Neurophysiological determination of lateral hypothalamic and lateral preoptic interconnections

Brain Research Bz&tin, Vol. 5, pp. 315-323. Printed in the U.S.A.

Neurophysiological Determination of Lateral Hypothalamic and Lateral Preoptic Interconnectionslyz~~ F. C. BARONE, Brain Research Laboratory,

M. J. WAYNER, Syracuse

W. I% TSAI AND F. E. BARASH

University, 601 University Avenue,

Received

15 November

Syracuse,

NY 13210

1979

BARONE, F. C., M. J. WAYNER, W. H. TSAI AND F. E. BARASH. Neurophysiological determination of lateral h~l~~thaia~~c and lateral preoptic interconnections. BRAIN RES. BULL. 5(3) 315323, 1980.-The effects of lateral hypothalamic (LH) stimulation on ipsilateral laterat preoptic area (LPA) neuronal activity were determined in anesthetized rats. The effects of LPA stimulation on LH neuronal activity were also determined. Recordings from 99 hypothalamic neurons indicate that reciprocal inhibitory relations exists between the LPA and LH. Following single rectangular pulse, 0.5 msec, O-500 @A, stimulation short latency decreases in activity occurred. Longer latency increases in activity were also observed. Dose response relations were established for 90% of the LPA neurons following LH stimulation and for 80?&of the LH neurons following LPA stimulation. Decreases and in a few cases increases in activity seemed to involve only one or two synapses. Antidromic responses revealed relatively slow conduction velocities of 0.4-0.9 mfsec. Results demonstrate a considerable degree of interconnectivity between the LPA and LH along the extent of the medial forebrain bundle. In addition to establishing the nature of these interconnections, the ~rossv~idation between the horseradish peroxidase neuroanatomicai technique and electrophysiological methods was discussed as a means of determining hypothalamic organization and function. Lateral hypothalamic area Hypothalamic interconnections

Lateral preoptic area

Medial forebrain bundie

NEUROPHYSIOLOGICAL and neuroanatomical relations of the lateral h~oth~~us (LH) and lateral preoptic area (LPA) in the control of spinal motor reflex excitability and behavior have been discussed previously [26,27]. The integrity of this part of the hypothalamus is necessary for normal ingestive behavior [ 1,6,9]. Neurons of the LH and LPA are sensitive to osmotic [2, 5, 21, 291 and visceral [2,3] stimulation associated with drinking Major portions of the LPA and LH form the bed nucleus of the medial forebrain bundle. Both ascending and descending MFB fibers terminate on these hypothalamic neurons which are characterized by extensive arborization 111, 12, 16, 181. A recent study utilizing the retrograde transport of horseradish peroxidase has elucidated some of the connections between the LPA and LH [28]. Although the effects of posterior hypothalamic and mesencephic MFB stimulation on hypothalamic neurons have been studied 113, 14, 20, 22, 231, little if any data are available on the effects of reciprocal LH and LPA stimulation. The present study was conducted to stimulate electrically LPA and LH neurons in order to determine the nature of the interconnections and to correlate these results with the data obtained in the previous study which utilized the retrograde transport of horseradish peroxidase [28]. Many of the

Single unit activity

interconnections are inhibitory and relatively facilitatory effects were observed. EXPERIMENT

few direct

1

This experiment was carried out to determine the effects of single rectangular pulse electrical stimulation of the LH on LPA neuronal activity. Results indicate that most LPA neurons are initially decreased by LH stimulation. Many neurons were also increased but usually with a sign~cantly longer latency. The decreases usually involve only one or two synapses. METHOD

Animals Male Long Evans rats weighing between 450 and 550 g were selected from our colony. Animals were housed in individual cages and were allowed to eat standard Purina Lab Chow and drink water ad libitum. Animals were kept on a constant Light-dark cycle. The 12 hr light phase began at 0600 and was followed by a 12 hr dark phase. Room temperature was maintained at 70?2”F.

