Influences of the limbic system on hypothalamo-neurohypophysial system

Brain Research, 264 (1983) 31-45 Elsevier Biomedical Press

31

Influences of the Limbic System on Hypothalamo-Neurohypophysial System HORACIO FERREYRA*, HIROSHI KANNAN** and KIYOMI KOIZUMI*** Department of Physiology, State University of New York, Downstate Medical Center, Brooklyn, N Y 11203 (U.S.A.)

(Accepted August 31st, 1981)

(1) Effects of stimulations of various limbic structures (the olfactory bulb, olfactory tubercle, prepyriform cortex, endopyriform nucleus and various parts of amygdaloid nuclei) on the neurosecretory neurons in the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus were studied. All regions stimulated received strong inputs from the olfactory bulb. (2) Out of 195 'identified' neurosecretory neurons tested one-half or more (49-74 ~ , depending on the structures stimulated) were inhibited by stimuli consisting of 1-3 short pulses. The inhibition occurred immediately after the stimulus in approximately onefifth of all inhibited neurons, in the remaining four-fifths inhibition occurred after more than 20 ms latency. Inhibition of neurosecretory neuron activity lasted for several hundred milliseconds, often followed by clear post-inhibitory excitation or rebound. (3) In 23 neurons, a distinct 'evoked' response of brief duration occurred with a 30 ms latency following stimulation of the lateral and medial amygdala, olfactory tubercle and prepyriform cortex. In another 17 neurons, a general increase in background activity with a longer latency (50-100 ms) occurred following stimulation of nearly all amygdaloid nuclei, olfactory tubercle and the pyriform cortex: lateral amygdala stimulation caused an excitation of the largest proportion of neurosecretory cells (30~) while none was excited by stimulation of the olfactory bulb and endopyriform cortex, except those occurring as post-inhibitory excitation. (4) There was a convergence of afferent impulses on single neurosecretory cells. A large proportion (42 ~o) of the neurons received inputs from 2 to 4 limbic regions. (5) Neurosecretory cells which were influenced by limbic stimuli were also inhibited by baroreceptor activation and excited by osmotic stimulation. 'Unidentified' neurons within SON and PVN and 'atypical neurosecretory cells' (those responding to pituitary stalk stimulation with varying latencies) were also affected by the forebrain stimulation; some of these were also affected by an osmotic stimulus. A part of this group may send their axons to the median eminence. INTRODUCTION T h e o l f a c t o r y system is k n o w n to influence a n d p a r t i c i p a t e in r e g u l a t i o n o f e n d o c r i n e functions in m a m m a l s a n d thus involved in b o t h sexual 3z a n d m a t e r n a l I behavior. D i r e c t evidence in s u p p o r t o f a role p l a y e d b y o l f a c t o r y structures is n e u r o e n d o crine c o n t r o l o f the a d e n o h y p o p h y s i s has been p r o v i d e d recently; release o f g o n a d o t r o p i c horm o n e s f r o m the a d e n o h y p o p h y s i s can be m o d i f i e d following electrochemical s t i m u l a t i o n o f the m a i n a n d accessory o l f a c t o r y b u l b s 4.

Less well k n o w n are possible ties between rostral olfactory regions o f the b a s a l f o r e b r a i n a n d the neur o h y p o p h y s i s . In goldfish, o s m o r e c e p t o r s are located in the o l f a c t o r y b u l b a n d n e u r o n s o f p r e o p t i c nuclei are affected directly b y o s m o t i c s t i m u l a t i o n o f the olfactory b u l b 2z. I n m a m m a l s , a l t h o u g h some evidence in f a v o r o f such a relationship has been p r o v i d e d in the p a s t (see ref. 19), no systemic study has yet been a t t e m p t e d with p h y s i o l o g i c a l techniques, n o r have possible f u n c t i o n a l connections between olfactory limbic regions a n d h y p o t h a l a m i c m a g n o c e l l u l a r n e u r o n s been investigated. This is

