Neuroscience Letters, 133 (1991) 125-128
125
© 1991 ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/91/$03.50 NSL 08211
Acetylcholine and muscarinic agonists increase synaptic ribbon numbers in the rat pineal Braj B.P. G u p t a * , R a i n e r Spessert a n d L u t z V o l l r a t h Department of Anatomy, Johannes Gutenberg-University, Mainz ( F.R.G.)
(Received21 June 1991;Revisedversion received26 August 1991;Accepted26 August 1991) Key words: Pineal; Synapticribbon; Muscarinicagonist; Muscarinicantagonist; N-Acetyltransferase
Mammalian pinealocytespossess synaptic ribbons (SR) which are commonly present in photoreceptor cells at synaptic junctions. Pineal SR numbers undergo a diurnal rhythm parallel to that of pineal N-acetyltransferase(NAT) activity and melatonin levels. Recent findings suggest that SR numbers, unlike NAT activity and melatonin synthesis and release, do not seem to be regulated by adrenergic mechanisms or neuropeptides in adult rats. Sincethe pineal gland also receivescholinergicnerve fibres, we have investigatedin vitro effectsof acetylcholine(ACh) and carbamyl-flmethylcholine(CBMC; a specificmuscarinic agonist) in the presence and absence of pirenzipine (a specificinhibitor of muscarinic M~ receptors). ACh and CBMC increased SR numbers significantly.Pirenzipineinhibited the CBMC-inducedincrease in SR numbers. On the basis of these findings, it is suggested that cholinergicagonists increase pineal SR numbers by acting through muscarinicM~ receptors.Hence muscarinicmechanisms may have a functional role in pineal physiology.
The mammalian pineal gland possesses synaptic ribbons (SR) which are a common feature in afferent synapses of photoreceptor and other sensory cells. Pineal S R undergo a pronounced diurnal rhythm (with high numbers during the dark-phase and low numbers during the light-phase) [23] similar to that of pineal melatonin synthesis [3]. The pineal gland is heavily innervated by sympathetic nerve fibres [40] which play a decisive role in regulating melatonin synthesis. Besides adrenergic mechanisms, also neuropeptides are involved [2, 4, 6]. In addition to the sympathetic fibres, the pineal gland receives a central nervous projection containing peptidergic and cholinergic nerve fibres [7, 16, 17, 22, 33]. As pineal SR regulation is still poorly understood, in the present study we have investigated the possible involvement of cholinergic mechanisms in the regulation o f SR numbers in the rat pineal. Male Sprague-Dawley rats (220-260 g b.wt.) were obtained from a commercial supplier and maintained for two weeks prior to the experimentation under standard
*Presentaddress: Department of Zoology,North-Eastern Hill University, Shillong-793014,India. Correspondence: R. Spessert, Department of Anatomy, Johannes Gutenberg-University,Saarstr. 19-21, D-6500 Mainz, F.R.G.
laboratory conditions (LD 12/12 h; lights on at 05.00 h; temperature 22 + 2°C) with food and water ad ~ibitum. Rats were decapitated between 10.00 h and 10.30 h. Pineal glands were quickly removed under sterile conditions and placed in culture dishes containing 1.2 ml BGjb medium Fitton Jackson modified (Gibco) with bovine serum albumin Fraction V (1 mg/~nl), ascorbic acid (0.1 mg/ml), CaCO3 (0.125 mg/ml), glutamate (2 mM) and penicillin (100 U)/streptomycin (0.1 mg/ml). For the intrapineal nerve fibres to degenerate, the pineal glands where pre-incubated for 48 h at 37°C under 95% 02 and 5% CO2. Culture medium was changed daily. The pineal glands were incubated further for 6 h with or without the drugs, the latter serving as controls. Uncultured pineal glands from rats killed at 11.00 h and 23.00 h served as additional controls. At the end of the incubations, pineal glands were divided into two halves. One half was fixed and processed for electron microscopic study of SR numbers. The second half o f the pineal was used for the measurement of N-acetyltransferase (NAT) activity as described by Seidel et al. [37]. The pineal glands were fixed by immersion and processed for electron microscopy as described earlier [37]. SR were counted under an electron microscope (EM 10, Zeiss) at a magnification of 20,000 ®. Tissue covering 5 randomly selected grid apertures of randomly selected sections were scanned for SR. Data were analysed statis-
126
L
f
1
~
t/1 20
~
n=7
oi,01
n=7
day night
culture 10-.M control ACh
n=13
IO-iM IO-~M IO'~M IO'~MCBMC I( - CBMC W +IO-WMPIR
Fig. 1. In vitro effectsof acetylcholine(ACh) and carbamyl-fl-methylcholine (CBMC) on synaptic ribbon (SR) numbers in the rat pineal gland. Values are means + S.E.M. *P
125
© 1991 ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/91/$03.50 NSL 08211
Acetylcholine and muscarinic agonists increase synaptic ribbon numbers in the rat pineal Braj B.P. G u p t a * , R a i n e r Spessert a n d L u t z V o l l r a t h Department of Anatomy, Johannes Gutenberg-University, Mainz ( F.R.G.)