‘Supported by a grant from the NINCDS USPHS No. 13543. 2Reprint requests to Dr. M. J. Wayner, Brain Research Laboratory, 601 University Avenue, Syracuse, NY 13210. “Some of these data were presented at the 30th Annual Meeting of the American Physiological Society, New Orleans, LA, October 15-19, 1979 and at the 9th Annual Meeting of the Society for Neuroscience, Atlanta, GA, November 2-6, 1979.

Copyright o 1980 ANKHO International

Inc .-0361-9230/80/030315-09$01.40/O

316

Rats were anesthetized with 1.4 g/kg body weight urethane. Rectal temperature was continuously monitored by a thermister and maintained at 37.0-~0.5”C by means of an electrically heated blanket. Animals were fixed in a stereotaxic inst~ment with the skuil horizontal, lambda and bregma in the same horizontal plane. A longitudinal incision was made in the scalp and the skull was exposed. A portion of the caivarium 4x6 mm, was removed on the left side over the hypothalamus according to a rat brain atlas [lS’& The meninges were removed and the exposed brain tissue was covered with parafFim oil. Recording electrode placements in the LPA-MFB were at the following coordinates. in mm, with the anterior coordinate relative to bregman, the later coordinate to the sag&al suture, and the ventral position measured from the surface of the brain: A= + 1.0 to 1.5. L- 1-3.0 to 3.2. Vs7.5 to 9.0. The recording electrode was driven into the brain at an 80” angle. A concentric bipolar stimulating electrode (Rhodes Medical Instruments, inc.) was placed in the LH-MFB at the following coordinates: A=4.5, L= + 1.0, V-9.8. The stimulating electrode was driven into the brain at a 70” angle. Micr~iectr~es were driven by a ~cromanipuiator to the predete~ined neural sites. Ext~a~iiul~ action potentials from spontaneously active ceils were recorded through one barrel glass capillary electrodes (Frederick Haer Co.) which had been pulled and broken to a tip diameter of 4-6 pm and filled with 2 M NaCi having a DC resistance of i-4 MfZ. Action potentials were amplified by a high input impedance ~re~pIi~er and audio amplifier and displayed on a conventional Tektronix oscilloscope. Potentials utilized for study exhibited a bipolar waveform. were usually observed over a 40-60 pm distance, and were identified as soma potentials according to previously established criteria [4,24j. A Grass SD 9 stimulator was utilized to eiectricaiiy stimuiate the brain. Single rectangular pulses, 0.5 msec in duration and usually from 0 to 500 PA in amplitude, were delivered to the LH-MFB at a rate of 0.5 Hz. The prepuise output of the stimulator triggered the horizontal beam of a Tektronix S103N storage oscilloscope. A Tektronix C-SB poiaroid camera was used to photo~ph the oscilloscope trace. Different current intensities were applied to the LH or MFB in order to determine dose response relations. Stimulation tests were always repeated in order to establish the reliability of a given effect. The number of sweeps utilized for a given trace was determined by the ongoing basal discharge frequency of the neuron under study. The amount of time monitory post stimulation was determined by the type, latency, and duration of effect exhibited by the ceil in response to the stimuli. Usually IO, 50 or 100 msec of post stimulation activity was stored as repetitive sweeps which represented a given current intensity of electrical stimulation. Photographs were analyzed and effects were considered signif=ant if the number of action potentials observed for a given post stimulation period was increased or decreased by 5O?Gcompared to baseline periods during which no stimulation was applied but the same number of sweeps were triggered. A threshold was determined for each affected neuron by averaging the current in PA which resulted in a si~i~~t effect (effective stimulation) with the next lowest current intensity tested which did not result in a significant effect. Latency was also determined for each affected neuron. Latency was the amount of time in msec from the offset of the effective stimulus pulse to the occurrence of a significant elect. Fi-

BAHONE

E’i ,.I!.