* Present address: Instituto de Investigacion Medica Mercedes Y Martin Ferreyra, Cordoba, Argentina. ** Present address: Department of Physiology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu City, Japan. *** To whom reprint requests should be addressed. 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

32 somewhat surprising, considering that: (a) posterior lobe hormones are known to be released, in addition to changes in osmotic pressure and volume of the blood, by stressful neural stimuli which most probably act via limbic system structures; and (b) more than 50 % of supraoptic nucleus afferents arise in rhinencephalic brain regions as demonstrated by electron microscopic studies 56. The present experiments were therefore designed to provide some answers to the problem of functional connections between rostral forebrain regions and identified neurosecretory cells in the supraoptic and paraventricular nuclei of the hypothalamus in cats. The following limbic regions were explored; the olfactory bulb, prepyriform cortex, endopyriform nucleus, olfactory tubercle and several amygdaloid nuclei. A short account of this work has appeared previously2L

the following regions; prepyriform cortex, endopyriform nucleus, olfactory tubercle, lateral, medial, central, cortical and basal amygdaloid nuclei (all on the right side). These were selected according to the particular experiment. The exact stimulating points within the forebrain structures were determined by finding the site where the largest evoked potential was obtained in response to a single pulse stimulus applied to the olfactory bulb (see Fig. 7D). Using the same electrodes these particular sites were then stimulated to examine their effects on the hypothalamic neurons. For this purpose 1-3 stimulating pulses at an intensity of 0.05-0.2 mA and 0.5 ms duration were given at 200 Hz. The stronger stimulus was used only for testing effects of increasing stimulus intensities or for certain olfactory bulb stimulations. In the latter instance, the stimulating electrode was placed on the surface of the bulb.

MATERIALS AND METHODS

Recording from neurosecretory neurons

Preparation Twenty-seven cats of either sex weighing 2.5-4.0 kg were anesthetized with pentobarbital sodium (Nembutal, 35 mg/kg given intraperitoneally). Supplemental anesthetic, Nembutal (2 mg/kg) or thiopental sodium (pentothal, also 2 mg/kg) was given intravenously when necessary. After tracheotomy, the femoral artery and vein were cannulated for blood pressure recording and fluid and drug administration. The rectal temperature was maintained at 37-38 °C. A hemispherectomy was performed and the right hypothalamic area was exposed23,27,2s, 52. The animal's head was then placed on a stereotaxic holder and a few small holes were made on the right skull for placement of stimulating electrodes. The right olfactory bulb was also exposed. Blood pressure (of the femoral artery) and heart rate (measured by a tachometer triggered by the blood pressure pulse) were displayed on a Grass Polygraph throughout the experiment.

Stimulation of forebrain A bipolar stainless steel electrode was placed visually on the olfactory bulb. Concentric stainless steel electrodes (tip diameter 100 #M, outside diameter 250/,M) were placed stereotaxically on 3 of

Detailed methods of recording from neurosecre. tory neurons have been described elsewhere23,27, 28, 52. In short, a bipolar metal electrode was placed on the pituitary stalk for stimulation. An Insl-x-coated tungsten microelectrode was inserted into supraoptic (SON) and paraventricular (PVN) nuclei through exposed hypothalamic surface2L Neurosecretory neurons were identified by antidromic action potentials recorded from these neurons following the pituitary stalk stimulation (single pulse, 0.5-2.0 mA, 0.5 ms) and by a collision test. Extracellularly recorded action potentials of single neurons were amplified and displayed on oscilloscopes and processed through a window discriminator. A counter displayed the pulse rate on the same polygraph on which the blood pressure and the heart rate were monitored. Post stimulus time histograms were constructed from 32-256 responses using Nicolet Series 1700 computer. Records were also stored on magnetic tape.

