.0 spikes/second. Post-third-infusion refers to the electrical activity of unit #7 occuring 20 to 30 minutes after the termination of the third infusion. The median average discharge frequency is 0.0 spikes/second. The semi-interquartile range of average discharge frequencies is 0.0 spikes/second. Pest-water-load refers to the electrical activity of unit #7 occuring 15 to 20 minutes after stomach loading 300000 of water. The median average discharge frequency is 2.26 spikes/ second. The semi-interquartile range is 0.48 spikes/second. Pro or‘hon hep r of I." +2!” v‘ '5 second PIC. 84 Figure 2| Pr: — mansion PEI-Sod LO LO 1.1. 1.9 i" afikcs/sec. first “\qu I'On Per-€04 LO Lb 1_1_ Seemed |}\‘Fu510n FEvn'eA l-O Lb Thfi‘d L'nfuelén Per- Rod Posf- wafer - lqu Per-i0 "' '-° '4' 1-1 La 3.4 Po 51’ - ‘Ffrsf- 'ln +ueion PEr-iod fi I I '4 no I-b Po sf— satand- {ngusfen Pe rKoA Posf - ‘H‘ird- fn‘usfon Per EDA 85 Figure 22 The histograms represent the effect of repeated infusions of hypertonic saline and stomach water load on the discharge frequency of neural unit #8. The neural unit activity was -recorded from goat #18. There were 1 series of three hyper- tonic saline infusions followed by water stomach loads pr- ior to the observations of this unit. This unit was 1 of 1 units recorded from the same electrode site. The electrode tip was estimated after histology to be in the preoptic region of the hypothalamus. Refer to Figure 13. Pre-infusion refers to the electrical activity of the neural unit #8 prior to beginning the series of infusions. The histogram represents 15 minutes of tape recorded electrical activity. The median average discharge frequency is 0.29 spikes/second. The semi-interquartile range is 0.16 spikes/ second. First infusion refers to the electrical activity of unit #8 during 15 minutes of infusion. The median average discharge frequency is 1.95 spikes/second. The semi-interquartile range is 0.92 spikes/second. Post-first-infusion refers to the electrical activity of unit #8 occuring 25 to 30 minutes after the termination of the first infusion. The median average discharge frequency is 0.53 spikes/second. The semi-interquartile range is 0.54 spikes/second. Second infusion refers to the electrical activity of unit #8 during 10 minutes of infusion. The median average discharge frequency is 2.23 spikes/second. The semi-interquartile range is 0.62 spikes/second. ' Fost-second-infusion refers to the electrical activity of unit occuring 20 to 30 minutes after the termination of the sec- ond infusion. The median discharge frequency is 0.92 spikes/ second. The semi-interquartile ran e is 0.19 spikes/secc 8. Third infusion refers to the electrfcal activity of unit # during 10 minutes of infusion. The median discharge fre- quency is 2.41 spikes/second. The semi-interquartile range is 0.90 spikes/second. Post-third-infusion refers to the electrical activity of unit #8 occuring 20 to 30 minutes after the termination of the third infusion. The median average discharge frequency is 0.76 spikes/second. The semi-interquartile range of average dis- charge frequencies is 0.28 spikes/second. Post-water-load refers to the electrical activity of unit #8 occuring 10 to 20 minutes after stomach loading 300000 of water. The median average discharge frequency is 5.91 spikes/ second. The semi-interquratile range is 0.56 spikes/second. 