- Email: [email protected]
Electrophysiological responses of hamster suprachiasmatic neurones to neuropeptide Y in the hypothalamic slice preparation
Neuroscience Letters, 80 (1987) 173 179 Elsevier Scientific Publishers Ireland Ltd.
173
NSL 04809
Electrophysiological responses of hamster suprachiasmatic neurones to neuropeptide Y in the hypothalamic slice preparation Robert M a s o n 1, Mary E. Harrington 2 and Benjamin Rusak 2 IDepartment (?f'Physiology & Pharmacology, Medical School, Queen's Medical Centre, Nottingham ( U. K. ) and 2Department of Psychology. Dalhousie University. Halifax, N.S. (Canada) (Received 16 April 1987; Revised version received 20 May 1987; Accepted 27 May 1987)
Key words: Circadian rhythm; Suprachiasmatic nucleus; Neuropeptide Y; Brain slice; Single unit activity; Visual entrainment The rate and pattern of neuronal discharge in the hamster suprachiasmatic nucleus (SCN) was studied using an in vitro hypothalamic slice preparation. The firing rate of hamster SCN neurones ( n - 183 cells) exhibited a circadian variation similar to that reported in the rat. SCN neurones tested for responses to pressure ejection of neuropeptide Y (NPY) (n=49) were either tonically excited (65%) or unresponsive (35%). There was a tendency for more NPY-responsive cells to be recorded during the light phase of the circadian light,lark cycle.
The suprachiasmatic nuclei (SCN) of the hypothalamus are central structures for the regulation of circadian rhythms [22]. The SCN receive visual information by the retino-hypothalamic tract and indirectly via the retino-geniculo-suprachiasmatic pathway (RGSP). The geniculo-suprachiasmatic projection originates in the intergeniculate leaflet (IGL) and adjacent portions of the lateral geniculate body: these regions receive a direct retinal projection [15]. The cells forming this projection are characterised by their immunoreactivity to neuropeptide Y (NPY) [3, 15]. The RGSP may play a role in the photic entrainment of circadian rhythms. Lesions at several points along the RGSP alter various aspects of photic responsiveness of hamster activity rhythms [13, 20, 21]. Electrical stimulation of the IGL [18] or microinjection of NPY into the SCN [1] produce phase shifts in free-running locomotor activity rhythms. The phase shifts obtained resemble the phase response profile induced by dark pulse presentation [2]. The present study was undertaken to examine the electrophysiological effects of NPY on hamster SCN neurones recorded in vitro. A preliminary abstract of this work has appeared [14]. Correspondence: R. Mason, Dept. of Physiology & Pharmacology, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K. 0304-3940/87'$ 03 50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
174
Male golden hamsters were exposed to daily cycles of 14 h of light alternating with 10 h of dark (LD 14:10). For one group of animals (LD group) lights were on from 05.00 to 19.00 h (Atlantic Daylight Time, ADT). For a second group of hamsters lights were on from 20.00 to 10.00 h ADT (DL group). Illumination intensity measured in the centre of the cage was 30-100 lux during the light phase and <0.1 lux under dim red light during the dark phase. To allow comparisons and pooling of the data from LD and DL groups, circadian time (CT) 00.00 h refers to the time of lights on. At random times during different phases of the circadian cycle animals were anaesthetised with halothane and decapitated. Coronal hypothalamic slices (400-500 /zm) containing the SCN were cut and transferred to a slice chamber. Animals obtained during the dark phase were prepared under dim red light. Slices were maintained at 35+0.5°C at the gas/fluid interface between warm humidified Or-CO: (95%-5%) and artificial cerebrospinal fluid (in mM: NaC1 124, KC1 3.3, KH2PO4 1.2, CaC12 2.5, MgSO4 1, NaHCO3 2.5, glucose 10) at pH 7.4. Extracellular recordings were made with single or multi-barrel micropipettes, the recording barrel contained 2% Pontamine sky blue in 1 M NaCI. In some experiments recordings were made via the NPY-barrel using a micropipette holder (Clark Electromedical Instruments, UK: type EH-2MR) which allowed simultaneous recording and pressure ejection of NPY. The effects of pressure-ejected NPY (Peninsula Labs.; 50-200/tM NPY dissolved in 0.9% NaCI with 1% bovine serum albumin (BSA), aliquoted and kept frozen until use) were observed on the discharge activity of SCN neurones. Spike amplitude and shape were monitored for any pressure-related effects; recordings containing gross artifacts (i.e. showing changes > 10%) were omitted from further analysis. Following recording sessions the brain slices were fixed in formalin and sectioned at 50 /tm, stained with Cresyl violet and examined for deposits of Pontamine blue dye marks to identify SCN recording sites. Neurones recorded in the hamster SCN exhibited regular (n = 53/183, 29%), irregular (n =95/183, 52%) or bursting (n = 35/183, 19%) firing patterns in spontaneous discharge activity. Similar spontaneous discharge patterns have been described in the rat SCN slice preparation [9, 10, 25, 26-28]. The discharge rate ranged between 0.2 and 19.0 Hz. The regular and irregular discharge types in the SCN showed a circadian variation in their firing rate which was higher (5.9+ 1.0 Hz, mean+S.E.M., n=48 cells) during the period CT 06.00-12.00 