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An intrinsic neuronal-like network in the rat pineal gland
Brain Research 823 Ž1999. 231–233
Short communication
An intrinsic neuronal-like network in the rat pineal gland J. Schenda, L. Vollrath
)
Department of Anatomy, Johannes Gutenberg-UniÕersity, Becherweg 13, D-55099, Mainz, Germany Accepted 26 January 1999
Abstract Recent studies have shown that in rat pineal glands kept in vitro action potential-producing cell clusters are demonstrable. To test whether the clusters interact, multiple-unit recordings were carried out simultaneously from different clusters, with or without electrical stimulation. Clusters with rhythmic burst activity exhibit highly synchronized firing and electrical stimulation of one cluster elicits an immediate response in another one, apparently involving synapses but not gap junctions. It is hypothesized that the interacting clusters form a network. As the firing is affected by norepinephrine, acetylcholine and Ca2q, the network may monitor the interstitial concentrations of these substances and spread this information in the gland, to modulate melatonin secretion. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Rat pineal; Electrophysiology; In vitro; Neuron-like network
The mammalian pineal gland contains action-potential producing cells, demonstrable both in vivo Žfor review, see Ref. w6x. and in vitro w7–9x. Recently, it was shown that the electrically active cells of the rat pineal gland are arranged in clusters of three to five cells and are antagonistically affected by norepinephrine and acetylcholine w7–9x. The aim of the present study was to test the hypothesis that the clusters interact to form a functional network. Male Sprague–Dawley rats of 150 to 200 g b.wt. were kept under 12 h light and 12 h dark Žlights on at 0600; room temperature 22 " 28C; water and food ad libitum. for at least 3 weeks. The pineal glands were excised and transferred to a temperature-controlled superfusion chamber w7–9x. Multi-unit recordings were made simultaneously from two clusters lying at least 0.4 mm apart, with or without alternating electrical stimulation through the recording electrodes Žrectangular pulses of 1 Hz, 0.3 mA, 0.1 ms.. To test the effects of different concentrations of Ca2q, CaCl 2 was added to the superfusion medium yielding concentrations of 1.5 mM, 2.0 mM or 2.5 mM. For synaptic blockade Ca2q was substituted by Mg 2q Ž6.5 mM MgCl 2 , w2x.. Gap junctions were blocked with halothane w5x.
)
Corresponding author. Fax: q49-6131-393719
The discharge patterns were analyzed by spectral analysis ŽSPSS on a VAX computer.. Changes in frequency were analyzed by the Mann–Whitney rank sum test for significant differences Ž p - 0.05.. The data of simultaneously recorded cell clusters were analyzed with cross-correlation tests for possible correlation ŽSPSS on a VAX computer.. In rat pineal glands in vitro, regularly firing cell clusters ŽREGs, 64%. and rhythmically bursting clusters ŽRHYs, 36%. exist w7–9x. Simultaneous recordings Ž n s 50. from two RHYs revealed a strong cross-correlation in the occurrence of the bursts Ž) than two standard errors., their peaks appearing within 10–70 ms. No correlation in firing existed between REGs and RHYs Ž n s 50. and REGs and REGs Ž n s 20.. RHYs that were electrically stimulated responded with statistically significant Ž p - 0.05. excitation Ž n s 42. or inhibition Ž n s 8.. REGs were excited Ž n s 12., inhibited Ž n s 1. or unaffected Ž n s 11.. The responses occurred after 1–2 ms and lasted for 3–10 ms. Single and repetitive stimulation yielded identical responses. Simultaneous recordings from two RHYs combined with alternate electrical stimulation consistently revealed interactions in both directions, the responses in the two RHYs being mostly identical Žexcitation, 15; inhibition, 5., whereas in 5 they were dissimilar. The responses occurred approximately 5 ms after the onset of electrical stimulation
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 1 9 9 - 3
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J. Schenda, L. Vollrathr Brain Research 823 (1999) 231–233
of one cluster affected other clusters. Assuming the clusters consist of electrically active pinealocytes w6x, the structural and functional link could be the long, thin pinealocyte processes equipped with ribbon synapses w11,12x. The time elapsing between stimulation of the first cluster and the response of the second is compatible with conduction velocities of small non-myelinated axons. The involvement of chemical synapses is suggested by the finding that after synaptic blockade the electrically stimulated first cluster was unable to affect the second cluster. Gap junctions w10x do not seem to play a role as halothane had no effects. As the firing of the clusters depends on norepinephrine w7,8x, acetylcholine w9x and Ca2q Žthis study., the clusters appear to be ideally suited to monitor the extracellular concentrations of these substances. The relevance of this is emphasized by the well established fact that norepinephrine plays a key role in regulating melatonin synthesis Žfor review, see Ref. w3x., and that norepinephrine release from intrapineal nerve fibers and melatonin synthesis are inhibited by acetylcholine w1x, the intrapineal synthesis of which undergoes a prominent day - night rhythm w13x. Moreover, acetylcholine depolarizes pinealocytes w4x, activates L-type Ca2q channels w4,15x and triggers L-glutamate exocytosis from pinealocytes which in turn leads to inhibition of melatonin synthesis w14,15x. Thus, several intrapineal modulatory processes exist in which the presently described network of RHY clusters seems eminently suited to participate. Fig. 1. Spikertime-histograms of two simultaneously recorded rhythmical clusters, one of which Ža. was electrically stimulated Ž1 Hz, 0.3 mA, 0.1 ms., revealing interactions between the clusters. It can be seen that the directly stimulated cluster Ža. was excited after a latency of 1.5 ms, whereas the parallel recorded cluster Žb. responded with a clear increase in discharge rate after a latency of 4 ms.