Histological identification of stimulating and recording sites At the end of each experiment all stimulation sites were marked by passing DC current to deposit iron. The brain was fixed in 10% formalin with 3 % ferroand ferricyanide solution; frozen sections, 40 #M thick, were stained with cresyl violet. Enlarged

33 drawings were made from each slide to identify stimulating positions. RESULTS

(a) Cell types and l~ring properties One-hundred and ninety-five hypothalamic neurons were recorded from supraoptic (SON) and paraventricular nuclei (PVN) and categorized as magnocellular neurosecretory cells by criteria described elsewhereZ3,27,2s,52. Fig. 1 shows an example of a collision test and high frequency stimulation

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cellular recordings from a single neuron in SON. Five superimposed tracings in all cases. Oscilloscope sweeps were triggered by spontaneously occurring orthodromic action potentials and the pituitary stalk was stimulated after certain intervals (indicated by an arrow). An antidromically conducted action potential was evoked in A, but not in B when the stalk stimulation was given earlier (interval between spike and the stimulus, 16 ms). In this case the antidromically conducted impulse in the axon collided with that originating from the neuron. This fact indicates that this SON neuron sends its axon to the stalk. C: three stimulating pulses at 100 Hz were applied to the pituitary stalk. The cell responded to the first two stimuli with full-size spikes and to the third with IS (initial-segment) spike only.

test used for identification of the neurosecretory cells. The terms 'neurosecretory neurons' or 'SON, PVN cells' are used here only when they were identified by the criteria. Accurate recording depth from the third ventricle surface could be established in 108 out of 195 neurosecretory neurons, since recording electrodes penetrated the hypothalamus diagonally from the exposed third ventricle surface. The histological locations of electrode tips, however, could not be exactly determined for all neurons. Thus, neurons situated between 0 and 2000 /~M from the third ventricle surface were tentatively designated as PVN cells (n = 7), and beyond 2000/~M as SON cells (n 101). Under the present experimental conditions, the majority of neurosecretory neurons in the SON and PVN showed a random or irregular pattern of discharge, as previously reported 53. Firing frequencies ranged generally between less than 1 to 12 per second. Another 105 neurons unresponsive to pituitary stalk stimulation but situated in or near the morphological boundaries of SON and PVN were also studied. These were classified as 'unidentified' neurons for convenience. Fifteen of these cells (11 ~ ) were found to lie within less than 100 # M from 'antidromically identified' neurons. In 25 neurons, action potentials evoked by the pituitary stalk stimulations showed latency variability (ranging between 2 and 7 ms) and no collision with spontaneous discharges was observed. Ten of these cells (40 ~ ) were recorded within 100 # M or less from an 'identified' neurosecretory cell. These neurons may be orthodromically26, 27 or antidromically activated through branched axons 3, though in the latter situation a collision test would be positive. For convenience, these were called 'atypical neurosecretory cells'. Locations of the above-mentioned two groups of neurons in relation to neurosecretory neurons were found to be different; the contingency test showed that 'atypical neurosecretory cells' were situated significantly closer to neurosecretory neurons than 'unidentified' neurons. As SON and PVN neurons firing with 'bursting patterns' were few in cats 53, analysis o f responses were not made on ceils showing the clear 'bursting pattern', because in such cases effects of single pulse

34

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stimulations were not easily assessed. Also, cells with spontaneous discharges at a rate less than 1/s were excluded from analysis, except when evoked responses in 'silent' neurons were examined.

(b) Inhibitory effects of forebrain stimulation on spontaneous discharges All areas in the stimulated forebrain structures received inputs from the olfactory bulb. This was first tested by stimulating the ipsilateral olfactory bulb and recording evoked potentials from various areas. The particular point from which the largest evoked potential could be recorded after the shortest latency was chosen as a site of stimulation (see Methods).