86 pre-ini‘uslon Petfiod _ of- n n+1 rvqls 3 'P‘Q proporhon +en second in 4: 7 spike: / sec. Fir-5+ Infusion ‘3 Peri cd Ll 3.3 'M’ 5.1 5 count! infusion 3 Pemot‘ c a M 3.3 15 a {ntusfon ' P2 r I'oA Th'ns Figure. 22 Post wafer—10m; -9 Pa rl'oé Pos+- first In ’Fuu'o n Period P0511 second - in'fusion Period Posf- ‘HMI'A- infusibn 3 PeriOd 87 Figure 23 The histograms represent the effect of repeated infu- sions of hypertonic saline and stomach water load on the discharge frequency of neural unit #9. The neural unit ac- tivity was recorded from goat #18. There were 1 series of three hypertonic saline infusions followed by water stomach loads prior to the observations of this unit. This unit was 1 of 2 units recorded from the same electrode site. The electrode tip was estimated after histology to be in the preoptic region of the hypothalamus. Refer to Figure 14. Pre-infusion refers to the electrical activity of the neural unit #5 prior to beginning the series of infusions. The histogram represents 15 minutes of tape recorded electrical activity. The median average discharge frequency is 9.20 spikes/second. The semi-interquartile range is 1.26 spikes/ second. First infusion refers to the electrical activity of unit #9 during 10 minutes of infusion. The median average discharge frequency is 0.0 spikes/second. The semi-interquartile range is 0.16 spikes/second. Post-first-infusion refers to the electrical activity of unit occuring 20 to 30 minutes after the termination of the first infusion. The median average discharge frequency is 0.35 spikes/second. The semi-interquartile range is 0.32 spikes/second. Second infusion refers to the electrical activity of unit #9 during 10 minutes of infusion. The median average discharge frequency is 0 spikes/second. The somi-interquartile range is 0 spikes/second. Post-second-infusion refers to the electrical activity of unit occuring 20 to minutes after the termination of the second infusion. The median discharge frequency is 1.39 spikes/second. The semi-interquartile range is 0.58 spikes/ second. Third infusion refers to the electrical activity of unit #9 during 10 minutes of infusion. The median discharge fre- quency is 0.0 spikes/second. The semi-interquartile range is 0.17 spikes/second. ‘ Post-third-infusion refers to the electrical activity of unit occuring 2 to 30 minutes after the termination of the third infusion. The median average discharge frequency is 0.0 spikes/second. The semi-interquartile range of average discharge frequencies is 0.0 spikes/second. Post-water-load refers to the electrical activity of unit #9 occuring 10 to 20 minutes after stomach loading 300000 of water. The median average discharge frequency is 2.95 spikes/ second. The semi-interquartile range is 3.35 spikes/second. 88 W Figure 23 ~I.° . . qt.) E prcr "\be swn 905+- wafEr- load a ' - 4E. Pernod 3 Pernod .g...‘ 7E 53.4 " o 0 Ln ° .2. o: s +- ‘ l I I ' V 'j— __ 5; 1.; 19' IL! 0.! 