naliy the duration of subsequent effects were determined. Duration was the amount of time in msec which elapsed from the beginning of a significant effect to when the activity was no longer different from baseline. When antidromic stimulation was suspected, neurons were then tested for constant latency to repetitive single pulse stimulation, unsent latency and response to high frequency stimulation (>500 Hz), and with double pulses delivered with short interpulse intervals I -1I .5 msec) to eliminate the second antidromic response due to the refractory period of the neuron [7,10]. Latencies were determined for antidramic effects and conduction velocities were estimated on the basis of the distance of the s~mulatjng electrode tip from the recording site. Recording sites were marked as follows. The recording electrode was maintained at the site from which data was collected. It was then emptied of saline, refilled with concentrated HCL, AND 3-15 PA of positive DC current was passed through the electrode tip for .S-15 min. This procedure resulted in the ejection of protons from the electrode tip and the formation of a small lesion [ 171. In addition. the glass capillary electrode was retained in place while the animals were perfused intracardiaily with normal saline foilowed by lQ% formal-saline. In this way the electrode tract was made more visible and the ~dent~cation of the small lesion which was approximately 200 pm in diameter was easier. Stimulation sites were marked by passing 15-40 PA of DC current between the two poles for 5-10 sec. Brains were removed and stored in 10% formal-saline for at least 24 hr prior to sectioning. Frozen serial sections, 50 pm, were made through the sites of the r~)~ing and stimniating iesions. The unstained sections were examined under a dissecting microscope and compared to places in the Kijnig and Klippei rat brain atlas 1151 to verify electrode tip locations. Sections were then stained with cresyi violet for permanent histoiogical records. Since only one HCi lesion was made along any one recording electrode tract, the locations of the other recording sites were estimated. RESUI.l”S

Data were coUected from 49 cells recorded in 16 animals. ~u~ent-~s~nse relations were established for all LPAMFB ceils which were affected by LH-MFB stimulation. Neural responses following single pulse stimulation consisted of increases in number of discharges, decreases, combinations of increases and decreases, or no effect. In all cases, increasi~ the stimulus current enhanced the effect observed. Table t is a summ~ of the effects in~~di~ threshold in PA, latency in msec, and duration in msec. For each of these measures the mcans?standard emor (SE), medians, and modes are listed respectively from top to bottom. In all cases the distributions are positively skewed and in some cases bi- and turnip. Thirty one netirons ~niti~y decreased (63%) at an average ~resho~d of 88 pA, with a mean latency and duration of 3 and 24 msec. Twelve ceils initially increased (24%) at a higher average threshold of 122 @A with a longer mean latency of 8 msec and a shorter durarion of 12 msec. In many cases decreases occurred with a latency too short to measure and were assigne$ values of one msec . For some ceils, dual effects following stirnn~~on were observed. In 10 cells (2%) the initial decrease which OCcurred with an average latency of 2 msec and endured for 17 msec was followed by an increase in the number of discharges. This increase had an average latency of 31 msec and

317

LH AND LPA INTERCONNECTIONS TABLE

1

EFFECTS OF LH STIMULATION ON LPA NEURON ACTIVITY

Effect

Number Of Cells

Threshold* (PA)

Latency* (msec)

Duration* (msec)

Cells Initially Decreasedi

31

88215 50 30

3-ti 1 1

2423 20 15

Cells Initially increased

12

122r32 78 50 and 290

S-+2 6 5

1224 5 4,s and 20

115r34 65 30, 50 and 80

251

17t2 14 13 and 25

128t35 80 30, 50 and 80

31”7 21 17 and 20

1324 9 5

Cells Initially Decreased 10 Followed By An Increase

412

158t 133 Cells Initially Increased 2 38k 12

Followed By A Decrease

Cells Not

1327

18t3

5

Affected *For each type of effect the Mean 2 SE, Median, and Mode is listed from top to bottom. tone cell decreased by LH stimulation also exhibited an initial antidromic response. One other cell, not listed here, was also affected antidromicahy. Based on latencies of 6 and 3 msec for these antidromic effects, conduction velocities were determined to be 0.4 and 0.9 mkec.