Spontaneous activity of neurosecretory neurons in SON and PVN were most often inhibited (4974 ~, depending on structures stimulated) by stimulation of one or several basal forebrain structures. Fig. 2 illustrates two types of inhibitory effect produced by stimulating amygdala medialis. In Fig. 2A, 10 superimposed oscilloscope tracings of spontaneous discharges of a SON neuron (top) and effect of stimulation are shown (bottom). The inhibition occurred almost immediately after the stimulus lasting for a few hundred milliseconds. Post-stimulus time histograms comprising 64 responses are shown below (Fig. 2B). As discussed below, this particular SON neuron was affected only by stimulation of amygdala medialis, but not by centralis or

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ceded by a brief excitatory period. The post-stimulus time histograms taken from 64 trials (lower trace) with the control response of the same number o f trials (upper) are shown in Fig. 2D. Fig. 3 from another SON neuron illustrates inhibitory effects to stimuli of increasing intensities. With an increase in stimulus strength the latency shortened from 100 to 50 ms and duration of the inhibitory effect increased greatly to several 100 ms. The immediate or short-latency inhibition (inhibition starting almost immediately after the stimulus artifact) caused by various limbic structures was found in 18 ~ of neurosecretory neurons. Its duration ranged between 90 and 480 ms. Some neurons showed rebound or 'post-inhibitory excitation' which lasted for another 100-310 ms. Delayed inhibition (inhibition of spontaneous discharges after latency over 20 ms) from limbic system stimulation was seen in the remaining 82 ~ ofneurosecretory neurons. Its latency ranged between 27 and 53 ms, and duration between 150 and 220 ms. For neurons responding to endopyriform nucleus stimulation only, a significant negative correlation (P < 0.5) existed between latency and duration of the inhibitory effect, i.e. shorter latencies corresponded to longer inhibitory periods. Delayed inhibition was also followed by 'post-inhibitory excitation' or rebound in many cells.

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In a small percentage of SON neurons (23 cells), distinct and repetitive discharges with relatively short latency were evoked by stimulating amygdala lateralis (15 cells), amygdala medialis (4 cells), olfactory tubercle (2 cells) and prepyriform cortex (2 cells). Such 'evoked' responses were observed even in 'silent' SON neurons. Fig. 4A illustrates that a SON neuron showing no spontaneous activity ('silent cells') discharged 3-5 spikes with a 30 ms latency after stimulation of amygdala lateralis. Similar evoked responses were obtained from another cell by stimulating the endopyriform nucleus (Fig. 4B) and olfactory tubercle (Fig. 4C). Latencies of the 'evoked' discharges from forebrain areas ranged between 19 and 35 ms and excitation was always brief, lasting less than 20 ms. This was often followed by inhibition of spontaneous activity. Latency of the response was longer (35.5 ~ 3.1 ms;

36 than stimulus-locked spikes ('evoked' response) was also observed in a small n u m b e r (17 cells) o f neurosecretory neurons as increased spontaneous dis-. charge frequencies after latencies o f 50-100 ms (Fig. 4D). Some neurosecretory as well as 'unidentified' neurons showed excitation after a very long latency which occurred following the inhibitory period and could be considered as a r e b o u n d or post-inhibitory excitation. Fig. 5 shows that inhibition o f neuron activity from stimulation o f the amygdala medialis was followed by a post-inhibitory excitation and that inhibitory and the excitatory phases alternated. In other neurons, excitation occurred following

-t- S.E.) following the a m y g d a l a lateralis stimulation than that o f the a m y g d a l a medialis (19.5 4- 3.8 ms; ~ 4- S.E.) (Table I). On the other hand, stimulations o f the olfactory bulb, endopyriform nucleus, central, cortical and basal a m y g d a l a p r o d u c e d no ' e v o k e d ' discharges. The percentage o f cells responding with stimuluslocked spikes to forebrain stimulation was higher in 'unidentified' and 'atypical' neurosecretory neurons (see (a) o f Results for definition) than identified neurosecretory cells (Table I). This difference between the two groups is statistically significant for each g r o u p (P < 0.003 ,~ 0.002). The excitatory effect o f forebrain stimuli other

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37 TABLE I

"Evoked' responses observed in 3 groups of neurons in SON and P VN Structures stimulated

Number tested

Number showing 'evoked' responses

Response latency (x ± S.E.)