0 us 3.: 5.5 1.! 9.! X SFIkQSISCC. 8 Fit-5f {ntusio-n PosT-‘Furs‘hm‘usmn ' Pei-(ed '9 Pat-(04 JI- 0 Is 3.: u 15 55W“ 1 Second Im‘FuSl'an Posf- second-Infusion 3 Ported .8 Per-(08 .‘h .‘f a is a '6. u Thfr‘d I'h‘guSlbn Pos‘r - fhird- u'n1usion '9' Per.“ .8 Pen'oA 89 Figure 24 The histograms represent the effect of repeated infu- sions of hypertonic saline and stomach water load on the dis- charge frequency of neural unit #10. The neural unit acti- vity was recorded from goat #18. There were 2 series of three hypertonic saline infusions followed by water stomach loads prior to the observations of this unit. This unit was 1 of 2 units recorded from the same electrode site. The electrode tip was estimated after histology to be in the preoptic region of the hypothalamus. Refer to Figure 15. Pro-infusion refers to the electrical activity of the neural unit #10 prior to beginning the series of infusions. The histogram represents 15 minutes of tape recorded electrical activity. The median average discharge frequency is 21.18 spikes/second. The semi-interquartile range is 2.51 spikes/ second. 'First infusion refers to the electrical activity of unit #10 during 10 minutes of infusion. The median average discharge frequency is 25.14 spikes/second. The semi-interquartile range is 4.32 spikes/second. Post-first-infusion refers to the electrical activity of unit #10 occuring 20 to 30 minutes after the termination of the first infusion. The median average discharge frequency is 12.5 spikes/second. The semi-interquartile range is 1.8 spikes/second. Second infusion refers to the electrical activity of unit during minutes of infusion. The median average dis- charge frequency is 23.14 spikes/second. The semi-inter- quartile range is 10.72 spikes/second. Post-second-infusion refers to the electrical activity of unit #10 occuring 25 to 30 minutes after the termination of the second infusion. The median discharge frequency is 8.64 spikes/second. The semi-interquartile range is 1.62 spikes/ second. Third infusion refers to the electrical activity of unit #10 during 15 minutes of infusion. The median discharge fre- quenci is 20.95 spikes/second. The semi-interquartile range is 5. 6 spikes/second. Post-third—infusion refers to the electrical activity of unit occuring 2 to 30 minutes after the termination of the third infusion. The median average discharge frequency is 5.18 spikes/second. The semi-interquartile range of average discharge frequencies is 1.20 spikes/second. Post-water-load refers to the electrical activity of unit #10 occuring 15 to 20 minutes after stomach loading 300000 of water. The median average discharge frequency is 9.6 spikes/ second. The semi-interquartile range is 1.06 spikes/second. ml: . of '?j econd mfer '0- when ’1 A Pro m 5.- 90 Pre- lifl‘FUJ {on PEriod Jim. .mr '11-, ‘ 1's; ‘r—3" 0‘ K spikes/sec. Fsrsf ih+u5lbh Perloti NJ 15‘ “S 313 ‘91.! if! SeCohd ihPuslon Per-(ad 9.: n: 25:: as 99.: 41.! There 'mi usio n Per-{ed M] I T‘ V V 1.! at 1525’ Figure 2 Li- '1 r P951”- wafer» '0ch .81 PEriod .q1 (a? (13 W Poe? - Hut. inguso'on 3" PErI'od { T} ”J {3' {Sr Ks f Pcsf- second:""{‘°'5'\ -"' PQPIO4 1 r! w [_ 337' 9‘: J Posf— 'HMHI - .hfum‘on -° 7; Per-.04 H- ‘L. Li J: 1:: Appendix C Raw Data 91 92 Unit 1 Each number represents the number of spikes per successive ten second interval (Read down columns) Second infusion infusion Post-first- First infusion Pro-infusion 1.4 26630176 91.. 