and lasted for 3–10 ms. Interactions were rare between REGs and RHYs and between REGs Ž3 out of 10. ŽFig. 1.. The gap junction blocker halothane Ž1 mM. affected neither the spontaneous activity nor the responses following electrical stimulation ŽRHYs, n s 10; REGs, n s 10.. Synaptic blockade with low-calciumrhigh-magnesium medium reduced the firing rates significantly Ž p - 0.05., without changing the discharge patterns. Synaptic blockade combined with electrical stimulation revealed that the directly stimulated cluster responded, but that this information did not reach the distant cluster ŽFig. 2.. Increasing the calcium concentration from 0.9 mM Žstandard. to 1.5 mM, 2.0 mM and 2.5 mM caused significant Ž p - 0.05. dose-dependent increases Ž n s 16. or decreases Ž n s 9. in firing frequency or had no effect Ž n s 4.. The essence of this study is that one type of electrically active cell cluster in the rat pineal gland, the RHYs, appears to be functionally linked. In RHYs, rhythmic burst activity was highly synchronized and electrical stimulation
Acknowledgements This study was carried out with equipment purchased with funds from the Volkswagen-Stiftung and the HertieStiftung.
References w1x W.J. Drijfhout, C.J. Grol, B.H.C. Westerink, Parasympathetic inhibition of pineal indole metabolism by prejunctional modulation of noradrenaline release, Eur. J. Pharmacol. 308 Ž1996. 117–124. w2x T. Hori, T. Kiyohara, T. Nakashima, M. Shibata, H. Koga, Multimodal responses of preoptic and anterior hypothalamic neurons to thermal and nonthermal homeostatic parameters, Can. J. Physiol. Pharmacol. 65 Ž1987. 1290–1298. w3x H.W. Korf, C. Schomerus, J.H. Stehle, The pineal organ, its hormone melatonin, and the photoneuroendocrine system, Adv. Anat. Embryol. Cell Biol. 146 Ž1998. 1–100, Springer: Berlin. w4x B. Letz, C. Schomerus, E. Maronde, H.W. Korf, C. Korbmacher, Stimulation of a nicotinic ACh receptor causes depolarization and activation of L-type Ca2q channels in rat pinealocytes, J. Physiol. Lond. 499 Ž1997. 329–340. w5x J. Mantz, J. Cordier, C. Giaume, Effect of general anesthetics on intercellular communications mediated by gap junctions between astrocytes in primary culture, Anesthesiology 78 Ž1993. 892–901. w6x S. Reuss, Electrical activity of the mammalian pineal gland, Pineal Res. Rev. 5 Ž1987. 153–189.