Neurosecretory neurons (identified) Amygdala lateralis 81 Amygdala medialis 52 Prepyriform cortex 28 Olfactory tubercle 23

15 4 2 2

(19~) (8~) (7 ~) (9 ~)

35.5 =c 3.1 ms 19.5 i 3.8 31.0 33.0

Unidentified neurons Amygdala lateralis Amygdala medialis Prepyriform cortex Olfactory tubercle

17 2 6 3

(25~o) (6 ~) (15~) (25~o)

28.9 ± 3.2 25.0 30.8 ± 6.2 24.0 5_ 4.1

7 (58 ~) 4 (40~) 0 0

30.8 ± 3.4 22.3 ± 6.6 ---

69 35 39 12

A typical neurosecretory neurons Amygdala lateralis 12 Amygdala medialis 10 Prepyriform cortex 6 Olfactory tubercle 2

long-lasting inhibition from stimulation of the endopyriform nucleus (Fig. 5B, latency 220-230 ms) and of the olfactory bulb (Fig. 5C, latency 140-150 ms). In all cases, the late excitatory response was again followed by an inhibitory period. Fig. 5 also shows that these particular neurons were not much affected by stimulation of other forebrain areas. Internal capsule (IC) stimulation as a control was also ineffective (Fig. 5A). No neurosecretory cell was excited antidromically by stimulation of limbic structures in this study, except in one neuron following stimulation of the amygdala medialis. (d) Comparison o f effects produced by stimulation o f various forebrain regions Of 195 neurosecretory cells tested, percentages responding to various forebrain stimuli were calculated. Fig. 6 shows the proportion of cells inhibited (black), excited (dotted and hatched) and unaffected (white). Both 'immediate' and 'delayed' inhibitory responses were similarly treated but cells showing 'evoked' discharges and 'overall' excitation were counted separately. Neurons showing distinct post-inhibitory rebound (Fig. 5) were included in 'inhibited' groups.

A m o n g various amygdala areas, stimulation of the lateral amygdaloid nucleus excited the largest

number of SON and PVN neurons ( 2 5 ~ of all tested) and inhibited the least number of cells (39 ~). Stimulation of amygdala medialis inhibited over 67 ~ of neurons and excited only 8 ~ . This suggests regional differences in connections to SON or PVN neurons. Similar observations were made in neurons of the ventromedial hypothalamic nucleus. They were excited by basolateral and inhibited by corticomedial amygdala stimulation u. As shown in Table II, the responses of 'unidentified' cells to forebrain areas did not greatly differ from those antidromically 'identified' neurons. It is not easy to know whether or not some of these 'unidentified cells' are in the category of neurosecretory cells, since axons of these 'unidentified' neurons may not reach the posterior pituitary; particularly cells in PVN. Some of these neurons were excited by intracarotid injection of hypertonic solution and therefore, it is likely that they contribute to the physiological functions of the hypothalamo-neurohypophysial system. (e) Convergence o f inputs to single neurosecretory neurons f r o m forebrain regions Many neurosecretory cells received inputs from more than one region of basal forebrain areas. Out of 195 neurosecretory cells, 79 (42 ~ ) received inputs from two or more limbic structures. Of these neu-