393638.403954113603 11 321 1112-421.010121301011u5001011u00 22262.4361241.40317132121022012 nzea1f44392711,3:J3(4939231011721111:dnu1$na)o171192 2110u0615030306u232270:41205221 100000100000023023376581-456 .1111Iioc922eJQKQhwfiiZoJ 000 1.000 026 O 21 1000001000000000000 00 13.4“25751000000000001210001011 888 2 2 395316 2““. 2566 32 553 2 film/Oh». 956 2 779.“. £41. 73m 284%!“ 7.78 51. 55.“ BR 2 .4 5661. 7566688.“.“34 738 3536 526.“ 28 7 93 Unit 1 (cont'd.) Post-second Third Post-Third Post-water infusion infusion infusion load 0 1 7 3 3 14 25 21 3 5 11 3 6 13 18 23 12 2 3 3 2 3 8 25 3o 35 1 3 1 0 1 17 49 14 18 O 3 O 1 4 11 66 13 28 2 1 O 3 O 8 103 23 27 3 2 2 1 2 5 20 24 7 3 4 O 1 7 4 3O 27 1 O 3 2 4 9 5 33 13 3 1 1 a 5 2 z 25 22 1 4 3 3 10 17 15 O 2 3 4 6 8 24 17 21 16 8 3 16 3 55 13 21 23 a 1 1 3 5 57 18 27 19 O 3 a 16 22 19 37 12 7 o 9 12 21 23 27 11 16 3 o 8 25 27 19 16 6 a 5 6 5 103 32 20 19 2 15 2 1 3 192 1 3 21 23 2 7 2 1 9 8 3 21 22 9 5 4 3 2 46 118 20 38 1 9 1 R 3 1 43 25 21 0 11 O z 2 12 19 17 O 31 O 0 50 24 17 2 16 0 6 7 32 7 24 18 0 17 O 2 1 4 12 25 23 1 8 O 1 6 11 8 10 25 2 9 2 O 6 3 28 17 25 0 3 0 2 9 9 25 32 23 3 1 2 5 13 21 5 21 3O Unit 2 94 Each number represents the number of spikes per successive ten second interval (Bead down columns) Pre-infusion First Post-first- Second infusion infusion infusion 100 92 98 89 83 77 7 8 1 0 0 96 101 103 86 86 85 6 13 2 3 0 98 100 102 87 78 93 6 3 4 3 0 99 106 103 81 82 87 13 4 z 0 1 92 10 5 101 84 77 95 16 4 2 5 9 103 102 78 79 85 7 4 4 1 9 102 10 83 83 87 9 z 2 3 4 100 104 1 80 72 75 12 2 l 2 95 111 101 86 79 83 4 4 3 l 0 96 107 109 84 80 81 5 7 1 1 5 96 107 99 91 81 81 3 7 4 0 2 97 112 103 90 79 90 17 9 2 0 3 99 104 100 ‘90 80 93 6 6 1 6 5 96 106 99 87 77 95 7 2 1 2 o 97 109 101 86 76 102 9 l 3 4 6 97 108 106 86 81 100 12 g 0 1 3 92 104 101 90 81 110 7 3 0 3 99 103 112 89 80 101 12 12 2 2 l 103 110 107 92 85 98 7 8 1 3 3 102 112 112 88 80 101 5 5 2 2 a 103 109 97 83 8 106 8 5 0 5 102 110 111 95 8 105 2 9 2 0 0 102 114 108 89 85 108 6 9 2 5 97 107 102 82 88 102 9 5 4 3 o 97 110 100 92 91 98 8 3 1 5 1 107 111 100 87 87 98 12 l 1 1 0 105 110 100 94 75 94 . 6 2 4 3 3 108 108 108 88 78 88 8 5 2 3 1 104 110 110 79 93 85 11 1 4 2 o 103 106 110 89 95 89 3 6 1 3 0 95 Unit 2 (cont'd.) Third Post-third Post-water infusion infusion load Post-second infusion 56776 35577u6360451647362643587 111111111111112.111111111111111 50022291070140412734.4808360233 11.11.11 .1 111111111111111121111 000010100ooooooooooooooooooooo 00000010000001.0000000000000001 000000101102003010.