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Exp Brain Res 39, 187-192 (1980)
9 Springer-Verlag 1980
Single Unit Recordings in the Rat Pineal Gland: Evidence for Habenulo-pineal Neural Connections O.K. Rc~nnekleiv 1, M.J. Kelly 2, and W. Wuttke Max Planck-Institut ftir biophysikalische Chemie, P.O. Box 968, D-3400 G6ttingen, Federal Republic of Germany
Summary. Extracellular potentials were recorded in the pineal gland of urethane-anesthetized rats. Two distinct populations of excitable pineal "cells" were found, the silent "cells" which were driven by habenula stimulation and the spontaneously active cells. In the former case 17 of the responses (median latency of 1.2 ms) showed a positive-negative potential, and 6 (about 1 ms latency) showed only positive potential of 1-2 ms duration. The remaining ceils (114), which could not be driven by habenula stimulation, exhibited spontaneous activity with a firing frequency from less than 1 Hz to greater than 100 Hz with a median firing frequency of 10 Hz. These experiments clearly demonstrate a direct habenulo-pineal fiber pathway and furthermore show that there are neuronal elements in the pineal which are only activated by habenula stimulation. Key words: Pineal - Single unit recording - Habenula stimulation - Habenulo-pineal pathway
In most mammals the pineal is considered a parenchymatous organ which contains pinealocytes with an exclusive secretory function and which is exclusively innervated by the peripheral autonomic nervous system (Kappers 1976). Habenular commissural fibers as well as posterior commissural fibers have been shown to penetrate into the proximal part of the pineal organ. However, in the monkey (Le Gros Clarke 1940) and in the rat (Kappers 1960), these fibers are believed to form "hairpin loops" and return to the brain without synaptic communication. 1 Present address: University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic, Pittsburgh, PA 15261, USA 2 University of Pittsburgh School of Medicine, Department of Physiology, Pittsburgh, PA 15251, USA Offprint requests to: O.K. RCnnekleiv (address see above)
Lately different investigators, using morphological techniques, have shown that substantial fiber tracts enter the pineal from the commissural area in the cat and the monkey (Nielsen and M011er 1975; Hayes et al. 1974). Aberration of the fibers was not observed in either case. Furthermore, David and Herbert (1973), using the ferret as experimental animal, observed degenerative changes in axon terminals ending on pineal ganglion cells after lesions of the habenular nuclei. Electrophysiological data also support the existence of central neural input to the pineal organ (Schapiro et al. 1971; Dafny et al. 1975; Semm and Vollratti 1978). Moreover, McClung and Dafny (1975) and Brooks et al. (1975) observed spontaneous spike discharges in the pineal which could indicate the existence of neuronal elements. The latter authors suggested that the discharges were from sympathetic fibers. Indeed, the pineal contains a dense network of sympathetic fibers (Owman 1965), some of which penetrate the basal lamina and come into close contact with the pinealocytes ("synapses a distance"), while others are located in the perivascular space (Kappers 1960; Arstila 1967). The pinealocyte originates from neuro-epithelium, but is not considered a real neuron (Kappers 1976); although lately the term paraneuron has been applied to this cell type (Ueck and Wake 1977). We decided to study the possible existence of a habenulo-pineal neural connection and the excitability of the pineal cells using conventional single unit recording techniques. Preliminary data have been presented to the German Physiological Society (R0nnekleiv et al. 1978). Materials and Methods
Twenty-one female Sprague-Dawleyrats were used in this study. Rats of 180-220 g body weight were injected with an anesthetic