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50 msec Fig. 5. Post-inhibitory excitation produced by forebrain stimulations in 'unidentified' cell (A) and neurosecretory cell (B and C). Oscilloscope tracings are 10 superimposed sweeps, all post-stimulus time histograms and their controls are taken from 64 trials. C, control; AM, amygdala medialis; IC, internal capsule, OB, olfactory bulb; END, endopyriform nucleus; PPC, prepyriform cortex. Note that the late excitatory period was again followed by an inhibition. Each neuron responded to only one forebrain structure. rons, 50 received inputs from two forebrain zones, 17 from three zones, and 4 from four limbic regions• Figs. 7 and 8 illustrate this. Fig. 7A and C are two neurosecretory neurons from the same animal. One neuron (Fig. 7A) was inhibited by stimulation of olfactory bulb as well as of the medial, central and

lateral amygdala. On the other hand, another neuron (Fig. 7C) was inhibited by the first 3 but not by the lateral amygdala stimulations. Evoked potentials recorded in three regions in amygdala following stimulation of the olfactory bulb are also shown in D. Fig. 8A is from another experiment. The cell was

39 inhibited by s t i m u l a t i o n o f the e n d o p y r i f o r m n u cleus, the olfactory b u l b as well as by the medial a n d lateral amygdala. C:(=

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A n o t h e r interesting finding relative to convergence was that the p a t t e r n of response of neurosecretory cells to forebrain stimuli differed a m o n g n e i g h b o r i n g neurons, as illustrated in Fig. 9. Neu-

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AMYGDALOID NUCLEI

Fig. 9C a n d D were taken from a n o t h e r animal. Fig. 6. Comparison of effects produced by stimulations of various forebrain structures on neurosecretory neurons (identified) in SON and PVN. N =: total numbers tested. OB, olfactory bulb; OTb, olfactory tubercle; End, endopyriform nucleus; PpC, prepyriform cortex. Among amygdaloid nuclei, amygdala medialis (Med), corticalis (Cort), centralis (Cent), lateralis (Lat) and basalis (Bas) were stimulated. Inhibited neurons include both groups showing 'immediate' and 'delayed' inhibition. The excitatory effects, however, were divided into two groups; neurons showing stimulus-locked spikes ('evoked') and those showing a general increase in rate of basic discharges ('overall').

Latencies of the a n t i d r o m i c potentials were again different (12.5 a n d 15.5 ms), as were the responses to a m y g d a l a stimulation. The level o f s p o n t a n e o u s activity also differed in two pairs of n e u r o n s ; histograms in A represent 128 responses, while B only 64 responses. This difference in the level of activity of these n e u r o n s m a y be the reason why the i n h i b i t o r y effect of a m y g d a l a s t i m u l a t i o n was strong in a moderately active cell b u t n o t o n its n e i g h b o r i n g

TABLE II Responses of 3 groups of neurons in S O N and P V N to forebrain stimulations

Definition of 3 groups of SON and PVN cells - - see Results (a). The same neuron was counted more than once when its response to stimulation of more than one forebrain structure was examined. Stimulation sites

Neurosecretory neurons (identified)

"Atypical' neurosecretory neurons

Unidentifiedneurons

Numbers lnhibited Excited No tested {overall] effect (evoked)

Total cells tested

lnhibited Excited No [overall] effect (evoked)

Numbers lnhibited Excited No tested [overall] effect (evoked)

Olfactory Bulb

75

50

Olfactory tubercle Endopyriform cortex Prepyriform cortex Amygdala Medialis

23

11

35

22

28

16

52

35

[0] (o) [2] (2) [0] (0) [2] (2)

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(4) Corticalis

16

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39

22

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81

30

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Fig. 7. Convergence of inputs from various forebrain structures to single neurosecretory neurons. A and C are taken from the same preparation but different neurosecretory cells. (Note difference in shapes and latencies of antidromic potentials - - 5 superimposed tracings). Stimulation sites in the amygdala nucleus were the same for both neurons. Evoked potentials recorded at these sites following stimulation of the olfactory bulb are shown in D (5 superimposed sweeps). B: an increased activity of a neurosecretory cell (the same neuron as shown in A) produced by an osmotic stimulus (0.2 ml of 0.5 M NaCI injected into the carotid artery). The upper tracing - - systemic blood pressure (BP); the lower tracing - - a number of action potentials counted every second (SON). All post-stimulus histograms are from 64 trials. OB, olfactory bulb; A M G , amygdala medialis (M), centralis (Ce) and lateralis (L).