“01001030211 000000000000000000000000000000 0010000100100001.000003000000110 01001000OOOOOOOOOOOOOOOOOOOOOO 0300u22311122012u2112u21201110 96 Unit 3 Each number represents the number of spikes per successive ten second interval (Bead down columns) Pre-infusion First Post-first- Second infusion infusion infusion 8 12 8 12 7 10 0 0 0 0 1 ll 10 11 9 8 Z 0 0 0 0 0 l4 8 7 9 1 0 1 3 0 0 12 7 15 9 6 10 0 l 0 0 0 12 8 10 10 6 5 1 0 0 0 1 9 3 11 6 8 l 0 0 1 0 0 9 10 8 8 6 4 1 0 0 0 1 7 7 8 9 7 4 0 0 5 0 0 7 11 6 9 6 1 0 0 0 0 0 8 11 8 8 9 0 0 1 1 0 0 14 10 9 9 7 0 0 0 0 0 0 9 13 12 6 10 0 1 0 o 0 0 7 7 10 ll 8 0 2 0 o 0 0 10 10 8 8 10 0 0 0 o 0 0 7 14 7 7 10 0 0 0 1 0 0 11 11 11 9 10 0 0 0 o 0 l 16 12 12 5 11 0 2 0 o 0 0 7 7 l4 8 6 0 1 1 1 0 0 11 7 7 11 9 0 0 0 o 0 2 8 10 8 11 9 0 0 0 o 0 0 9 8 6 6 8 0 0 0 o 0 0 12 4 7 10 9 0 0 0 o 0 0 7 l4 5 6 9 0 0 0 o 0 0 10 6 11 11 14 0 0 0 1 0 0 10 13 6 9 6 0 0 0 o 1 0 11 8 11 9 4 0 0 0 1 o 0 4 6 3 9 15 0 0 1 1 o 0 13 8 13 8 9 0 0 0 o o 1 10 11 7 10 6 0 0 0 o o 0 12 12 12 12 11 0 0 0 1 o l Post-water load 9? Post—third infusion Unit 3 (cont'd.) Third infusion Post-second infusion 0862668146.“.67924888702628):?070 7.8 1 .1 .1 1 1 .11 1 1 .1 460486684092278 1:1 .111 .1 8m“. 98067914661 22 1.1 .1 O10211001110001.000000000000000 100030100100000000010000000000 000000000000100011011000000000 0000000000OOOOOOOOOOOOOOOOOOOO 000000000000000000000000000000 000000000000100000000010000000 00000000000OOOOOOOOOOOOOOOOOO0 98 Unit 4 Each number represents the number of spikes per successive ten second interval (Bead down Columns) Pro-infusion First Post-first- Second infusion infusion infusion 28% 2°? 22 22“ 0 5 5 7 2 37 46 45 ’28 33 o 12 55 53 87 25 35 43 47 44 32 o 12 5o 58 75 13 38 46 43 42 33 o 10 51 6 73 22 41 44 45 45 32 o 15 48 5 67 29 38 45 47 38 33 0 13 54 57 64 33 43 46 48 23 30 o 13 57 47 53 3o 43 49 44 26 o 8 57 3 2 31 41 9 45 4o 24 10 o 57 2 3 33 39 5 45 44 26 17 3 55 45 32 1 38 4o 45 1 27 14 8 50 5 29 2 3 4o 41 1 28 8 5 6o 6 18 31 g 45 42 46 28 1 9 51 56 14 1 43 46 41 23 4 11 52 5o 13 3g 32 46 45 4o 30 9 19 6 56 16 35 38 42 45 46 27 3 23 2 57 5 35 5 45 42 57 10 o 28 7 57 6 31 o 48 46 46 o 1 34 5 4 32 43 43 45 49 0 1 6 60 51 9 37 42 41 41 48 o 2 6 50 8 9 35 41 42 54 46 o 1 51 50 7 12 37 1 47 50 41 o 2 58 64 42 18 36 2 40 9 47 0 1 53 49 53 23 5 42 50 5 46 o 4 57 59 52 2 o 45 3 42 o o 3 55 68 62 2 39 41 7 42 3 o 7 9 63 51 18 26 42 50 46 42 o 6 o 58 62 14 35 45 o 46 42 o 6 45 51 62 17 35 41 3 46 39 o 7 4o 60 61 16 39 42 99 Unit 4 (cont'd.) Post-second Third Post-third Post-water infusion infusion 1nfusion load 56 5O 49 4 18 1 O 15 39 6O 20 29 O 21 O l 16 17 56 15 18 O 10 0 O 17 24 63 32 27 3 16 O O 16 23 67 2 21 1 16 2 O 14 26 57 23 18 2 21 O O 18 3O 2 E3 12 2 l3 0 5 13 35 8 26 1 11 l O 17 20 57 47 7 2 16 1 O 16 3O 52 48 5 1 17 1 O 19 36 56 23 11 2 20 O 2 16 2 2 9 8 1 14 O 2 l9 1 7 47 12 3 13 O 3 24 26 2 52 12 6 15 O O 13 35 1 44 10 2 16 O 1 15 23 6 1 8 5 16 0 o 37 36 6 6 7 6 16 O 1 20 37 41 53 8 3 18 1 l 20 27 fig 50 4 9 18 O 2 43 31 6 3 6 17 O 2 25 24 43 O 2 4 19 1 0 3O 37 4O 41 O 5 20 O 0 16 16 46 44 2 10 23 1 0 30 41 4O 47 O 10 21 l 1 21 14 5O 46 O 12 20 O O 31 44 l 52 2 15 19 O 0 19 31 7 41 3 11 23 O 0 33 50 8 52 1 13 21 l O 23 27 9 78 O 10 22 0 1 26 26 52 51 1 17 15 0 O 14 22 100 un1t 5 Each number represents the number of spikes per successive ten second interval (Read down columns) Ere-infusion First Post-first- Second infusion infusion infusion 