J. Schenda, L. Vollrathr Brain Research 823 (1999) 231–233
233
Fig. 2. Spikertime-histograms of two rhythmical clusters demonstrating both synchronized firing and the effects of electrical stimulation with and without synaptic blockade by superfusion of low Ca2qrhigh Mg 2q artificial cerebro-spinal fluid ŽACSF.. It can be seen that direct electrical stimulation Žthick arrow. of one cluster evokes a similar response in the distant cluster Žthin arrow. and that the interactions are bi-directional. Under synaptic blockade, the clusters respond to direct electrical stimulation, but this information is not conveyed to the distant cluster.
w7x J. Schenda, L. Vollrath, Nitric oxide inhibits electrically active units in the rat pineal gland, J. Neural. Transm. 104 Ž1997. 53–58. w8x J. Schenda, L. Vollrath, Demonstration of action potential producing cells in the rat pineal gland in vitro and their regulation by norepinephrine and nitric oxide, J. Comp. Physiol. A 183 Ž1998. 573–581. w9x J. Schenda, L. Vollrath, Modulatory role of acetylcholine in the rat pineal gland, Neurosci. Lett. 254 Ž1998. 13–16. w10x R. Taugner, A. Schiller, E. Rix, Gap junctions between pinealocytes. A freeze-fracture study of pineal glands in rats, Cell Tissue Res. 218 Ž1981. 303–314. w11x L. Vollrath, Synaptic ribbons of a mammalian pineal gland. Circadian changes, Z. Zellforsch. 145 Ž1973. 171–183. w12x L. Vollrath, The Pineal Organ, in: A. Oksche, L. Vollrath ŽEds..,
Handbuch der mikroskopischen Anatomie des Menschen, Vol. VIr7, Springer, Berlin, 1981, pp 1–665. w13x I. Wessler, T. Reinheimer, F. Bittinger, C.J. Kirkpatrick, J. Schenda, L. Vollrath, Day–night rhythm of acetylcholine in the rat pineal gland, Neurosci. Lett. 224 Ž1997. 173–176. w14x H. Yamada, A. Yamamoto, M. Takahashi, H. Michibata, H. Kumon, Y. Moriyama, The L-type Ca2q channel is involved in microvesicle-mediated glutamate exocytosis from rat pinealocytes, J. Pineal Res. 21 Ž1996. 165–174. w15x H. Yamada, A. Yamamoto, S. Yodozawa, S. Kozaki, M. Takahashi, M. Morita, H. Michibata, T. Furuichi, K. Mikoshiba, Y. Moriyama, Microvesicle-mediated exocytosis of glutamate is a novel paracrinelike chemical transduction mechanism and inhibits melatonin secretion in rat pinealocytes, J. Pineal Res. 21 Ž1996. 175–191.
Short communication
An intrinsic neuronal-like network in the rat pineal gland J. Schenda, L. Vollrath
)
Department of Anatomy, Johannes Gutenberg-UniÕersity, Becherweg 13, D-55099, Mainz, Germany Accepted 26 January 1999
Abstract Recent studies have shown that in rat pineal glands kept in vitro action potential-producing cell clusters are demonstrable. To test whether the clusters interact, multiple-unit recordings were carried out simultaneously from different clusters, with or without electrical stimulation. Clusters with rhythmic burst activity exhibit highly synchronized firing and electrical stimulation of one cluster elicits an immediate response in another one, apparently involving synapses but not gap junctions. It is hypothesized that the interacting clusters form a network. As the firing is affected by norepinephrine, acetylcholine and Ca2q, the network may monitor the interstitial concentrations of these substances and spread this information in the gland, to modulate melatonin secretion. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Rat pineal; Electrophysiology; In vitro; Neuron-like network
The mammalian pineal gland contains action-potential producing cells, demonstrable both in vivo Žfor review, see Ref. w6x. and in vitro w7–9x. Recently, it was shown that the electrically active cells of the rat pineal gland are arranged in clusters of three to five cells and are antagonistically affected by norepinephrine and acetylcholine w7–9x. The aim of the present study was to test the hypothesis that the clusters interact to form a functional network. Male Sprague–Dawley rats of 150 to 200 g b.wt. were kept under 12 h light and 12 h dark Žlights on at 0600; room temperature 22 " 28C; water and food ad libitum. for at least 3 weeks. The pineal glands were excised and transferred to a temperature-controlled superfusion chamber w7–9x. Multi-unit recordings were made simultaneously from two clusters lying at least 0.4 mm apart, with or without alternating electrical stimulation through the recording electrodes Žrectangular pulses of 1 Hz, 0.3 mA, 0.1 ms.. To test the effects of different concentrations of Ca2q, CaCl 2 was added to the superfusion medium yielding concentrations of 1.5 mM, 2.0 mM or 2.5 mM. For synaptic blockade Ca2q was substituted by Mg 2q Ž6.5 mM MgCl 2 , w2x.. Gap junctions were blocked with halothane w5x.