endured for 13 m&c. Only 2 cells (4%) exhibited initial increases followed by decreases in activity. The initial increase occurred with a meau latency of 2 msec and endured for 4 msec. The decrease occurred with a mean latency of 13 msec

and endured for 18 msec on the average. Again, in these two neurons, the decrease occurred at a significantly lower mean current of 38 @A when compared to the 158 PA required for increases in the same neurons. Five LPA cells (10%) were not affected by LH stimulation. One LPA cell which was decreased by LH stimulation also exhibited an initial antidromic response. One other cell, not listed in Table 1, was also antidromically activated. Based on latencies of 6 and 3 msec for these antidromic responses, conduction velocities were estimated to be 0.4 and 0.9 m/set. Figure 1 illustrates some typical responses of LPA neurons to single pulse stimulation of the LH. The solid bar at the lower left of each photo indicates 20 msec of time. Part A of Fig. 1 illustrates an LPA neuron which increased following stimulation of the LH. The photo is a composite of 10 sweeps prior to and after LH stimulation which is indicated by the artifact at the center of the photo. The top trace illustrates the response to no current or baseline. The middle

trace illustrates an increase in the number of action potentials following 180 PA of stimulating current. The bottom trace is the response to 500 PA. As current was increased the number of discharges increased and latency and variability decreased. In Part B, a decrease in an LPA neuron following LH stimulation is illustrated. Each trace is composed of 40 sweeps immediately following stimulation of the LH. The top trace depicts the response to 50 PA of stimulating current. The middle trace illustrates the response to 200 PA. The bottom trace is the response to 360 PA. As stimulation current was increased the latency decreased and the duration increased. Parts C and D depict decreases followed by increases in two different LPA neurons. In Part C, each trace is composed of 20 sweeps immediately following LH stimulation. The traces from top to bottom depict the responses to the following LH stimulating current: no current or baseline, 20 yA, 50 PA, 200 @A, and 500 PA. In Part D, each trace is composed of 10 sweeps immediately following LH stimulation. The top trace depicts the response to 200 PA of stimulating current. The middle illustrates the response to 180 PA. The bottom is the response to 280 PA. For both neurons depicted in Parts C and D increasing stimulation

C

FIG. I. Response of I-PA neurons to single pulse stimulation of the LH. PART A: LPA neuron initially excited. 10 ~weepsltrace. Stimulation indicated by artifact at center of photo. Top=baseline. Middle= I80 ,uA. Bottorn=SOO~A. PART B: Initia) decrease. 40 sweeps/trace. Each sweep immediately follows stimulation. Top=50 &A. ~ddIe~2~ @A. Bottom=360 @A. PART C: Initial decrease followed by increase. 20 ~weep~t~ce. Top=baseline. Second from top=ZO&A. M~die~SO~A. Second from ~~orn~~~A~ Botforn~~~~A. PARTD: Same effect as in 12 for different neuron. 10 sweeps/trace. Top=20 &A. Middle= 180 &A. Bottom=%0 PA. Solid bar at lower left of each phato indicates 20 msec. Part A retouched to improve contrast

current decreased the latency and increased the duration of the initial decrease and increased the amount and decreased the variability of the later increase in activity. Fi8ure 2 illustrates the results of histological examination of LH stimulation and LPA recording sites on numbered serial sections adapted from the Konig and KIippel rat brain atlas 1151. The top row of sections indicates LPA neuron locations from which data were collected. Neurons are depicted as follows: Initial increases by solid triangles, antidromic activation by open triangles, decreases by solid circles. and no effects by solid squares. The bottom row of sections indicates the locations of LH stimulation sites by solid circles. These results indicate that stimula~on of the LH results in significant changes in activity of many LPA neurons. Initial decrease in activity occur with a short latency of 3 msec and with a threshold of 88 @A. Of these 31 neurons, 32% with an average latency of 2 msec and threshold of I 15&A displayed a later increase with a latency of 21 msec but required a higher threshold of 128 &A. Of the 49 neurons tested, 24% displayed an initial increase with a latency of 8 msec and an average threshold of 122 PA. Of these 12 neurons, 2 or 17% displayed a later decrease. The initial increase in these 2 cells occurred with an average latency of 2 msec and threshold of