h, in the late projected light phase, and was minimal (2.9___0.9 Hz, n---24 cells) between CT 15.00 and 21.00 h, in the early projected dark phase. The peak firing rate occurred in the middle of the projected light phase (CT 07.00-10,00 h: 7.3_+ 1.3 Hz, n=31 cells). There was no evidence of a circadian variation in discharge activity in the bursting type neurones. The rat SCN in vitro is also reported to exhibit a circadian variation in firing rate which peaks during the projected light phase [8, 9, 24]. The majority of SCN neurones tested (32/49, 65%) showed an increase in discharge rate following pressure-ejection of NPY. The remaining neurones tested were unresponsive to NPY (n = 17, 35%). The excitatory responses were characteristically sustained throughout the duration of the ejection (Fig. 1) and the elevated discharge was often maintained (for up to 60 min) beyond the termination of the NPY ejection
175
SCNvL celt 31 13 (CT02 45hr) ~ IH] ] I~I IIItllHIlllnl|JiHIIIlflll~lgllll~llnsltdllmIHI!1fli LI]III
A
I]]IIIIIIIIIIIImlIIIHtHWIIIIHIIIHIINIIII[II]Ilil J
~____
NPY (4psi 100~M) t__l
B NPY (3psi: 20min)
20
Ssec // //i
NPY sT
~1S LJ~ ~10
L
I 2rain
G Hamster
LD14:10 (tiqhts-on I T 0 0 0 0
1400
Fig. 1. A: NPY-evoked discharge activity of a regularly discharging neurone recorded in the ventrolateral division of the SCN. B: integrated firing rate histogram for the same SCN neurone accumulated over successive 5-s epochs illustrating the prolonged elevation of discharge activity induced by pressure ejection of NPY (3 psi).
period. Control ejections of the BSA vehicle alone were without effect on all cells tested (n = 21). Some neurones (n = 6) originally showing an irregular discharge pattern exhibited a regular pattern, without any appreciable increase in spike rate, following NPY ejection. The SCN neurones exhibiting a bursting discharge pattern (n = 5) were unresponsive to NPY. NPY-activated neurones were found throughout the rostrocaudal extent of the SCN and there was no evidence of regional differences within the SCN with respect to NPY sensitivity. Of the 23 SCN cells tested during the projected light phase of the circadian cycle (CT 00.00-14.00 h), all were responsive to NPY. Only 9 o f the 26 SCN neurones were recorded during the projected dark period (CT 14.00-24.00 h) responded to NPY ejection. In contrast, extrasuprachiasmatic hypothalamic neurones remained responsive to NPY when recorded during the projected dark phase (n = 27 cells; 25/27 excited and 2 cells inhibited by NPY) or during the projected light phase (n = 32 cells; 31/32 excited and 1 cell inhibited by NPY). Nine SCN neurones were recorded for a sufficient duration (2 8 h) to enable an examination of the temporal effects of repeated NPY ejections. SCN neurones (n = 5) recorded during the transition between the projected light to dark phase (CT 13.00~ 16.40 h) showed a loss of responsiveness to NPY (Fig. 2). Whereas SCN neurones (n = 4) recorded during the late dark phase exhibited an emergence of responsiveness to NPY (Fig. 3). The loss of NPY excitation could be attributable to desensitisation of the cell to NPY, although it is suggestive that loss of NPY responsiveness was generally found at the transition between the projected light and dark phases. The
176
SENvm cell 360/,
(OHamsfer
lights-onCTOO.OO-1/..OOhr)
40 30 20 CL
E
!0 0
I
m
t
I CT 15.30hr
20
L
~
_
i
NPY (15psi)
10 0 20
i
J T
'
' NPY
ET 16.30hr
10 0 I
2rnin
J '
' NPY
Fig. 2. Integrated firing rate record for a neurone in the ventromedial SCN (cell 36.04) recorded during the projected l i g h t . l a r k phase transition period (from CT 13.05 to 16.40 h). NPY was repeatedly ejected (1.5 psi). Initially the neurone exhibited an excitatory response to NPY whereas later during the recording no NPY-induced response was obtained. Note the reduction in discharge rate found at the projected lightdark transition of the circadian cycle.
emergence of NPY responsiveness cannot be explained as the recovery of a previously blocked electrode since NPY-responsive cells were recorded outside the boundaries of the SCN (paraventricular nucleus area) with the same electrode earlier in the recording session. It appears that SCN neurones show some circadian variation in their response to NPY; previous studies have reported circadian changes in responsiveness of SCN neurones to iontophoresed serotonin [17] and of lateral hypothalamic neurones to systemically administered agents [23]. The present study has demonstrated that the SCN, a target of NPY fibres originating from the IGL [3, 15], contains neurones responsive to exogenously applied NPY. The observation that NPY responsive neurones were recorded throughout the SCN is consistent with the widespread distribution of NPY immunoreactivity found in the SCN [3, 15, 30]. Immunocytochemical studies at the electron microscopic level have demonstrated the localisation of NPY in axodendritic asymmetrical synaptic contacts within the SCN of the rat [4]; interestingly asymmetrical synapses have been associated with excitatory contacts [29]. SCN neurones appear to be more responsive to NPY applied during the projected light phase than during the projected dark phase. This observation may be related to the finding that NPY microinjection and RGSP stimulation generate the largest phase shifts during the subjective day [1, 19].