neuron which showed a rather high rate of discharge. In some neurosecretory cells, effects of baroreceptor excitation51, 5z and osmotic stimulus were tested as well as forebrain stimulation. Of a total of 21 cells examined, one was unresponsive but 20 ( 9 5 ~ ) responded with inhibition of spontaneous discharges to inflation of a balloon located in the carotid sinus region (Fig. 8B). All of 10 neurons tested for effects of intracarotid hypertonic saline injections (0.2 ml of 0.5 M NaC1) increased their discharge rate significantly (Fig. 7B). Some cells were also responsive to painful stimuli (pinching the skin of the contralateral leg); 8 cells were excited, 2 inhibited and 8 were unresponsive (total ---- 18).

DISCUSSION

(1) Functional consideration of limbic influence on the hypothalamo-neurohypophysial system The results presented above demonstrate that many neurosecretory cells in the SON and PVN of the cat can be subjected to inhibitory and/or excitatory influences from a variety of basal forebrain regions in which short latency-large evoked responses can be recorded following stimulation of the olfactory bulb. Many of these neurosecretory cells showed positive responses to baroreceptor and osmotic stimulation and possibly are vasopressinproducing neurons, particularly those in SON of the cat 5. Thus, we may speculate that the olfactory bulb

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Fig. 8. A and B: a neurosecretory cell was inhibited by stimulation of 4 areas (A) and also by baroreceptor excitation (B). The latter was caused by distension of a small balloon situated at the carotid sinus region• In B systemic blood pressure (BP), heart rate (HR) and counted action potentials per second (SON) are shown• Note that an increase in blood pressure occurring after the stimulus as a rebound inhibited the SON cell again due to the secondary baroreceptor excitation. All post-stimulus histograms are from 64 trials. OB, olfactory bulb; AMG-M and -L, amygdala medialis and lateralis; END, endopyriform nucleus. a n d related forebrain regions can partially m o d u l a t e release o f vasopressin from neurosecretory cells. I n

thirst ~4 a n d that osmoreceptors are present in the olfactory b u l b o f dogs ~4 a n d catsZZ, in the prepyri-

this regard, it has been shown that olfactory b u l b deafferentation results in diabetes insipidus a n d

form cortex of cats 14 a n d in the olfactory tubercle a n d a m y g d a l a of cats a n d rabbitsZO,~0. The olfactory

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42 bulb may also influence drinking behavior in response to NaCl loading or deprivation s,4s. In addition, increased plasma vasopressin occurs following electrical stimulations of the prepyriform cortex in the dog 54, of the amygdala in the rat 46, monkey is and man 10 and of the olfactory tubercle in the monkey 19. Since the rate of discharges in neurosecretory neurons have been positively correlated with vasopressin secretion, it seems pertinent to point out that in our study forebrain stimulation produced stimuluslocked discharges in some neurosecretory cells, or a general increase in the neuron activity as well as an excitatory rebound following an initial inhibition. Thus, our finding of excitatory influences of most limbic structures on some SON and PVN neurons seems to support the result of behavioral and endocrine studies. On the other hand, we also found that a large proportion of neurosecretory cells in SON and PVN examined were initially inhibited by various forebrain stimuli. The discrepancy between this observation and the result of the endocrine studies discussed above may be partly due to the type of stimulation employed. In our study we used stimuli consisting of a single pulse or a short train of pulses, while in endocrine studies repetitive and longerlasting stimulations were employed. In many neurosecretory neurons, the activity was first inhibited by forebrain stimulation, but this inhibitory period was followed by an increase in their basal discharge rate. I f a neuron shows a biphasic pattern of response to a single stimulus, repetitive stimulation of the same neuron produces more complex reactions that may differ from the initial response. Thus, the overall effect produced by repetitive stimulation of various forebrain structures on many neurosecretory neurons may not be simply an inhibition of their activity. Since only some P V N neurons were identified (see Results (a)) and their pattern of response to stimulation of forebrain structures was similar to that of SON neurons, cells in both nuclei were treated together for our data analysis. Some of the neurosecretory neurons studied, therefore, were oxytocinproducing cells or belong to a group which releases both oxytocin and vasopressin in the cat ~. It has been shown that an inhibition of oxytocin release was obtained following electrical stimulation of the pre-