(Received21 June 1991;Revisedversion received26 August 1991;Accepted26 August 1991) Key words: Pineal; Synapticribbon; Muscarinicagonist; Muscarinicantagonist; N-Acetyltransferase
Mammalian pinealocytespossess synaptic ribbons (SR) which are commonly present in photoreceptor cells at synaptic junctions. Pineal SR numbers undergo a diurnal rhythm parallel to that of pineal N-acetyltransferase(NAT) activity and melatonin levels. Recent findings suggest that SR numbers, unlike NAT activity and melatonin synthesis and release, do not seem to be regulated by adrenergic mechanisms or neuropeptides in adult rats. Sincethe pineal gland also receivescholinergicnerve fibres, we have investigatedin vitro effectsof acetylcholine(ACh) and carbamyl-flmethylcholine(CBMC; a specificmuscarinic agonist) in the presence and absence of pirenzipine (a specificinhibitor of muscarinic M~ receptors). ACh and CBMC increased SR numbers significantly.Pirenzipineinhibited the CBMC-inducedincrease in SR numbers. On the basis of these findings, it is suggested that cholinergicagonists increase pineal SR numbers by acting through muscarinicM~ receptors.Hence muscarinicmechanisms may have a functional role in pineal physiology.
The mammalian pineal gland possesses synaptic ribbons (SR) which are a common feature in afferent synapses of photoreceptor and other sensory cells. Pineal S R undergo a pronounced diurnal rhythm (with high numbers during the dark-phase and low numbers during the light-phase) [23] similar to that of pineal melatonin synthesis [3]. The pineal gland is heavily innervated by sympathetic nerve fibres [40] which play a decisive role in regulating melatonin synthesis. Besides adrenergic mechanisms, also neuropeptides are involved [2, 4, 6]. In addition to the sympathetic fibres, the pineal gland receives a central nervous projection containing peptidergic and cholinergic nerve fibres [7, 16, 17, 22, 33]. As pineal SR regulation is still poorly understood, in the present study we have investigated the possible involvement of cholinergic mechanisms in the regulation o f SR numbers in the rat pineal. Male Sprague-Dawley rats (220-260 g b.wt.) were obtained from a commercial supplier and maintained for two weeks prior to the experimentation under standard
*Presentaddress: Department of Zoology,North-Eastern Hill University, Shillong-793014,India. Correspondence: R. Spessert, Department of Anatomy, Johannes Gutenberg-University,Saarstr. 19-21, D-6500 Mainz, F.R.G.
laboratory conditions (LD 12/12 h; lights on at 05.00 h; temperature 22 + 2°C) with food and water ad ~ibitum. Rats were decapitated between 10.00 h and 10.30 h. Pineal glands were quickly removed under sterile conditions and placed in culture dishes containing 1.2 ml BGjb medium Fitton Jackson modified (Gibco) with bovine serum albumin Fraction V (1 mg/~nl), ascorbic acid (0.1 mg/ml), CaCO3 (0.125 mg/ml), glutamate (2 mM) and penicillin (100 U)/streptomycin (0.1 mg/ml). For the intrapineal nerve fibres to degenerate, the pineal glands where pre-incubated for 48 h at 37°C under 95% 02 and 5% CO2. Culture medium was changed daily. The pineal glands were incubated further for 6 h with or without the drugs, the latter serving as controls. Uncultured pineal glands from rats killed at 11.00 h and 23.00 h served as additional controls. At the end of the incubations, pineal glands were divided into two halves. One half was fixed and processed for electron microscopic study of SR numbers. The second half o f the pineal was used for the measurement of N-acetyltransferase (NAT) activity as described by Seidel et al. [37]. The pineal glands were fixed by immersion and processed for electron microscopy as described earlier [37]. SR were counted under an electron microscope (EM 10, Zeiss) at a magnification of 20,000 ®. Tissue covering 5 randomly selected grid apertures of randomly selected sections were scanned for SR. Data were analysed statis-
126
L
f
1
~
t/1 20
~
n=7
oi,01
n=7
day night
culture 10-.M control ACh
n=13
IO-iM IO-~M IO'~M IO'~MCBMC I( - CBMC W +IO-WMPIR
Fig. 1. In vitro effectsof acetylcholine(ACh) and carbamyl-fl-methylcholine (CBMC) on synaptic ribbon (SR) numbers in the rat pineal gland. Values are means + S.E.M. *P