43 46 47 16 1 2 2 8 6 1 38 44 38 14 2 3 2 15 1 2 39 3 44 10 2 3 3 2 13 1 51 7 44 12 3 6 3 l 6 3 2 49 51 37 19 2 2 3 O 6 2 11 1 49 34 1 O 2 2 6 9 5 2 46 3O 1 2 Z 2 1 ~9 6 8 45 46 38 14 3 3 1 11 2 9 48 46 33 12 2 4 2 3 9 6 8 47 48 9 18 2 2 3 3 8 3 8 41 45 6 19 4 5 3 2 2 O 4 4O 38 28 10 0 3 1 1 2 3 3 51 50 35 8 5 z 3 l 2 3 6 36 2 31 12 l 2 1 4 1 2 50 9 8 11 1 5 1 3 4 2 2 43 41 0 9 1 5 3 2 3 8 2 46 2 4O 7 1 7 3 2 3 O 2 24 1 39 7 1 6 2 2 3 3 3 43 28 7 O 3 2 2 1 3 4 41 43 fig 3 1 8 Z 1 2 8 2 46 1 2 2 5 2 3 6 4 47 9 38 5 l 8 2 2 8 3 2 43 37 6 1 1 4 2 3 O 5 4 36 37 0 2 3 4 2 3 l 2 4 £4 46 48 3 0 6 1 l O 2 5 46 37 1 1 5 2 2 6 6 13 22 4O 33 O 1 3 2 0 6 5 4 40 32 2 0 8 1 O 5 2 2 35 37 32 0 O 2 3 2 0 1 4 39 32 25 2 2 4 3 1 1 8 4 101 un1t 5 (cont'd.) Post-second Third Post-third Post-water infusion infusion infusion load 139 153 1 1 135 147 138 141 142 139 1&3 125 1 1 132 141 124 128 147 1 8 138 137 133 148 1 2 142 137 131 161 1 8 138 149 156 142 155 150 :2 133 122 138 141 136 149 132 140 1 2 145 136 140 1 6 141 121 142 1 o 138 146 1 7 139 138 HOONOONOHONHHOOOHHHONHONOI—‘NNON HHOOUNOONNHUONNNNHHNOOOHONUOON OOOOOOOOOOOI—‘I—‘ONOOOOOOOOHOHO\NO\U‘ COOOOOOOOOOOOOOOOOOOOOOOOOOCOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOHO OOOOOOOOOOOOI-‘OOI-‘OOOOOOOOHOOOOO COOOOOOOOOHOOOOOOOOOOOOOOHOCOO 102 Unit 6 Each number represents the number of spikes per successive ten second interval (Read down columns) Post—first- Second infusion infusion First infusion Pre-infusion 011002100000010101000001003011 013101111313211103033021200100 200030110200011115122311020231 oouulazuz2&1502021111410132232 312520300032052301003200012010 239253331513345025211552325342 5O5531225233242244321364451341 2053313357323242506220207350 8 32 53731271u2621332£747u556u3 gum/096 78872678 76562Z£6558hfi33 103 Unit 6 (cont'd.) Post-third Post-water infusion load Third infusion Post-second infusion O00001302201011010310211212100 100010010100001000001010000011 000000000000000000000010000100 00001000OOOOOOOOOOOOOOOOOOOOOO 110000000OOOOOOOOOOOOOOOOOOOOO 000000000000000000000000000000 0000OOOOOOOOOOOOOOO00000000000 000100000000000010000000000000 0001011220021.41120000011011000 104 Unit 7 Each number represents the number of spikes per successive ten second interval (Bead down columns) Pre-infusicn First Ecst-first- Second infusion infusion . infusion 16 26 23 3 0 14 14 4 3 3 O 1 27 20 o 0 10 9 6 3 2 0 2 26 22 0 0 6 6 7 3 3 0 12 24 20 2 0 12 ll 8 1 1 O 13 22 19 O O 11 9 13 2 0 0 1 14 11 O O 15 10 Z 4 l O 16 23 12 0 1 15 7 O 1 2 22 25 20 l 3 18 6 5 2 3 0 18 25 23 4 9 1 5 O 0 l7 l6 9 2 17 1 ll 5 3 1 O 20 25 21 2 19 19 9 6 2 6 O 20 19 17 2 16 17 10 8 3 4 O 1 17 15 2 16 15 7 10 5 4 O l 20 17 3 l6 15 11 6 2 5 1 19 21 14 O 15 13 8 7 3 2 1 21 25 17 O 11 20 11 6 ll 2 O 20 21 16 O 10 20 9 3 4 3 0 22 19 20 2 11 19 10 3 10 0 0 19 22 17 O 16 20 14 g 11 2 O 25 17 19 1 16 15 6 12 O 2 22 2 11 0 10 19 10 13 15 0 O 21 2 14 1 10 9 7 12 O O 16 20 18 2 9 1 7 11 15 1 O 1 20 17 3 6 l 12 11 12 O O 2 24 12 1 11 22 4 9 11 O 0 19 22 14 0 11 18 9 1 O O 22 20 15 2 7 22 6 4 ll 0 O 25 l6 l5 2 17 16 7 7 8 O O 18 1 1 1 17 10 9 O 0 O 21 2 1 O 10 17 10 11 2 O O Post-water load 105 