177
SCNvt cell 36-07 (G.Hamsfer
[ighfs-on CTOO.OO-14.0Ohr)
lET 19.50hr 3O cf]
\20 QJ vl
-5 I0 E
0
I,
i
t
2min
30f
J NPY (20psi)
l C T 20.50hr
20
10
c
O
20 10
O
f
J NPY
.
'
c
i Np Y
.
.
.
.
,/~0/
,
' NPY
Fig. 3. Integrated firing rate records for a neurone in the ventrolateral SCN (cell 36.07) recorded during the middle of the projected dark phase of the circadian cycle. Repeated ejections of NPY (2 psi) were applied. In the initial part of the recording (CT 19.30-21.00 h) the neurone was unresponsive to NPY. After approximately 120 rain (CT 21.10 h) NPY ejection evoked an increase in discharge. This cell was recorded following an electrode penetration in the paraventricular area where two cells were recorded (cells 36.05/36.06, not shown) which responded to NPY. The m a j o r i t y o f I G L n e u r o n e s in the h a m s t e r t o n i c a l l y increase their firing rate while the eye is i l l u m i n a t e d [12]. W e h y p o t h e s i s e t h a t increased firing o f I G L n e u r o n e s in response to o c u l a r i l l u m i n a t i o n releases N P Y in the S C N a n d affects the responses o f S C N n e u r o n e s to p h o t i c stimuli [18]. N P Y is r e p o r t e d to be c o l o c a l i s e d in n o r a d r e n e r g i c nerve terminals [6], a n d n o r a d r e n a l i n e - i n d u c e d e x c i t a t o r y responses o f s u p r a o p t i c nucleus n e u r o n e s have been r e p o r t e d to be a t t e n u a t e d by N P Y [5]. W h i l e b o t h N P Y a n d c a t e c h o l a m i n e s are f o u n d in the S C N , c o l o c a l i s a t i o n has n o t been r e p o r t e d in the S C N as yet [6, 30]. It m a y be n o t e w o r t h y t h a t N P Y p r e f e r e n t i a l l y excited S C N n e u r o n e s which were actively d i s c h a r g i n g (i.e. the r e g u l a r a n d i r r e g u l a r d i s c h a r g e p a t t e r n types), suggesting t h a t the effects o f N P Y m a y be d e p e n d e n t on the level o f excitability o f the target neurone. This level o f excitability m a y be the result o f i n p u t s f r o m afferents a n d local i n t e r n e u r o n s o r a f u n c t i o n o f the p o s t s y n a p t i c t a r g e t n e u r o n e itself. T h e m e c h a n i s m
178 o f action o f N P Y is u n k n o w n , a l t h o u g h cyclic nucleotides m a y be involved as N P Y is reported to i n h i b i t cyclic a d e n o s i n e m o n o p h o s p h a t e ( c A M P ) a c c u m u l a t i o n [7, 1 i], while c A M P suppresses the discharge activity of S C N n e u r o n e s [16]; thus it is conceivable that N P Y m a y e n h a n c e S C N firing via i n h i b i t i n g the c A M P system. N P Y i n d u c e d responses are also d e p e n d e n t o n extracellular calcium [7] a n d d u r i n g the present study N P Y was often observed to shift the firing o f a n S C N n e u r o n e from an irregular to a regular discharge pattern; the opposite shift has been s h o w n following r e d u c t i o n of extracellular calcium [26, 28] ( u n p u b l i s h e d observations). Studies of possible c o t r a n s m i t t e r i n v o l v e m e n t in the responses o f S C N n e u r o n e s to N P Y a n d whether the N P Y - i n d u c e d excitation o f S C N n e u r o n e s is m e d i a t e d pre- or p o s t s y n a p tically are c u r r e n t l y in progress. C o n d u c t o f this research was m a d e possible by a Scientific Exchange G r a n t from the R o y a l Society ( U K ) a n d N S E R C ( C a n a d a ) , a n d was s u p p o r t e d by G r a n t s A0305 ( N S E R C ) , M A - 8 9 2 9 ( M R C o f C a n a d a ) , a n d the D a l h o u s i e Research Developm e n t F u n d in the Sciences. W e are grateful to Patricia D i c k s o n a n d T i m D e l a n e y for their technical assistance.