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Fig. 1. Midline sagittal view of rat brain with schematic drawing of recording set-up. A Wood's metal electrode was lowered through a hole in the cranium and stereotaxically positioned in the medial habenular nucleus. Recording electrode of sturdy glass-insulated tungsten wires was stereotaxically lowered into the pineal body through the superior sagittal sinus. Abbreviations: OC = optic chiasm; SCN = Suprachiasmatic nucleus; ARC = arcnate nucleus; AC = anterior commissure; PC = posterior commissure; TCC = corpus callosum; HI = hippocampus; MH = medial habenula; PVR = periventricular nucleus; SGPV = periaqueductal central gray
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dose of urethane (1.2 g/kg body weight) and a jugular vein was cannulated for anesthetic supplementation. Each animal was placed in a stereotaxic frame (David Kopf Instruments), and a hole was drilled for placement of the stimulating electrode in the medial habenular nucleus (AP 3.5 ram, Lateral 0.3 mm and Depth 0.6-0.8 mm from interaural zero). The bifurcation of the superior sagittal sinus was exposed so that the pineal gland could be penetrated from the dorsal surface by the electrode coming through the sinus, which was evident by a change in the noise of the recording electrode. A schematic drawing of the recording setup is shown in Fig. 1. Stimulating electrodes were prepared from single barrel glass micropipettes of 40-60 ~tm tip diameter which were filled with Wood's metal (Merck). The recording electrodes were prepared from tungsten wires of 0.01 mm diameter which were etched to tip diameters of.1-2 ~m (etching solution of 7.1% NaNOJ3.4% KOH). The wires were insulated by glass which was heated and pulled around the wire leaving 1-5 ~m of the tip exposed. The resistances of ideal recording electrodes were 8-14 Mf~ as measured by a 5 nA AC current in physiological saline. Throughout the recording period single anodal pulses of 50-200 ~ts duration were delivered to the habenula complex via a Tektronix Stimulus Isolation Unit which delivered constant current pulses of 0.001-0.1 mA strength. Extracellular AC recordings were made in the pineal gland, amplified by conventional means, displayed on a storage oscilloscope and recorded on tape. The response to the stimulus was measured by leading the stimulus artifact and the signal through a window discriminator and then computing on-Iine with a PDP-11 computer the post-stimulus histogram (PSTH) with a bin width of 2 ~s for a total duration of 2 ms. The signal which triggered the stimulus also was used to trigger the computer. A light was flashed on the retina of the rats to see if crude light stimulation could evoke changes in the firing frequency of the recorded units. The EEG was recorded also in order to monitor the state of alertness of the animal. When activity was encounted, either evoked by habenula stimulation or spontaneous, a small lesion was made in the pineal gland by passing 0.1 mA anodal current for 2-3 s through the tungsten electrode
after recording the single unit. This did not damage the tip. Penetrations were made from above the sinus to depth of 3.0-3.5 mm. At the end of a recording session, a lesion was placed in the habenula nucleus by passing a 0.1 mA anodal current for 2-3 s through the Wood's metal electrode. Afterwards, the rat was perfused via the left ventricle With a cold mixture of 2.5% gluteraldehyde/2% paraformaldehyde in 0.1 M Na-cacodylate buffer (pH 7.4). The brain was carefully removed from the cranium in order to leave the pineal intact. The whole brain was left overnight in the perfusion mixture and then placed in a cacodylate buffer (pH 7.4) before cutting on a cryostat. Twenty micron thick sections were cut from the habenula through the pineal, stained with cresyl violet, and visualized under a light microscope for the recording and stimulating sites (the respective lesions).