Post-third infusion Unit 7 (cont'd.) Third infusion Post-second infusion~ 9 21 14 24 10 27 1o 26 11 38 23 22 23 25 22 25 13 20 3 7 22 1o 22 24 23 23 22 26 23 25 25 21 11 17 11 15 16 21 27 31 25 20 25 28 23 27 21 28 29 27 27 27 25 31 23 29 10 27 o00000000ooooooooooooooooooooo 0000000000000OOOOOOOOOOOOOOOOO o 00000000200oooooooooooooooooo 0 00000000000000000000000000000 o 01000000100000000001000000000 0 OOOOOOOOOOOOOOOOOOOOO00000000 o 00000000000000000000000000000 106 Unit 8 Each number represents the number of spikes per successive ten second interval (Bead down Columns) Pro-infusion First Post-first Second infusion infusion infusion 4 2 o 6 34 12 2 10 5 42 10 7 3 o 2 36 15 10 3 7 41 11 5 6 o 1 3o 16 z 2 9 4o 9 5 3 2 4 34 5 3 6 38 13 2 z z 1 31 11 2 Z 6 25 10 3 2 25 11 9 5 31 6 o 2 3 a 22 14 5 9 10 34 7 1 5 2 1 12 3 10 14 26 2 5 3 1o 1 12 5 3 21 21 4 Z 8 4 12 29 9 3 5 9 30 3 3 2 19 13 8 2 11 16 2 6 1 o 10 16 7 6 20 21 6 1 1 3 11 1 10 5 5 23 26 4 1 1 1 12 2 10 3 6 22 25 3 5 1 6 12 23 7 10 11 22 23 3 3 1 o 20 19 11 6 12 24 15 9 a 3 1 21 13 6 5 30 22 6 o 3 4o 16 10 8 7 29 24 6 1 3 3 47 18 8 6 5 27 2“ 5 2 o 2 42 23 6 7 9 27 12 7 1 2 2 1 25 1o 2 1 22 17 9 1 4 3 3 22 4 4 Z 24 23 3 5 3 2 48 24 1 5 24 13 g 3 o 49 21 12 10 12 23 15 3 6 3 45 20 9 10 5 26 2 5 2 3 1 27 11 5 8 5 30 2 5 z 1 2 o 16 5 4 1 29 16 3 2 o 32 15 2 4 6 21 15 9 g 2 2 37 18 7 5 11 39 23 z 6 2 33 17 3 5 10 37 16 107 Unit 8 (cont'd.) Post-second Third Post-third Post-water infusion infusion infusion load 3 1 3 39 9 12 8 23 69 2 2 4 38 22 18 5 66 3 3 2 33 11 19 11 75 54 3 2 5 20 17 23 5 58 65 7 10 3 19 15 21 9 59 61 3 4 11 20 12 4 9 56 63 1 g 18 22 8 7 9 70 56 3 23 22 11 8 8 56 64 7 3 23 24 g 11 15 58 53 5 2 28 24 5 11 70 53 3 3 28 11 5 7 9 59 72 2 2 31 11 8 l6 7 7 62 0 3 34 2 9 6 5 7 44 3 3 33 2 7 8 2 62 79 6 5 3O 15 2 l6 2 32 62 3 6 36 22 2 3 4 57 65 6 7 36 17 13 15 2 53 55 a 5 1 23 7 10 6O 63 5 2 15 3 8 5 50 58 5 7 38 16 5 5 7 55 88 Z 3 37 2 6 7 1 71 74 3 32 2 g 6 10 53 52 6 5 7 11 3 6 59 77 8 1 3 16 7 10 9 57 2 5 3 42 16 6 3 5 70 5 3 5 40 2 4 11 6 63 66 g 3 41 2 3 8 7 50 5O 3 5 16 5 4 1o 54 63 1 6 5 24 8 7 8 59 6o 1 3 37 19 4 10 7 76 58 108 Unit 9 Each number represents the number of spikes per successive ten second interval (Bead down columns) Pro-infusion First Post-first- Second infusion infusion infusion 74 76 94 65 0 2 4 3 O 2 1 111 88 100 19 0 4 2 1 0 0 2 82 97 102 20 0 2 3 1 0 1 2 103 113 97 31 0 2 0 O 0 2 109 73 9 15 0 Z l O 0 0 6 93 81 1 7 0 1 2 0 1 1 63 99 83 2 l 4 2 3 1 0 6 61 72 95 1 0 7 1 o o o 4 22 78 80 0 0 6 2 2 O 0 4 62 78 108 O O 2 2 2 O 0 2 6 68 111 0 0 2 0 O 0 2 8 63 112 0 O 7 2 1 O 1 2 109 32 .86 O 0 9 2 1 0 O O 103 94 3 O 6 l 1 O O 0 69 73 91 0 0 10 2 O 0 0 2 67 99 100 0 0 4 2 1 O 0 2 87 93 105 0 O 6 O 2 O 0 l 89 69 93 0 0 5 2 O l 0 2 86 95 9 1 l 2 1 1 0 O O 96 135 8 2 1 8 1 0 O 0 1 93 127 67 0 1 4 4 O 0 0 l 60 97 99 o o 2 o o o o 2 56 62 99 O 1 2 1 0 0 0 0 55 94 107 O O 4 1 1 O 0 O 96 105 109 0 1 5 O 1 1 0 0 126 113 105 1 O 1 1 1 0 1 O 104 11 100 0 0 2 1 0 1 O 0 9? 