1 Albers, H.E. and Ferris, C.F., Neuropeptide Y: role in light-dark cycle entrainment of hamster circadian rhythms, Neurosci. Lett., 50 (1985) 163-168. 2 Boulos, Z. and Rusak, B., Circadian phase response curves for dark pulses in the hamster, J. Comp. Physiol., A146 (1982) 411-417 3 Card, J.P. and Moore, R.Y., Ventral lateral geniculate efferents to the rat suprachiasmatic nucleus exhibit avian pancreatic polypeptide-likeimmunoreactivity, J. Comp. Neurol., 206 (1982) 390-396. 4 Card, J.P. and Moore, R.Y., Immunocytochemical localization of neuropeptide Y in the rat suprachiasmatic nucleus, Soc. Neurosci. Abstr., 11 (1985) 1139. 5 Day, T.A., Jhamandas, J.H. and Renaud, L.P., Comparison between the actions of avian pancreatic polypeptide, neuropeptide Y and norepinephrine on the excitability of rat supraoptic vassopressin neurons, Neurosci. Lett., 62 (1985) 181-185. 6 Everitt, B.J., H6kfelt, T., Terenius, L., Tatemoto, K., Mutt, V. and Goldstein, M., Differential coexistence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat, Neuroscience, 11 (1984) 443-462. 7 Fredholm, B.B., Jansen, I. and Edvinsson, L., Neuropeptide Y is a potent inhibitor of cyclic AMP accumulation in feline cerebral blood vessels, Acta Physiol., Seand., 124 (1985) 467-469. 8 Green, D.J. and Gillette, R., Circadian rhythm of firing rate recorded from singlecells in the rat suprachiasmatic brain slice, Brain Res., 245 (1982) 198-200. 9 Groos, G.A. and Hendriks, J., Regularly firing neurones in the rat suprachiasmatic nucleus, Experientia, 35 (1979) 1597-1598. 10 Groos, G.A. and Hendriks, J., Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro, Neurosci. Lett., 34 (1982) 283-288. 11 H/irfstrand, A., Fredholm, B. and Fuxe, K., Inhibitory effects of neuropeptide Y on cyclic AMP accumulation in slices of the nucleus tractus solitaris region of the rat, Neurosci. Lett., 76 (1987) 185-190. 12 Harrington, M.E. and Rusak, B., Visual responses of neurones in the intergeniculate leaflet and ventral lateral geniculate nucleus of the hamster, Soc. Neurosci. Abstr., 12 (t986) 1036. 13 Harrington, M.E. and Rusak, B., Lesions of the thalamic intergeniculate leaflet alter circadian rhythms, J. Biol. Rhythms, I (1986) 309-325. 14 Harrington, M.E., Mason, R. and Rusak, B., The effects of neuropeptide-Y (NPY) on hamster suprachiasmatic neurones, J. Physiol. (London), 386 (1987) 91P.
179 15 Harrington, M.E., Nance, D.W. and Rusak, B., Neuropeptide Y immunoreactivity in the hamster geniculo-suprachiasmatic tract, Brain Res. Bull., 15 (1985) 465~,72 16 Liou, S.Y., Shibata, S., Shiratsuchi, A. and Ueki, S., Effects of dibutryl cyclic adenosine monophosphate and dibutryl guanosine monophosphate on neuron activity of suprachiasmatic nucleus in rat hypothalamic slice preparation, Neurosci. Lett., 67 (1986) 339-343. 17 Mason, R., Circadian variation in sensitivity of suprachiasmatic and lateral geniculate neurones to 5hydroxytryptamine in the rat, J. Physiol. (London), 377 (1986) I 13. 18 Meijer, J.H., Groos, G.A. and Rusak, B., Luminance coding in a circadian pacemaker: the suprachiasmatte nucleus of the rat and the hamster, Brain Res., 382 (1986) 109-118. 19 Meijer, J.H., Rusak, B. and Harrington, M.E., Geniculate stimulation phase shifts hamster circadian rhythms, Soc. Neurosci. Abstr., 10 (1984) 502. 20 Pickard, G.E., Ralph, M.R. and Menaker, M., The intergeniculate leaflet partially mediates the effects of light on circadian rhythms, J. Biol. Rhythms, in press. 21 Rusak, B. and Boulos, Z., Pathways for photic entrainment of mammalian circadian rhythms~ Photochem. Photobiol., 34 (1981)267 273. 22 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449 526. 23 Schmitt, M., Circadian rhythmicity in responses of cells in the lateral hypothalamus, Am. J. Physiol., 225 (1973) 1096 1101. 24 Shibata, S., Oomura, Y., Kita, H. and Hattori, K., Circadian rhythmic changes of neuronal activity in the suprachiasmatic nucleus of the rat hypothalamic slice, Brain Res., 247 (1982) 154-158. 25 Shibata, S., Liou, S.Y., Ueki, S. and Oomura, Y., Influence of environmental light dark cycle and enucleation on activity of suprachiasmatic neurones in slice preparations, Brain Res., 302 (1984) 75 8 I. 26 Shibata, S., Shiratsuchk A., Liou, S,Y. and Ueki, S., The role of calcium ions in circadian rhythm of suprachiasmatic nucleus neuron activity in rat hypothalamic slices, Neurosci. Lett., 52 (1984) 181 184. 27 Shibata, S., Oomura, Y., Liou, S.Y. and Ueki, S., Electrophysiological studies of the development of suprachiasmatic neuronal activity in hypothalamic slice preparations, Dev. Brain Res., 13 (1984) 29 35. 28 Thompson, A.M., Slow, regular discharge in suprachiasmatic neurones is calcium dependent, in slices of rat brain, Neuroscience, 13 (1984) 761 767. 29 Uchizono, K., Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat, Nature (London) 207 (1965) 642~43. 30 Van den Pol, A.N. and Tsujimoto, K.L., Neurotransmitters of the hypothalamic suprachiasmatic nucleus: immunocytochemical analysis of 25 neuronal antigens, Neuroscience, 15 (1985) 1049 1086.
