Neurophysiological properties of the pineal body

Pergamon Press

Life Sciences Vol. 16, pp. 611-620 Printed in the U.S.A.

NEUROPHYSIOLOGICAL PROPERTIES OF THE PINEAL BODY I. Field Potentials N. Dafny, R. McOung, and S. I. Strada The University of Texas Medical School at Houston Houston, Texas 77025 (Received in final form January 17, 1975)

Freely behaving and acutely anesthetized rats were used to study electrophysiologically the neuronal input from the amygdala complex (Amyg), acoustic click stimuli (AC), optic tract (OT) and superior cervical ganglion (SCg) to the pineal body (PB). Monopolar and bipolar recordings were used in the present study to record the field potentials with semimicro electrodes (50JZ). Depth profile recordings through the PB were also obtained. In monopolar recordings, the PB responses to all the modalities were of short latencies. In bipolar recordings, Amyg stimulations appeared to demonstrate unequivocal responses in the PB. In recent years the pineal body (PB) of the rat has been the subject of numerous anatomical and biochemical investigations {I). Despite this fact, there have been only a few studies concerned with

the electrophysiological characteristics (EEG

activity) of this structure (2,3). The

electrophysiological studies have demonstrated that photic input can modify EEG activities in the PB independent of its effects on visual cortical areas. The present study further investigates the electrical activity of the pineal in relationship to central and sensory input.

MATERIALS AND METHODS

The experiments were performed on 12 male albino rats weighing 250-350 gm. Two preparations were used: 1) freely behaving rats with implanted electrodes, and 2) depth profile studies which employed

acute

animal

preparations. The animals were anesthetized

by

intraperitoneal injections of pentobarbital (50 mg/Kg) in the chronic preparations, and urethane ( 1.25 gm/Kg) in the acute preparations. Semimicro electrodes

(SO~t)

of diamel insulated nichrome

wire were implanted stereotaxically (4). Recording electrodes in chronic animals were placed in pineal body (PB), anterior hypothalamus (AH), and ventromedial hypothalamus (VMH). A stimulating electrode, consisting of two twisted SO~t wires with the bare ends separated (0.1 0.2 mm) was implanted in the amygdaloid complex (Amyg). Electrodes were fixed to the skull with acrylic cement and were attached to terminals of a nylon plug. In the acute animals, stimulating electrodes were placed in the Amyg and optic tract (OT) in the same manner. The superior cervical ganglia (SCg) were exposed and two silver electrodes were placed on the postganglionic fibers. The PB was exposed by removal of the overlying portion of the skull. Bipolar 611

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recording electrodes, with a configuration identical to the stimulating electrodes, were placed on the superior surface of the PB to allow bipolar and monopolar recording. Electrical stimulation was produced by a Device Digitimer through its isolation unit, (square pulses of 0.2 msec (l.Q.3.0V=SCg; 4.0-S.SV=OT; and 3.0-4.5V=Amyg) every 1.1 sec; the voltages were determined by the criteria of starting stimulation· at 0.1 V and increasing in increments of 0.1 V until the smallest detectable responses were observed (threshold), then the voltages were increased to establish the intensity of 1.5 times the threshold at each site of stimulation), while acoustic stimuli (AC) were produced by a Grass ultra-linear audiomonitor with a remote speaker located 25 em from the animals and triggered by the Device Digitimer. Four to six days after chronic implanation of electrodes, the animals were placed in a plastic cage within a Faraday chamber. Electrodes were connected to the recording apparatus through a counter balanced commutator which allowed the animal freedom of movement. The amplified (Grass PSI I preamplifiers with their emitter followers, filters set at 0.3 Hz. and l KHz.) electrical activity was displayed on a storage oscilloscope interfaced with a signal averaging computer (NIC 1070) to average evoked responses. In acute depth profile experiments, bipolar and monopolar evoked responses were recorded on FM, magnetic tapes (Sangamo Model 3618, 3-3/4 ips, 400 Hz - 50kHz); the electrode was advanced through the PB in 0.4 mm increments and responses were evaluated off line. Responses were photographed and evaluated from film in both types of preparations. Latencies were measured in msec from the point of initiation of the stimuli to the different peak responses; amplitudes were measured from base line to the first peak (P 1), and the subsequent components from peak to peak in pV. At the conclusion of each experiment, animals were anesthetized with pentobarbital and D.C. current was passed through the electrodes for localization of the electrode tip. Animals were perfused with 10% formalin and 3% potassium ferrocyanide. Frozen sections were serially cut ( 40p) and were stained with hematoxylin and eosin. Electrode positions were verified by location of the Prussian blue spots. Except for a few instances in which the OT electrode was present in the optic chiasma (OC), and in two animals where the PB electrode was in the occipital cortex, the electrodes were properly implanted.