1 75 O O 3 4 1 1 O O 93 78 81 1 O 1 2 O 0 0 O 91 101 86 0 O 2 l 1 1 0 1 109 Unit 9 (cont'd.) Post-second Third Post-third Post-water infusion infusion infusion load 38 9 O 1 4 O 0 92 10 20 17 4 1 4 o o 9 17 12 3o 1 O 0 O O 114 28 16 28 O 0 11 O O 113 26 20 31 1 1 8 0 0 119 48 19 23 1 0 17 O O 112 88 11 16 l 0 28 0 0 113 26 7 38 O O 26 0 O 107 97 17 lO 0 0 48 0 0 100 26 15 8 0 l 14 O O 100 50 18 22 0 O 10 O O 99 63 12 22 1 1 2 O O 107 71 15 1 0 O O O 88 28 10 8 O O 10 O O 107 21 11 10 O O l O O 93 24 23 11 O O 10 O O 97 16 12 18 O O 4 O O 86 14 1a 6 o o 7 o o 86 33 1 5 1 0 10 O 0 89 22 l 1 0 O 6 O O 80 30 17 9 0 O 4 O O 47 38 29 g 1 1 16 O O 71 23 14 1 o 24 o 0 63 16 31 17 l O 6 O O 50 28 9 8 4 0 14 O O 26 31 21 7 1 O 16 O O 31 17 23 6 O l 10 0 0 38 30 11 13 1 O 2 0 O 22 13 8 4 O 1 O O 17 29 16 2 O 0 2 O O 38 17 110 Unit 10 Each number represents the number of spikes per successive ten second interval (Bead down columns) Pre-infusion First Post-first- Second infusion infusion infusion 22% 254 217 319 279 186 129 111 114 274 123 3 239 187 339 280 17 120 122 101 265 117 231 206 162 311 273 117 116 130 223 118 225 207 245 292 287 176 128 108 156 211 126 263 220 196 319 272 186 127 125 254 238 109 258 209 168 288 291 184 108 110 58 215 122 229 227 157 316 252 170 129 109 02 212 101 247 217 191 92 287 1 8 147 112 88 206 107 272 191 211 56 262 178 113 115 08 200 115 220 219 180 480 274 154 123 10 432 193 10 242 185 173 550 255 166 129 10 42 200 82 231 173 228 557 280 163 121 144 411 186 111 188 210 267 140 121 132 401 193 102 258 183 209 300 149 133 125 437 201 108 256 l 221 3% 280 158 96 107 425 20 97 186 2 8 214 246 162 132 125 436 19 102 226 2:3 182 439 23% 123 116 132 450 196 109 200 2 180 401 2 1 110 132 422 216 106 230 262 20 388 224121135 124 42 191 105 289 224 17 324 246 1 5 13 114 432 182 111 228 211 17 335 242 146 11 128 419 185 86 234 226 22 321 237 148 120 145 401 216 96 282 216 174 313 235 133 102 131 379 150 96 182 204 203 281 231 139 103 127 368 194 101 191 201 197 305 226 132 115 119 298 192 89 206 190 187 280 224 130 118 283 178 88 258 170 180 299 199 142 101 114 305 153 82 252 185 192 282 207 160 110 131 260 153 77 280 212 184 266 209 168 124 124 290 129 103 259 228 194 241 233 123 123 128 289 140 107 111 Unit 10 (cont'd.) A Post-second Third Post-third Post-water infusion infusion infusion load 102 83 95 234 204 46 47 98 87 97 97 91 242 223 9 43 103 85 105 70 114 208 226 9 47 105 89 103 66 101 233 21 57 50 87 91 98 70 103 205 21 3 2 116 82 103 78 97 227 231 9 5 95 101 g3 84 106 226 2:6 5% 2; 110 100 71 95 237 2 2 102 9 84 93 104 233 241 £6 2 100 8; 84 88 93 239 219 6 5 95 101 83 82 8 236 213 1 O 116 100 90 52 101 255 212 65 32 103 96 72 89 93 227 185 40 33 95 89 85 76 98 267 227 76 39 105 91 67 80 102 258 216 38 58 111 85 84 69 144 238 216 36 39 86 88 67 78 221 238 216 5 1 92 102 75 84 153 255 215 7 2 112 101 66 73 161 235 205 38 39 93 96 74 67 178 2 3 209 5 32 107 90 76 72 181 260 194 7 34 118 92 87 71 170 233 207 44 32 105 81 99 100 219 231 198 44 36 105 105 76 83 213 213 188 46 35 89 98 9o 82 193 246 186 43 38 99 106 86 75 210 228 188 50 36 91 91 86 77 221 237 194 53 32 89 105 64 65 196 251 162 6 28 87 103 96 91 195 221 216 8 37 93 99 86 60 226 223 195 45 46 103 103 93 13177 5368 I|| H a“ LI)| " T” H H “ A|I| III] 312 lIHIHWIU