173
NSL 04809
Electrophysiological responses of hamster suprachiasmatic neurones to neuropeptide Y in the hypothalamic slice preparation Robert M a s o n 1, Mary E. Harrington 2 and Benjamin Rusak 2 IDepartment (?f'Physiology & Pharmacology, Medical School, Queen's Medical Centre, Nottingham ( U. K. ) and 2Department of Psychology. Dalhousie University. Halifax, N.S. (Canada) (Received 16 April 1987; Revised version received 20 May 1987; Accepted 27 May 1987)
Key words: Circadian rhythm; Suprachiasmatic nucleus; Neuropeptide Y; Brain slice; Single unit activity; Visual entrainment The rate and pattern of neuronal discharge in the hamster suprachiasmatic nucleus (SCN) was studied using an in vitro hypothalamic slice preparation. The firing rate of hamster SCN neurones ( n - 183 cells) exhibited a circadian variation similar to that reported in the rat. SCN neurones tested for responses to pressure ejection of neuropeptide Y (NPY) (n=49) were either tonically excited (65%) or unresponsive (35%). There was a tendency for more NPY-responsive cells to be recorded during the light phase of the circadian light,lark cycle.
The suprachiasmatic nuclei (SCN) of the hypothalamus are central structures for the regulation of circadian rhythms [22]. The SCN receive visual information by the retino-hypothalamic tract and indirectly via the retino-geniculo-suprachiasmatic pathway (RGSP). The geniculo-suprachiasmatic projection originates in the intergeniculate leaflet (IGL) and adjacent portions of the lateral geniculate body: these regions receive a direct retinal projection [15]. The cells forming this projection are characterised by their immunoreactivity to neuropeptide Y (NPY) [3, 15]. The RGSP may play a role in the photic entrainment of circadian rhythms. Lesions at several points along the RGSP alter various aspects of photic responsiveness of hamster activity rhythms [13, 20, 21]. Electrical stimulation of the IGL [18] or microinjection of NPY into the SCN [1] produce phase shifts in free-running locomotor activity rhythms. The phase shifts obtained resemble the phase response profile induced by dark pulse presentation [2]. The present study was undertaken to examine the electrophysiological effects of NPY on hamster SCN neurones recorded in vitro. A preliminary abstract of this work has appeared [14]. Correspondence: R. Mason, Dept. of Physiology & Pharmacology, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K. 0304-3940/87'$ 03 50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
174
Male golden hamsters were exposed to daily cycles of 14 h of light alternating with 10 h of dark (LD 14:10). For one group of animals (LD group) lights were on from 05.00 to 19.00 h (Atlantic Daylight Time, ADT). For a second group of hamsters lights were on from 20.00 to 10.00 h ADT (DL group). Illumination intensity measured in the centre of the cage was 30-100 lux during the light phase and <0.1 lux under dim red light during the dark phase. To allow comparisons and pooling of the data from LD and DL groups, circadian time (CT) 00.00 h refers to the time of lights on. At random times during different phases of the circadian cycle animals were anaesthetised with halothane and decapitated. Coronal hypothalamic slices (400-500 /zm) containing the SCN were cut and transferred to a slice chamber. Animals obtained during the dark phase were prepared under dim red light. Slices were maintained at 35+0.5°C at the gas/fluid interface between warm humidified Or-CO: (95%-5%) and artificial cerebrospinal fluid (in mM: NaC1 124, KC1 3.3, KH2PO4 1.2, CaC12 2.5, MgSO4 1, NaHCO3 2.5, glucose 10) at pH 7.4. Extracellular recordings were made with single or multi-barrel micropipettes, the recording barrel contained 2% Pontamine sky blue in 1 M NaCI. In some experiments recordings were made via the NPY-barrel using a micropipette holder (Clark Electromedical Instruments, UK: type EH-2MR) which allowed simultaneous recording and pressure ejection of NPY. The effects of pressure-ejected NPY (Peninsula Labs.; 50-200/tM NPY dissolved in 0.9% NaCI with 1% bovine serum albumin (BSA), aliquoted and kept frozen until use) were observed on the discharge activity of SCN neurones. Spike amplitude and shape were monitored for any pressure-related effects; recordings containing gross artifacts (i.e. showing changes > 10%) were omitted from further analysis. Following recording sessions the brain slices were fixed in formalin and sectioned at 50 /tm, stained with Cresyl violet and examined for