Results W e w e r e a b l e to d i s c r i m i n a t e a n d i s o l a t e s i n g l e u n i t a c t i v i t y f r o m a t o t a l o f 114 s p o n t a n e o u s l y f i r i n g cells w i t h i n t h e p i n e a l g l a n d s o f 21 a n i m a l s . T h e f i r i n g f r e q u e n c y v a r i e d f r o m less t h a t 1 H z t o g r e a t e r t h a n 100 H z w i t h a m e d i a n f i r i n g f r e q u e n c y o f 10 H z . N e i t h e r t h e s l o w n o r t h e fast f i r i n g cells w e r e l o c a l i z e d to a n y p a r t i c u l a r p a r t o f t h e p i n e a l g l a n d (i.e., dorsal versus ventral), and the activity could be r e c o r d e d in all a r e a s o f t h e g l a n d . F i g u r e s 2 a n d 3 a r e r e p r e s e n t a t i o n s of t h e d i f f e r e n t a c t i v i t i e s r e c o r d e d in the pineal gland. A small percentage of the slower firing cells s h o w e d a r a n d o m f i r i n g f r e q u e n c y as t h e cell d e m o n s t r a t e d in F i g . 2a, w i t h its f i r i n g f r e q u e n c y h i s t o g r a m in F i g . 2b s h o w i n g a v a r i a b l e r a t e o f i to 20
O.K. ROnnekleiv et al.: Single Unit Recordings in the Rat Pineal Gland
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Fig. 2. a AC recording of spontaneous activity of a cell in the pineal body. The cell was not driven by habenula stimulation. The firing frequency varied from 1-20 Hz. Spikes have been retouched. b Frequency histogram displaying the heterogenous firing of the cell in Fig. 2a over about an 80 s period. Frequency in spikes/s is depicted along the ordinate and the elapsed time in seconds along the abscissa cE.L 91-3
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Fig. 3. Frequency histogram of fast firing pineal cell. The firing frequency was about 20 Hz. Fig. 2b for further explanation
Hz. However, the majority of cells showed a very regular firing pattern as shown in the histogram of another cell in Fig. 3. On a few occasions we were able to impale cells in the pineal, but we were never able to record from them intracellularly for periods longer than several seconds. None of the spontaneously firing cells could be driven orthodromically and/ or antidromically by habenula stimulation. Further-
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Fig. 4, Constant latency response to habenula stimulation. Beginning of stimulus artifact was cut off by oscilloscope sweep but PSTH revealed a latency of 1 ms for this cell. The end of the stimulus artifact is marked by a dot, The arrow indicates the start of the potential. The picture shows three superimposed sweeps
more, crude light stimulation had no effect on the activity. Another distinct population of cells did not show any spontaneous activity (silent) but did respond to stimulation of the habenular area. A representative response is exhibited in Fig. 4. The PSTH for this cell exhibited a constant latency response at 1 ms for 50 repetitive stimuli. The latencies for the 17 cells varied from 1 ms to 4 ms with a median latency of 1.2 ms, but the response was extremely constant for any given cell. Increasing the stimulus strength rarely shortened the latency for the response. Sometimes, there appeared to be an inflection of the initial rising positive potential of the spike (Fig. 4), but we never observed the separation of the spike into the "A potential" and the "B potential" at higher stimulation frequencies (Novin et al. 1970). Although the responses were able to follow stimulation frequencies up to 10 Hz (the maximum tried), we were unable to apply the collision test (Novin et al. 1970) as a further verification of an antidromic response. Also, extracellular positive potentials were recorded from six other sites following low intensity habenula stimulation. The potentials were constant latency ( - i ms) biphasic positive potentials for 1-2 ms duration. We were never able to impale any of the silent cells. An example of a lesion placed in the recording area is shown in Fig. 5. The electrode tract is visable; however, the microlesion as revealed by the clotting of blood on the original tissue section, cannot be clearly visualized in this black and white photograph. Such histological verification assured us that the single unit activity which we recorded was within the pineal gland. Figure 6 shows a representative lesion produced by our stimulating electrode at the end of the experiment. The identification of the site confirmed the placement of the electrode, and at the low stimulation intensities which we were using, gave us some certainty that we were activating fibers originating or passing through the habenular complex.