)
Corresponding author. Fax: q49-6131-393719
The discharge patterns were analyzed by spectral analysis ŽSPSS on a VAX computer.. Changes in frequency were analyzed by the Mann–Whitney rank sum test for significant differences Ž p - 0.05.. The data of simultaneously recorded cell clusters were analyzed with cross-correlation tests for possible correlation ŽSPSS on a VAX computer.. In rat pineal glands in vitro, regularly firing cell clusters ŽREGs, 64%. and rhythmically bursting clusters ŽRHYs, 36%. exist w7–9x. Simultaneous recordings Ž n s 50. from two RHYs revealed a strong cross-correlation in the occurrence of the bursts Ž) than two standard errors., their peaks appearing within 10–70 ms. No correlation in firing existed between REGs and RHYs Ž n s 50. and REGs and REGs Ž n s 20.. RHYs that were electrically stimulated responded with statistically significant Ž p - 0.05. excitation Ž n s 42. or inhibition Ž n s 8.. REGs were excited Ž n s 12., inhibited Ž n s 1. or unaffected Ž n s 11.. The responses occurred after 1–2 ms and lasted for 3–10 ms. Single and repetitive stimulation yielded identical responses. Simultaneous recordings from two RHYs combined with alternate electrical stimulation consistently revealed interactions in both directions, the responses in the two RHYs being mostly identical Žexcitation, 15; inhibition, 5., whereas in 5 they were dissimilar. The responses occurred approximately 5 ms after the onset of electrical stimulation
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 1 9 9 - 3
232
J. Schenda, L. Vollrathr Brain Research 823 (1999) 231–233
of one cluster affected other clusters. Assuming the clusters consist of electrically active pinealocytes w6x, the structural and functional link could be the long, thin pinealocyte processes equipped with ribbon synapses w11,12x. The time elapsing between stimulation of the first cluster and the response of the second is compatible with conduction velocities of small non-myelinated axons. The involvement of chemical synapses is suggested by the finding that after synaptic blockade the electrically stimulated first cluster was unable to affect the second cluster. Gap junctions w10x do not seem to play a role as halothane had no effects. As the firing of the clusters depends on norepinephrine w7,8x, acetylcholine w9x and Ca2q Žthis study., the clusters appear to be ideally suited to monitor the extracellular concentrations of these substances. The relevance of this is emphasized by the well established fact that norepinephrine plays a key role in regulating melatonin synthesis Žfor review, see Ref. w3x., and that norepinephrine release from intrapineal nerve fibers and melatonin synthesis are inhibited by acetylcholine w1x, the intrapineal synthesis of which undergoes a prominent day - night rhythm w13x. Moreover, acetylcholine depolarizes pinealocytes w4x, activates L-type Ca2q channels w4,15x and triggers L-glutamate exocytosis from pinealocytes which in turn leads to inhibition of melatonin synthesis w14,15x. Thus, several intrapineal modulatory processes exist in which the presently described network of RHY clusters seems eminently suited to participate. Fig. 1. Spikertime-histograms of two simultaneously recorded rhythmical clusters, one of which Ža. was electrically stimulated Ž1 Hz, 0.3 mA, 0.1 ms., revealing interactions between the clusters. It can be seen that the directly stimulated cluster Ža. was excited after a latency of 1.5 ms, whereas the parallel recorded cluster Žb. responded with a clear increase in discharge rate after a latency of 4 ms.
and lasted for 3–10 ms. Interactions were rare between REGs and RHYs and between REGs Ž3 out of 10. ŽFig. 1.. The gap junction blocker halothane Ž1 mM. affected neither the spontaneous activity nor the responses following electrical stimulation ŽRHYs, n s 10; REGs, n s 10.. Synaptic blockade with low-calciumrhigh-magnesium medium reduced the firing rates significantly Ž p - 0.05., without changing the discharge patterns. Synaptic blockade combined with electrical stimulation revealed that the directly stimulated cluster responded, but that this information did not reach the distant cluster ŽFig. 2.. Increasing the calcium concentration from 0.9 mM Žstandard. to 1.5 mM, 2.0 mM and 2.5 mM caused significant Ž p - 0.05. dose-dependent increases Ž n s 16. or decreases Ž n s 9. in firing frequency or had no effect Ž n s 4.. The essence of this study is that one type of electrically active cell cluster in the rat pineal gland, the RHYs, appears to be functionally linked. In RHYs, rhythmic burst activity was highly synchronized and electrical stimulation
Acknowledgements This study was carried out with equipment purchased with funds from the Volkswagen-Stiftung and the HertieStiftung.