158 PA. The decrease in these 2 cells occurred with an average latency of 13 m.sec and a much lower thresha~ of 38 FA. Thresholds for producing decreases in activity were less than thase thresholds for producing increases. Usually decreased activity was associated with a much shorter latency than increased activity. Also, the duration of increases was always shorter than the duration of the decreases. Of the 49 LPA cells tested. only 5 or 10% were not affected by LH stimulation. EXPER1MENT

2

This experiment was carried out to determine the effects of shrgle rectanguhu pulse electrical sultan of the LPA on LH neuronal activity. Results are similar td Experiment 1 and indicate that predominately inhibitory interactions occur between the LPA and LH.

The animals and procedure were exactly the same as in Experiment 1 except for the placements of the stimulating and recording electrodes. Record@ electrode placements were in the LH-MFB at the following coordinates, in mm,

319

LH AND LPA INTERCONNECTIONS

FIG. 2. Results of histoloaical verification of LH Klilppel rat brain atlas [cl. Top row indicates \ , .. . . . ~ . , ..* decrease (sona cn-cles), ana no (sona squares)

stimulation and LPA recording sites illustrated on serial sections adapted from the Kiinig and LPA neuron locations where-initial increase (solid triangles), antidromic (open triangles), .-a-_ ~I~_ 1 ~.._. ~~~~~ .~>!..l__ .L- I___.. . . . .f,,T _I.~..,..f _._~_I~ ,.I enecrs were onservea. ~~otrorn row molcaux me locauons 01 em srnnurauon snes (sona

circles).

with the anterior coordinate relative to bregma, the lateral coordinate relative to the saggital suture, and the ventral position measured from the surface of the brain: A=O.O to -2.0, L=+2.8 to 2.9, V=7.5 to 9.5. The recording electrode was positioned in the brain at an 80” angle. The stimulating electrode was placed in the LPA-MFB at the following coordinates: A= -2.2, L= + 1S, V=7.8. The stimulating electrode was positioned in the brain at a 78” angle. RESULTS

Current response relations were established for all LH-MFB cells which were affected by LPA-MFB stimulation. Similar effects to those observed in Experiment 1 were observed following single pulse stimulation. In all cases, increasing the stimulating current enhanced the effect observed. Table 2 is a summary of the effects including threshold in PA, latency in msec, and duration in msec. For each of these measures the means?standard error (SE), medians, and modes are listed respectively from top to bottom. In many cases the distributions are positively skewed. Twenty three neurons (or 46%) were initially decreased at an average of 168 PA of current, with a mean latency and duration of 2 and 24 msec. Data were collected

from 50 cells in 16 animals.

Fifteen cells (or 30 %) were initially increased at a higher average current of 292 PA with a longer mean latency of 12 msec and a shorter duration of 17 msec. As observed in Experiment 1, in many cases decreases were observed immediately after stimulation and a latency of one was assigned. Only in a few cases were dual effects observed. In 2 cells (or 4%) the initial decrease occurred with a mean latency of 2 msec and lasted an average of 6 msec. This was followed by an increase in the number of discharges which occurred at a mean latency of 16 msec and had an average duration of 4 msec. In these two neurons the decrease occurred at a signi~can~y lower threshold current of 30 PA when compared to the 120 @A required for the increase of the same neurons. Only one cell (or 2%) exhibited an initial increase followed by a decrease in activity. The initial increase occurred at a latency of 3 msec and endured for only 4 msec. The decrease occurred at a latency of 7 msec and endured for 20 msec. Both effects occurred at the same threshold current of 290 yA. Ten LH cells (or 20%) were not affected by LH stimulation. Two other cells, not listed here, were affected antidromically. Based on latencies of 6.5 and 3.5 msec for these antidromic effects, conduction velocities were estimated to be 0.4 and 0.8 misec.