RESULTS

In freely behaving animals, the average evoked response (AER) following 32 repetitive Amyg stimulations demonstrated a typical tri-phasic pattern in VMH, AH, and PB that consisted of an initial positive deflection (P 1) followed by a negative (N 1), and positive (P2) wave (Fig. I). The acoustic stimulation evoked a five component pattern which consists of initial positive deflections (PJ) followed by alternating negative and positive deflections (Fig. I). The latencies to the various peaks are summarized in Table I .

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--.,-

-c:;

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Pineal Body: Field Potentials

613

TABLE 1 Component

Latency (msec) AH

PB 1.3 2.2 2.9 4.0 6.3

24.9 41.4 74.0 94.6 118.9

VMH 22.0 42.0 86.0 112.0 141.0

Table 1. Average latencies of components of evoked response to acoustic stimulation. PB: pineal body; AH: anterior hypothalamus; VMH: ventromedial hypothalamus, P1 : first positive component; N 1: flfSt negative component. Figure

Amygdala stim

Acoustic stim

~VMH

L

-£AH 100}JV

~ L 100}JV

100msec

100}JV

L

100msec

PB

SO~N

L Smsec

Average evoked responses from the VMH, AH, and from the PB following 32 repetitive acoustic and amygdala stimulation. The onset of stimulus is shown by "i". Latencies of the AH and VMH AER following acoustic stimulation are in the range of previously described values (5). However, the PB had much shorter latencies to all five components of the response (Table I). The responses to Amyg stimulation (Fig. I) were in the range reported previously by Gloor (6) for AH and VMH, and were of longer latencies in the PB (Table 2). Responses in two animals were not observed following either mode of stimulation; histological examination revealed placement of the electrode in the occipital cortex instead of the PB. In acute experiments, monopolar recording from the surface of the PB showed responses to all four modalities of stimulation (Amyg, SCg, OT, and AC). Reversing the polarity of the stimulus resulted in an inversion of the stimulus artifact while not altering the components of the responses (Fig. 2). Only minor fluctuations were seen in amplitudes and latencies.

Pineal Body: Field Potentials

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TABLE 2 Latency (msec)

Component

Pl Nl p2

PB

AH

VMH

6.0 12.6 21.6

3.8 8.7 16.8

5.0 10.3 19.7

Table 2. Average latencies of components of evoked response to amygdala stimulation. Figure 2 Amygdala stim

Optic chiasma stim

PB

IOO)JYL 5msee

Acoustic stim

Single sweeps of evoked responses in the PB following normal and following reversal of the stimulus polarity. The shape of the response was not affected. Stimulus artifact suppressor was used in these experiments; thus "i" represents the onset of stimulation. SCg indicates stimulation of postganglionic fibers of superior cervical ganglion. Monopolar recordings made while the electrode was advanced through the PB in 0.4 mm increments were similar in the first three recordings (0.0-0.8 mm below surface) following Amyg, OT and AC stimulation respectively. 1.2 mm below the surface, changes in the pattern of the responses occurred (Fig. 3 and 4). Bipolar recording following Amyg stimulation exhibited a similar pattern through all the recording points (Fig. 3); OT stimulation produced small responses at some levels (Fig. 4). No responses were observed in bipolar recording following AC stimulation. However, following SCg stimulation the PB exhibit a similar pattern of responses through all the recording points and increasing the strength of the stimulus brought out latent responses in monopolar recording. Even though the strength of stimulation was five times the stimulus

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615

Figure 3

Amygdala Stim

0.0

0.4 0.8

1.6

2.0 2.4

2.8 3.2

so..uvL

10m sec

Mono

so..uvL

so)JvL

Mono

Bipo

10m sec

10m sec

Depth profile recording of pineal responses with single and S superimposed traces to Amyg stimulation ( 1.5 X threshold). The number on the left side of the figure indicates the site of the recording in mm from the superior surface (0.0) of the PB. The abbreviations Mono and Bipo indicate monopolar and bipolar recording respectively; dotted lines mark the onset of stimulation.

616

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Pineal Body: Field Potentials

Figure 4

Optic tract stim

d.~

0.0 lv~

0.4~

Optic tract stim

Acoustic stim

I

I o.sr 1.2~ 1.6~

2.0~ 2.4~

I I I

r

I 2.8~ 3.2~

so)JvL 10m sec

Mono

I! - -

50)JL

2o)JvL

10m sec

Bipo

Smsec

Mono

Depth prof'lle recording of pineal response to optic tract (1.5 X threshold) and acoustic stimulation.