pyriform cortex and basolateral amygdala in the rabbit 2 and exposure to a certain odor in the rat 16. In another study the urine odor of female rats in estrus was found to affect significantly the discharge rate of'antidromically identified neurons' in PVN of males55. Macrides 29 speculates that 'the odor of pups promotes release of oxytocin in lactating rodents'. It is known that in many species the mothers lick or sniff to make frequent naso-anogenital contacts with their young during nursing 15. Thus, we may suggest that olfactory stimuli can produce changes in release of neurohypophysial hormone which are relevant to reproductive and maternal behavior. In addition to 'identified' neurosecretory cells in SON and PVN, there were two additional neuron groups which were also found to be affected by the same stimulation of forebrain structures. One group was 'atypical neurosecretory cells' which were excited by the stalk stimulation with varied latencies; the other, 'unidentified' cells, which were situated in or near the histological boundaries of SON and PVN but were not antidromically excited by stimulation of the pituitary stalk. Moreover, the pattern of responses of these two groups of neurons to the same stimulus was not markedly different from that of'identified' neurosecretory cells. At least, a part of these cells are likely to send their axons to the area other than the posterior lobe, possibly to the median eminence or to the medulla a5,49. In this connection, it is interesting to note that cells producing gonadotropin-releasing hormone have been demonstrated recently to lie within the SON and that their axons are directed to the median eminence 30. L H - R H (luteinizing hormone-releasing hormone) producing cells and fibers have been identified all along the central olfactory pathways to the hypothalamus TM 43 It is also known that L H secretion can be enhanced or inhibited following electrochemical stimulation of the accessory olfactory bulb and main olfactory bulb, respectively 4. Thus, the well-known olfactory influence on reproductive behavior in mammals may also include participation of neurons in SON or PVN. In the literature most studies of limbic influences on neurosecretory neurons in SON and PVN are limited to the effect of stimulation of the amygdala or septal regions. Stimulation of the central amygda-

43 loid nucleus in the cat was found to have an inhibitory effect on 6 4 ~ of PVN neurosecretory cells, but contrary to our findings in SON cells, lateral amygdala stimulation was ineffective 3~. In another study conducted in the rat z7 only 1 0 ~ of SON neurons were inhibited by amygdala or hippocampus stimulation. Only inhibitory effects on SON neurons following septal stimulation were reported in the monkey 43 and rat z6, whereas both excitatory and inhibitory effects were observed in the cat27, ~3 and in the dog z8. Taken together, it is clear that the study of the olfactory influences on the hypothalamo-neurohypophysial system is still at its early stage, and we do not yet have sufficient knowledge to suggest a clear functional role of this system. ( 2 ) Morphological connections between the olfactory bulb and S O N

It is known that functional monosynaptic connections exist in fish between the olfactory bulb and the preoptic nuclei. Stimulation of the bulb induces EPSPs and action potentials on preoptic neurons iv, z2. Electrical stimulation of the olfactory bulb depletes preoptic nucleus neurons from stainable neurosecretory granules 21. In the rat, Scalia and Winans 41 have described a fiber fascicle running medially from the lateral olfactory tract towards the supraoptic nucleus and ending between the latter and the medial amygdaloid nucleus. Furthermore, Carlsen and de Olmos 7 using terminal degeneration, horseradish peroxidase anterograde transport and autoradiographic techniques have confirmed these findings in the guinea pigs. In addition, the olfactory system can be linked to

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