320

BAKONE t,‘l’Af ‘TABLE 2 EFFECTS

OF LPA ~~~LAT~~N

Effect

Number Of Cells

ON L>H NEURON

Threshold* +A)

Latency* (msec)

Duration* tmsec) 24-3 22 30

CkllS

Initially Decreased

Cells Initially Increasedt

ACTlVfTY

I5

292~64 290 290

17-6 x Saud 10

6-I Cells Initially Decreased Followed By An Increase

12o-tro

290 Cells Initially increased

4 _.

290

Followed By A Decrease

Cells Not Affected

4:. I

10

20

.

*For each type of effect the Mean r SE, Median, and Mode is listed from top to bottom. tTwo other cells, not Iisted here, were affected an~ic~ly. Based on latencies of 6.5 and 3.5 msec for these antidromic effects. conduction yelocities were determined to be 0.4 and 0.8 m/set.

Figure 3 illustrates some typical responses of LH neurons to single pulse stimulation of the LPA. The solid bar at the lower left of each photo indicates 10 msec. Parts A and B of Fig. 3 illustrates an LH neuron which increased foLiowing stimulation of the LPA area, a composite of 25 sweeps immediately following LPA stimulation. The top trace in Part A illustrates the response to no current or baseline. The bottom trace ilhrstrates the response to 180 fl, The top trace in Part B illustrates an increase in the number of action potentials followin 280 JLA of stimulating current. The bottom trace is the response to 500 PA. Increasing the stimulation current decreased the latency and variability of the increase. In Part C, a decrease in an LH neuron following LPA stimulation is illustrated. Each trace is composed of 15 sweeps immediately following stimulation of the LPA. The top trace depicts the response to no current or baseline. The middle trace ihustrates the response to 180 PA. The bottom trace is the response to 500 &A. A similar decrease for a different LH neuron is depicted in Part D. Each trace is compsed of 10 sweeps ~medi~ely following LPA stimulation. The top trace is the response to 50 JLA of si@e pulse suction. The middle and bottom traces are the responses to 180 and 280 &A respectively. For both neurons depicted in Parts C

and D increasing stimulation current decreases the latency and increases the duration of the &crease in activity. Part E illustrates a short latency synaptic activation of an LH

LH neuron. Each trace consizcof 5 sweeps and illustktes the cell response to pulse pairs tsf equal current. The top by 3 msec trace is the response to twin pukes se delay or at 333 Hz. An ~tidro~ both pulses in each sweep with no variability in latency. The middle trace is the response to t by 2 inah msec or at 5OQHz. An rtntidromis 8 not sweeps but one where the respons observed. The bottom trace is the response to twin pukes separated by 1.5 msec or at 667 Hz. No response is ob@rved to the second pulse for any sweep due to the rektory period of the neuron. examination Fire 4 illustrates the results ofhistd of LPA stimulation and LH recording sites illustrated on

LH AND LPA INTERCONNECTIONS

321

FIG. 3. Response of LH neurons to single pulse stimulation of the LPA. PART A: LH neuron initially excited. 25 sweeps/trace. Each sweep immediately follows stimulation. Top=baseline. Bottom= 180 PA. PART B: Same cell as in A. 25 sweeps/trace. Top=280 WA. Bottom=500 PA. PART C: Initial decrease 1.5sweeps/trace. Top=baseline. Middle= 180 PA. Bottom=500 PA. PART D: Same effect as in C for different neuron. 10 sweeps/trace. Top=50 PA. Middle= 180 PA. Bottom=280 PA. PART E: Short latency increase. 5 sweeps/trace. Top= 180 PA. Middle=290 PA. Bottom=500 PA. PART F: Antidromic response. Each trace consists of 5 sweeps and illustrates the cell response to 0.5 msec pulse pairs of equal current. Top=3 msec delay between pulses or 333 Hz. Middle=2 msec delay or 500 Hz. Bottom= 1.5 msec delay or 667 Hz. Solid bar at lower left of each photo indicates 10 msec.

322

BARONE El- .4I.