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Pineal Body: Field Potentials

617

Figure 5

SCg Stim

O.Odf\.sZ:~

0.4~ 0.8~~ 1.2~~~ ~. 16 . J/. :''~;..-} ....

./

,___....

.... ~ 2.0 --~~-~-: ~~ xr ~"'"-

24~ 2.8~ 3.2~

J~-~ ~- '-''"'~::-J/\ ~\ ~~ /:'-:

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~~~

~ ~ >i~ ~

T

~--x

1~

~

~eo

o

.

~

~

r---~ i~

~

so).JvL 10m sec

Mono 1.5

Mono 5.0

Bipo 5.0

Depth profile recording of pineal body response to SCg stimulation. 1.5 and 5 indicate 1.5 and 5 times threshold stimulation intensities. threshold no responses were observed following SCg stimulation in bipolar recording (Fig. 5).

DISCUSSION

The experiments demonstrate for the first time the recording of evoked responses in pineal body following stimulation of some neural pathways. Previous electrophysiological studies measured only EEG activities in response to photic stimuli (2,3). In freely behaving rats, the latencies to acoustic input (with monopolar recording) to the AH and VMH were in ranges previously reported (5). The acoustic response in the PB was not anticipated when the study was initiated and the short latencies, which are in the range of t-2 synapses, were striking. Monopolar recordings in the PB of acute anesthetized animals demonstrate

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Pineal Body: Field Potentials

Vol. 16, No. 4

amplitudes different from those observed in the chronic animal experiments. The reasons for this discrepancy may involve methodological differences in the procedure of the evaluation (computer averaged versus superimposed and/or possibly an effect of urethane anesthesia). Bipolar responses were not observed following AC stimulation even below the PB, at the level of the colliculi. The lack of responses in bipolar recording in these areas indicates the need for further investigation of possible acoustic inputs to the PB and/or spread of current from other remote structures. The present study indicates that an Amyg to PB pathway exists. To rule out spread of current through adjacent structures (OT or OC) as one possible cause of the observed responses, the visual pathway was stimulated and recordings were made from PB. The data indicate that the results observed were not due to spread of current but do represent an Amyg contribution to the PB. At 1.2 mm below the PB surface, the patterns of the responses in mono polar recording following Amyg, OT and AC were changed. It is possible that at this point the electrodes cross the PB. However, reversal in polarity was not obtained following Amyg stimulation with bipolar recordings. The reason the phenomenon was missed could be the configuration of the electrodes (twisted and not concentric) (7) or because of the large increments (0.4 mm) used in advancing the electrodes (7). Stimulation of the SCg attempted to evoke a response through the classical pathway of pineal innervation (8). While 1.5 times the threshold of Amyg stimulation was sufficient to evoke a monopolar recording, no response was observed in bipolar recording with as much as 5 times the SCg threshold stimulation. Pharmacological and neurochemical studies have demonstrated that long bursts of SCg stimulation or continuous SCg stimulation (1-3 hours) are necessary to bring about biochemical changes in the PB (9,10). In this regard, we were unable to find consistent changes in cyclic AMP levels in the pineal body using short bursts of SCg stimulation (II), even though cyclic nucleotide levels are elevated following in vitro or in vivo administration of the presumptive neurotransmitter norepinephrine ( 12). Thus it is possible to assume that the stimuli used in this study were of too low frequency or too short duration to elicit responses. The present observations provide evidence for a major input from the Amyg to the PB and possibly from AC and OT. In order to substantiate this pathway, further investigation is necessary at higher levels of resolution, i.e., the single unit level.

REFERENCES I.

J. Axelrod, Sci. 184, 1341 (1974).

2.

A. N. Taylor and R. Wilson, Experimentia. 26, 261 (1970).

3.

S. Shapiro and M. Salas, Brain Res. 28, 41 (1971).

4.

J. Konig and R. Klippel, The Rat Brain - A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brainstem, Robert E. Krieger Publishing Co., Huntington, N. Y. (1967).

S.

N. Dafny, Electroenceph. and Clin. Neurophysiol. 36, 123 (1974).

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6.

P. Gloor, Electroenceph. and Gin. Neurophysiol. 7, 223 (1955).

7.

J. Schlag, Bioelectric Recording Techniques: Part A, p. 273, R. Thompson and M. Patterson Eds., Academic Press, New York (1973).

8.

J. Kappers, Z. Zel/forsch. 52, 163 (1960).

9.

M. Brownstein and A. Heller, Science 162, 367 (1968).

10.

P. Volkman and A. Heller, Science 173, 839 (1971).

II.

R. McClung, S. J. Strada and N. Dafny, unpublished observations.

12.

S. J. Strada, D. C. Klein, J. Weller and B. Weiss, Endocrinology 90, 4, 1972.