deposits of Pontamine blue dye marks to identify SCN recording sites. Neurones recorded in the hamster SCN exhibited regular (n = 53/183, 29%), irregular (n =95/183, 52%) or bursting (n = 35/183, 19%) firing patterns in spontaneous discharge activity. Similar spontaneous discharge patterns have been described in the rat SCN slice preparation [9, 10, 25, 26-28]. The discharge rate ranged between 0.2 and 19.0 Hz. The regular and irregular discharge types in the SCN showed a circadian variation in their firing rate which was higher (5.9+ 1.0 Hz, mean+S.E.M., n=48 cells) during the period CT 06.00-12.00 h, in the late projected light phase, and was minimal (2.9___0.9 Hz, n---24 cells) between CT 15.00 and 21.00 h, in the early projected dark phase. The peak firing rate occurred in the middle of the projected light phase (CT 07.00-10,00 h: 7.3_+ 1.3 Hz, n=31 cells). There was no evidence of a circadian variation in discharge activity in the bursting type neurones. The rat SCN in vitro is also reported to exhibit a circadian variation in firing rate which peaks during the projected light phase [8, 9, 24]. The majority of SCN neurones tested (32/49, 65%) showed an increase in discharge rate following pressure-ejection of NPY. The remaining neurones tested were unresponsive to NPY (n = 17, 35%). The excitatory responses were characteristically sustained throughout the duration of the ejection (Fig. 1) and the elevated discharge was often maintained (for up to 60 min) beyond the termination of the NPY ejection
175
SCNvL celt 31 13 (CT02 45hr) ~ IH] ] I~I IIItllHIlllnl|JiHIIIlflll~lgllll~llnsltdllmIHI!1fli LI]III
A
I]]IIIIIIIIIIIImlIIIHtHWIIIIHIIIHIINIIII[II]Ilil J
~____
NPY (4psi 100~M) t__l
B NPY (3psi: 20min)
20
Ssec // //i
NPY sT
~1S LJ~ ~10
L
I 2rain
G Hamster
LD14:10 (tiqhts-on I T 0 0 0 0
1400
Fig. 1. A: NPY-evoked discharge activity of a regularly discharging neurone recorded in the ventrolateral division of the SCN. B: integrated firing rate histogram for the same SCN neurone accumulated over successive 5-s epochs illustrating the prolonged elevation of discharge activity induced by pressure ejection of NPY (3 psi).
period. Control ejections of the BSA vehicle alone were without effect on all cells tested (n = 21). Some neurones (n = 6) originally showing an irregular discharge pattern exhibited a regular pattern, without any appreciable increase in spike rate, following NPY ejection. The SCN neurones exhibiting a bursting discharge pattern (n = 5) were unresponsive to NPY. NPY-activated neurones were found throughout the rostrocaudal extent of the SCN and there was no evidence of regional differences within the SCN with respect to NPY sensitivity. Of the 23 SCN cells tested during the projected light phase of the circadian cycle (CT 00.00-14.00 h), all were responsive to NPY. Only 9 o f the 26 SCN neurones were recorded during the projected dark period (CT 14.00-24.00 h) responded to NPY ejection. In contrast, extrasuprachiasmatic hypothalamic neurones remained responsive to NPY when recorded during the projected dark phase (n = 27 cells; 25/27 excited and 2 cells inhibited by NPY) or during the projected light phase (n = 32 cells; 31/32 excited and 1 cell inhibited by NPY). Nine SCN neurones were recorded for a sufficient duration (2 8 h) to enable an examination of the temporal effects of repeated NPY ejections. SCN neurones (n = 5) recorded during the transition between the projected light to dark phase (CT 13.00~ 16.40 h) showed a loss of responsiveness to NPY (Fig. 2). Whereas SCN neurones (n = 4) recorded during the late dark phase exhibited an emergence of responsiveness to NPY (Fig. 3). The loss of NPY excitation could be attributable to desensitisation of the cell to NPY, although it is suggestive that loss of NPY responsiveness was generally found at the transition between the projected light and dark phases. The
176
SENvm cell 360/,
(OHamsfer
lights-onCTOO.OO-1/..OOhr)
40 30 20 CL
E
!0 0
I
m
t
I CT 15.30hr
20
L
~
_
i
NPY (15psi)
10 0 20
i
J T
'
' NPY
ET 16.30hr
10 0 I
2rnin
J '
' NPY
Fig. 2. Integrated firing rate record for a neurone in the ventromedial SCN (cell 36.04) recorded during the projected l i g h t . l a r k phase transition period (from CT 13.05 to 16.40 h). NPY was repeatedly ejected (1.5 psi). Initially the neurone exhibited an excitatory response to NPY whereas later during the recording no NPY-induced response was obtained. Note the reduction in discharge rate found at the projected lightdark transition of the circadian cycle.