FIG. 4. Results of histological verification of LPA stimulation and LH recording sites illustrated on serial sections adapted from the Kijnig and Klippel rat brain atlas [IS]. Top row indicates LH neuron locations where initial increase (soiid. trian@e$, anti&or& (open triangles), decrease (solid circles), and no (solid squares) effects were observed. Bottom row indicates the locations of LPA stimulation sites (solid circles).

numbered serial sections adapted from the Kiinig and Klippel rat brain atlas [15]. The top row of sections indicates LH neuron locations from which data was collected. Neurons are depicted as follows: Initial increases by solid triangles, antidromic activation by open triangles, decreases by solid circles, and no effect by solid squares. The bottom row of sections indicates the locations of LPA stimulation sites by solid circles. These results are similar to those in Experiment 1. Stimulation of the LPA results in significant changes in activity of most LH neurons. Initial decreases in activity occurred in 63% of the neurons studied with a short latency of 2 msec and with a threshold of 168 PA. Of these 23 neurons, 9% with an average latency of 2 msec and threshold of 30 @A displayed a later increase with a latency of 16 msec but required a h&er threshold of 120 PA, Of the 50 neurons tested, 3@% displayed an initial increase with a latency of 12 msec and an average threshold of 191 PA. Of these 15 neurons, 1 or 7% displayed a later decrease. The results are similar to those in Experiment 1. Thresholds for producing decreases in activ-

ity were less than those for producing increases. Usually, decreased activity was associated with a much shorter latency thiul increased activity. Als& the da&on Of the increases are usually shorter than the duration of the decreases. Of the 50 LH cells tested, only 10 or 20% were not affected by LH stimulation. GENERAL

DISCUSSION

Results of Experiments 1 and 2 demonstrate reciprocal relations between LH and LPA neuronal activity. Similar decreases followed by increases occur in both regions. These results are similar to related data collected from the hypothalamus of awake animals [14,20]. Unlike piev&~us work [l/20], effects were unrelated to the proximity of stimulating and recording electrodes as illustrated in Pig. 2 and 4. The decreases in neuronal activity associated -with relatively short late&es are probably caused by direct inhibitory connections or involving no more than two synapses.

Increases

in neuronal

activity

are more d&CUlt to

323

LH AND LPA INTERCONNECTIONS interprete. The occassional increases which occurred with short latencies were probably the result of the stimulation of direct excitatory connections. However, such effects were observed infrequently. The increases which followed periods of decreased activity associated with long latencies apparently result from the excitation of extrahypothalamic structures which in turn are excitatory. The direct inhibitory effects are also associated with lower stimulating thresholds and always endure for a longer period. Inhibitory interactions between the LPA and LH were suggested earlier [25]. Intrahypothalamic inhibition has been attributed to short fiber and recurrent collateral interconnections [ 18,191. The excitation which typically follows neuronal decreases could easily be extrahypothalamic and also mediated by the ascending and descending fibers of the MFB [ 13,141. The considerable presynaptic convergence which occurs within the LH and involves interactions with limbic forebrain and midbrain structures substantiates such an explanation [ 19, 26, 271. Observed conduction velocities confirm those previously reported for the hypothalamus [8,22]. The slow conduction

velocities might be attributed to the small diameter unmyelinated axons of the neuropil. The direct connections between the LPA and LH are confirmed by the results obtained with retrograde transport of horseradish peroxidase [28]. A large number of cells in either the LPA or the LH appear to project either to the LH or LPA. There was no obvious difference in the number of cells labelled in the LPA and LH. However, it is interesting to note that direct effects when stimulating the LH had in general a lower threshold than in the LPA. There were always more spontaneously active neurons in the LPA as compared to the LH under these experimental conditions. The low cell density of spontaneously active and glutamate excited (unpublished results) LH neurons indicates that electrical stimulation of the LH probably resulted in the activation of some noradrenergic ascending fibers which terminate in the LPA. Therefore, the present data demonstrate not only the nature of LPA and LH interconnections but also establish the crossvalidity of the HRP anatomical technique and neurophysiological methods in the determination of hypothalamic organization and function.

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