References w1x W.J. Drijfhout, C.J. Grol, B.H.C. Westerink, Parasympathetic inhibition of pineal indole metabolism by prejunctional modulation of noradrenaline release, Eur. J. Pharmacol. 308 Ž1996. 117–124. w2x T. Hori, T. Kiyohara, T. Nakashima, M. Shibata, H. Koga, Multimodal responses of preoptic and anterior hypothalamic neurons to thermal and nonthermal homeostatic parameters, Can. J. Physiol. Pharmacol. 65 Ž1987. 1290–1298. w3x H.W. Korf, C. Schomerus, J.H. Stehle, The pineal organ, its hormone melatonin, and the photoneuroendocrine system, Adv. Anat. Embryol. Cell Biol. 146 Ž1998. 1–100, Springer: Berlin. w4x B. Letz, C. Schomerus, E. Maronde, H.W. Korf, C. Korbmacher, Stimulation of a nicotinic ACh receptor causes depolarization and activation of L-type Ca2q channels in rat pinealocytes, J. Physiol. Lond. 499 Ž1997. 329–340. w5x J. Mantz, J. Cordier, C. Giaume, Effect of general anesthetics on intercellular communications mediated by gap junctions between astrocytes in primary culture, Anesthesiology 78 Ž1993. 892–901. w6x S. Reuss, Electrical activity of the mammalian pineal gland, Pineal Res. Rev. 5 Ž1987. 153–189.
J. Schenda, L. Vollrathr Brain Research 823 (1999) 231–233
233
Fig. 2. Spikertime-histograms of two rhythmical clusters demonstrating both synchronized firing and the effects of electrical stimulation with and without synaptic blockade by superfusion of low Ca2qrhigh Mg 2q artificial cerebro-spinal fluid ŽACSF.. It can be seen that direct electrical stimulation Žthick arrow. of one cluster evokes a similar response in the distant cluster Žthin arrow. and that the interactions are bi-directional. Under synaptic blockade, the clusters respond to direct electrical stimulation, but this information is not conveyed to the distant cluster.
w7x J. Schenda, L. Vollrath, Nitric oxide inhibits electrically active units in the rat pineal gland, J. Neural. Transm. 104 Ž1997. 53–58. w8x J. Schenda, L. Vollrath, Demonstration of action potential producing cells in the rat pineal gland in vitro and their regulation by norepinephrine and nitric oxide, J. Comp. Physiol. A 183 Ž1998. 573–581. w9x J. Schenda, L. Vollrath, Modulatory role of acetylcholine in the rat pineal gland, Neurosci. Lett. 254 Ž1998. 13–16. w10x R. Taugner, A. Schiller, E. Rix, Gap junctions between pinealocytes. A freeze-fracture study of pineal glands in rats, Cell Tissue Res. 218 Ž1981. 303–314. w11x L. Vollrath, Synaptic ribbons of a mammalian pineal gland. Circadian changes, Z. Zellforsch. 145 Ž1973. 171–183. w12x L. Vollrath, The Pineal Organ, in: A. Oksche, L. Vollrath ŽEds..,
Handbuch der mikroskopischen Anatomie des Menschen, Vol. VIr7, Springer, Berlin, 1981, pp 1–665. w13x I. Wessler, T. Reinheimer, F. Bittinger, C.J. Kirkpatrick, J. Schenda, L. Vollrath, Day–night rhythm of acetylcholine in the rat pineal gland, Neurosci. Lett. 224 Ž1997. 173–176. w14x H. Yamada, A. Yamamoto, M. Takahashi, H. Michibata, H. Kumon, Y. Moriyama, The L-type Ca2q channel is involved in microvesicle-mediated glutamate exocytosis from rat pinealocytes, J. Pineal Res. 21 Ž1996. 165–174. w15x H. Yamada, A. Yamamoto, S. Yodozawa, S. Kozaki, M. Takahashi, M. Morita, H. Michibata, T. Furuichi, K. Mikoshiba, Y. Moriyama, Microvesicle-mediated exocytosis of glutamate is a novel paracrinelike chemical transduction mechanism and inhibits melatonin secretion in rat pinealocytes, J. Pineal Res. 21 Ž1996. 175–191.