emergence of NPY responsiveness cannot be explained as the recovery of a previously blocked electrode since NPY-responsive cells were recorded outside the boundaries of the SCN (paraventricular nucleus area) with the same electrode earlier in the recording session. It appears that SCN neurones show some circadian variation in their response to NPY; previous studies have reported circadian changes in responsiveness of SCN neurones to iontophoresed serotonin [17] and of lateral hypothalamic neurones to systemically administered agents [23]. The present study has demonstrated that the SCN, a target of NPY fibres originating from the IGL [3, 15], contains neurones responsive to exogenously applied NPY. The observation that NPY responsive neurones were recorded throughout the SCN is consistent with the widespread distribution of NPY immunoreactivity found in the SCN [3, 15, 30]. Immunocytochemical studies at the electron microscopic level have demonstrated the localisation of NPY in axodendritic asymmetrical synaptic contacts within the SCN of the rat [4]; interestingly asymmetrical synapses have been associated with excitatory contacts [29]. SCN neurones appear to be more responsive to NPY applied during the projected light phase than during the projected dark phase. This observation may be related to the finding that NPY microinjection and RGSP stimulation generate the largest phase shifts during the subjective day [1, 19].
177
SCNvt cell 36-07 (G.Hamsfer
[ighfs-on CTOO.OO-14.0Ohr)
lET 19.50hr 3O cf]
\20 QJ vl
-5 I0 E
0
I,
i
t
2min
30f
J NPY (20psi)
l C T 20.50hr
20
10
c
O
20 10
O
f
J NPY
.
'
c
i Np Y
.
.
.
.
,/~0/
,
' NPY
Fig. 3. Integrated firing rate records for a neurone in the ventrolateral SCN (cell 36.07) recorded during the middle of the projected dark phase of the circadian cycle. Repeated ejections of NPY (2 psi) were applied. In the initial part of the recording (CT 19.30-21.00 h) the neurone was unresponsive to NPY. After approximately 120 rain (CT 21.10 h) NPY ejection evoked an increase in discharge. This cell was recorded following an electrode penetration in the paraventricular area where two cells were recorded (cells 36.05/36.06, not shown) which responded to NPY. The m a j o r i t y o f I G L n e u r o n e s in the h a m s t e r t o n i c a l l y increase their firing rate while the eye is i l l u m i n a t e d [12]. W e h y p o t h e s i s e t h a t increased firing o f I G L n e u r o n e s in response to o c u l a r i l l u m i n a t i o n releases N P Y in the S C N a n d affects the responses o f S C N n e u r o n e s to p h o t i c stimuli [18]. N P Y is r e p o r t e d to be c o l o c a l i s e d in n o r a d r e n e r g i c nerve terminals [6], a n d n o r a d r e n a l i n e - i n d u c e d e x c i t a t o r y responses o f s u p r a o p t i c nucleus n e u r o n e s have been r e p o r t e d to be a t t e n u a t e d by N P Y [5]. W h i l e b o t h N P Y a n d c a t e c h o l a m i n e s are f o u n d in the S C N , c o l o c a l i s a t i o n has n o t been r e p o r t e d in the S C N as yet [6, 30]. It m a y be n o t e w o r t h y t h a t N P Y p r e f e r e n t i a l l y excited S C N n e u r o n e s which were actively d i s c h a r g i n g (i.e. the r e g u l a r a n d i r r e g u l a r d i s c h a r g e p a t t e r n types), suggesting t h a t the effects o f N P Y m a y be d e p e n d e n t on the level o f excitability o f the target neurone. This level o f excitability m a y be the result o f i n p u t s f r o m afferents a n d local i n t e r n e u r o n s o r a f u n c t i o n o f the p o s t s y n a p t i c t a r g e t n e u r o n e itself. T h e m e c h a n i s m
178 o f action o f N P Y is u n k n o w n , a l t h o u g h cyclic nucleotides m a y be involved as N P Y is reported to i n h i b i t cyclic a d e n o s i n e m o n o p h o s p h a t e ( c A M P ) a c c u m u l a t i o n [7, 1 i], while c A M P suppresses the discharge activity of S C N n e u r o n e s [16]; thus it is conceivable that N P Y m a y e n h a n c e S C N firing via i n h i b i t i n g the c A M P system. N P Y i n d u c e d responses are also d e p e n d e n t o n extracellular calcium [7] a n d d u r i n g the present study N P Y was often observed to shift the firing o f a n S C N n e u r o n e from an irregular to a regular discharge pattern; the opposite shift has been s h o w n following r e d u c t i o n of extracellular calcium [26, 28] ( u n p u b l i s h e d observations). Studies of possible c o t r a n s m i t t e r i n v o l v e m e n t in the responses o f S C N n e u r o n e s to N P Y a n d whether the N P Y - i n d u c e d excitation o f S C N n e u r o n e s is m e d i a t e d pre- or p o s t s y n a p tically are c u r r e n t l y in progress. C o n d u c t o f this research was m a d e possible by a Scientific Exchange G r a n t from the R o y a l Society ( U K ) a n d N S E R C ( C a n a d a ) , a n d was s u p p o r t e d by G r a n t s A0305 ( N S E R C ) , M A - 8 9 2 9 ( M R C o f C a n a d a ) , a n d the D a l h o u s i e Research Developm e n t F u n d in the Sciences. W e are grateful to Patricia D i c k s o n a n d T i m D e l a n e y for their technical assistance.