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Fig. 5. Frontal section from the rat pineal body (20 ~m) which shows an electrode tract through the midsection of the organ (arrowheads). Clotted blood reveals the microlesion (0.1 mA for 3 s) (arrow) produced by the recording electrode following recording in an area where spontaneous activity was found. The microlesion cannot be clearly distinguished in this black and white photograph. Cresyl violet, x 150
Discussion
We have shown that there are two distinct excitable neuronal elements in the pineal, the silent "cells" which are driven by habenular stimulation and the spontaneously active cells which could not be influenced by either electrical stimulation of the habenular area or by light stimulation of the eyes. The constant latency of the habenula-activated pineal response may indicate that the response is antidromic. However, neuronal elements in the pineal gland of the rat which send processes down to the habenula have not been described. Another possibility is that we were activating axons passing up the pineal stalk into the pineal gland. This latter interpretation would be consistent with the findings that small lesions confined to the medial habenula nucleus cause degenerative changes in axonal boutons within
the pineal gland (R0nnekleiv and M011er 1979). Also, the evoked fast positive potentials recorded from six other sites in the pineal following habenular stimulation could be fiber activity. Assuming a distance of 6 mm between the stimulating and recording electrodes, the median latency (1.2 ms) would yield a conduction velocity of 5.0 m/s, a value indicative of myelinated fibers (Patton 1965). Myelinated fibers in the pineal stalk or the pineal proper have been observed in several species such as the rat (Kappers 1960; Arstila 1967), the hamster (Ueck et al. 1977), the ferret (David and Herbert 1973), the guinea pig (McNeil et al. 1979), the cat and the monkey (Nielsen and M011er 1975). In the mouse large bundles of myelinated fibers can be observed in the deep pineal running to or from the pineal gland (MOller, pers. commun.). Earlier reports stated that these fibers leave the pineal without synaptic contact and therefore have no functional significance in the pineal (Kappers 1960, 1965). However, in the present study 23 of 137 neuronal elements (17%) responded to right habenular area stimulation. This is in accordance with the morphological data showing that small unilateral lesions of the habenular area caused degeneration of about 15% of pineal nerve terminals (Ronnekleiv and MOller 1979). The pineal organ in the rat is situated immediately below the bifurcation of the sagittal and the transverse sinuses. Therefore, the investigator is faced with the problem of penetrating the sinuses with the fragile recording electrode or approaching the gland by other means. To date this has mostly resulted in recordings with large (50 ~m) tip electrodes (McClung and Dafny 1975; Dafny 1977) or the use of a pseudo in vivo preparation with impaired blood flow due to removal of the sinus (Schapiro et al. 1971). We circumvented these problems by using sturdy glass-insulated tungsten electrodes. Hence, we could penetrate the sinus without breaking the small electrode tip (less than 2 ~tm); the penetration caused no bleeding and we were able to discriminate and isolate (spontaneous) single unit activity within the pineal organ. Spontaneous activity partially comparable to the one observed in the present study has been found by Brooks et al. (1975). These authors assumed that they were recording action potentials from nerve terminals since the spontaneous activity was increased in frequency by stimulation of the cervical sympathetic trunk. The possibility exists that we also were recording spontaneous sympathetic fiber activity, for some of the spontaneous activity was of quite high frequency (100 Hz), and we were not successful in influencing the spontaneous activity by habenular area stimulation. However, in several instances we impaled the cell which we were record-
O.K. R0nnekleivet al.: Single Unit Recordingsin the Rat Pineal Gland
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Fig. 6. Dark field photograph of frontal section through the epithalamic area of a rat brain showing lesion (arrow) produced by the stimulatingelectrode in the vicinityof the right medialhabenular nucleus (mh). Stimulationat this site evoked constant latencyresponses in the pineal. (lh = lateral habenula, cp = choroid plexus. Cresyl violet, x 150
ing extracellularly; this resulted in injury discharges typical of injured cells with leaking membranes. This would imply that at least on some of the occasions we were recording cellular and not fiber activity. McClung and Dafny (1975) observed activity only in the first 200 ~m of the pineal organ, while they were not able to record from the center region. This was not the case in the present study. There seemed to be an even distribution of spontaneously active cells and cells which responded to habenular area stimulation. Our electrophysiological results are consistent with our data on the distribution of degenerating boutons following lesions (R0nnekleiv and M01ler 1979). The discrepancy with McClung and Dafny's data can as yet not be explained. However, we feel that the microlesions enable us to localize the activity and responses to different pineal areas. To reiterate, there are two distinct populations of excitable pineal cells, but crude light stimulation did not influence the activity of either population. Brooks et al. (1975) did not see any effect of sectioning the optic nerve on activity within the rat pineal. They
related this to the lack of sympathetic discharge to the pineal gland during the day as concluded by Wurtman and Axelrod (1966), and Brownstein and Axelrod (1974). Our experiments were performed in a dark room and activity was usually obtained after 1800 h, and under those conditions no effect of light on single cells was obtained. In contrast, Taylor and Wilson (1967) and McClung and Dafny (1975) found spontaneous pineal activity influenced by similar light stimulation (surgical lamp). The discrepancy in the data could be due to differences in the electrode size, 2-3 ,am in the present experiments versus 400 ~m and 50 ~tm respectively in theirs. With a large electrode tip one might pick up activity from adjacent areas such as the stiperior colliculus. Since neurons have not been described anatomically in the pineal gland of the rat, the activity should be of pinealocyte origin. Gland cells of neuroectoderm origin have been found to exhibit spontaneous spiking (Taraskevich and Douglas 1977, 1978; Dufy et al. 1979), and perhaps the pinealocytes, also endocrine gland cells of neuroectoderm origin (Kap-
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p e r s 1976), s h o w e l e c t r i c a l activity in t h e f o r m of action potentials. However, only intracellular recordi n g of t h e s e cells will r e v e a l t h e n a t u r e ( C a + 2 - s p i k e ? ) of this e x t r a c e l l u l a r activity, a n d w h e t h e r it is r e l a t e d to t h e s e c r e t o r y f u n c t i o n o f t h e p i n e a l o c y t e .
Acknowledgements. Dr. O.K. R~nnekleiv was a fellow of the Alexander von Humboldt Stiftung and Dr. M.J. Kelly was a fellow of the National Institutes of Communicative Diseases and Stroke. The authors wish to thank U. Mecke for histological technical assistance.
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Kappers JA (1976) The mammalian pineal gland, a survey. Acta Neurochir 34:109-149 Konig JFR, Klippel A (1967) The rat brain. A stereotaxic atlas of the forebrain and lower parts of the brain stem. Krieger, New York Le Gros Clark WE (1940) The nervous and vascular relations of the pineal gland. J Anat (Lond) 74:471-492 McClung R, Dafny N (1975) Neurophysiological properties of the pineal body. 2. Single unit recording. Life Sci 16:621-628 McNeill ME, Whitehead DS (1979) The synaptic ribbons of the Guinea pig pineal gland in sterile, pregnant, and fertile but nonpregnant females and in reproductively active males. J Neural Transm 45:149-164 Nielsen JT, M011er M (1975) Nervous connections between the brain and the pineal gland in the cat (Felis catus) and the monkey (Cercopithecus aethiops). Cell Tiss Res 161:293-301 Novin D, Sudsten JW, Cross BA (1970) Some properties of antidromically activated units in the paraventricular nucleus of the hypothalamus. Exp Neurol 26:330-341 Owman CH (1965) Localization of neuronal and parenchymal monoamines under normal and experimental conditions in the mammalian pineal gland. Progr Brain Res 10:423-453 Patton HD (1975) Special properties of nerve-trunks and tracts. In: Ruch TC, Patton HD (eds) Physiology and biophysics. Saunders, Philadelphia, pp 75-85 R0nnekleiv OK, Kelly MJ, M011er M, Wuttke W (1978) Electrophysiological and morphological evidence of direct central innervation of the pineal gland. Pflfigers Arch [Suppl] 373:54 R0nnekleiv OK, MNler M (1979) Brain-pineal nervous connections in the rat: An ultrastructure study following habenula lesion. Exp Brain Res 37:551-562 Schapiro S, Salas M (1971) Effects of age, light and sympathetic innervation on electrical activity of the rat pineal gland. Brain Res 28:47-55 Semm P, Vollrath L (1978) Electrophysiological properties of single cells of the guinea-pig epiphysis cerebri. Pflfigers Arch [Suppl] 373:55 Taraskevich PS, Douglas WW (1977) Action potentials occur in cells of the normal anterior pituitary gland and are stimulated by the hypophysiotropic peptide thyrotropin-releasing hormone. Proc Natl Acad Sci USA 74:4064--4067 Taraskevich PS, Douglas WW (1978) Catecholamines of supposed inhibitory hypophysiotrophic function suppress action potentials in prolactin cells. Nature 276:832-834 Received July 16, 1979