1 Albers, H.E. and Ferris, C.F., Neuropeptide Y: role in light-dark cycle entrainment of hamster circadian rhythms, Neurosci. Lett., 50 (1985) 163-168. 2 Boulos, Z. and Rusak, B., Circadian phase response curves for dark pulses in the hamster, J. Comp. Physiol., A146 (1982) 411-417 3 Card, J.P. and Moore, R.Y., Ventral lateral geniculate efferents to the rat suprachiasmatic nucleus exhibit avian pancreatic polypeptide-likeimmunoreactivity, J. Comp. Neurol., 206 (1982) 390-396. 4 Card, J.P. and Moore, R.Y., Immunocytochemical localization of neuropeptide Y in the rat suprachiasmatic nucleus, Soc. Neurosci. Abstr., 11 (1985) 1139. 5 Day, T.A., Jhamandas, J.H. and Renaud, L.P., Comparison between the actions of avian pancreatic polypeptide, neuropeptide Y and norepinephrine on the excitability of rat supraoptic vassopressin neurons, Neurosci. Lett., 62 (1985) 181-185. 6 Everitt, B.J., H6kfelt, T., Terenius, L., Tatemoto, K., Mutt, V. and Goldstein, M., Differential coexistence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat, Neuroscience, 11 (1984) 443-462. 7 Fredholm, B.B., Jansen, I. and Edvinsson, L., Neuropeptide Y is a potent inhibitor of cyclic AMP accumulation in feline cerebral blood vessels, Acta Physiol., Seand., 124 (1985) 467-469. 8 Green, D.J. and Gillette, R., Circadian rhythm of firing rate recorded from singlecells in the rat suprachiasmatic brain slice, Brain Res., 245 (1982) 198-200. 9 Groos, G.A. and Hendriks, J., Regularly firing neurones in the rat suprachiasmatic nucleus, Experientia, 35 (1979) 1597-1598. 10 Groos, G.A. and Hendriks, J., Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro, Neurosci. Lett., 34 (1982) 283-288. 11 H/irfstrand, A., Fredholm, B. and Fuxe, K., Inhibitory effects of neuropeptide Y on cyclic AMP accumulation in slices of the nucleus tractus solitaris region of the rat, Neurosci. Lett., 76 (1987) 185-190. 12 Harrington, M.E. and Rusak, B., Visual responses of neurones in the intergeniculate leaflet and ventral lateral geniculate nucleus of the hamster, Soc. Neurosci. Abstr., 12 (t986) 1036. 13 Harrington, M.E. and Rusak, B., Lesions of the thalamic intergeniculate leaflet alter circadian rhythms, J. Biol. Rhythms, I (1986) 309-325. 14 Harrington, M.E., Mason, R. and Rusak, B., The effects of neuropeptide-Y (NPY) on hamster suprachiasmatic neurones, J. Physiol. (London), 386 (1987) 91P.
179 15 Harrington, M.E., Nance, D.W. and Rusak, B., Neuropeptide Y immunoreactivity in the hamster geniculo-suprachiasmatic tract, Brain Res. Bull., 15 (1985) 465~,72 16 Liou, S.Y., Shibata, S., Shiratsuchi, A. and Ueki, S., Effects of dibutryl cyclic adenosine monophosphate and dibutryl guanosine monophosphate on neuron activity of suprachiasmatic nucleus in rat hypothalamic slice preparation, Neurosci. Lett., 67 (1986) 339-343. 17 Mason, R., Circadian variation in sensitivity of suprachiasmatic and lateral geniculate neurones to 5hydroxytryptamine in the rat, J. Physiol. (London), 377 (1986) I 13. 18 Meijer, J.H., Groos, G.A. and Rusak, B., Luminance coding in a circadian pacemaker: the suprachiasmatte nucleus of the rat and the hamster, Brain Res., 382 (1986) 109-118. 19 Meijer, J.H., Rusak, B. and Harrington, M.E., Geniculate stimulation phase shifts hamster circadian rhythms, Soc. Neurosci. Abstr., 10 (1984) 502. 20 Pickard, G.E., Ralph, M.R. and Menaker, M., The intergeniculate leaflet partially mediates the effects of light on circadian rhythms, J. Biol. Rhythms, in press. 21 Rusak, B. and Boulos, Z., Pathways for photic entrainment of mammalian circadian rhythms~ Photochem. Photobiol., 34 (1981)267 273. 22 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449 526. 23 Schmitt, M., Circadian rhythmicity in responses of cells in the lateral hypothalamus, Am. J. Physiol., 225 (1973) 1096 1101. 24 Shibata, S., Oomura, Y., Kita, H. and Hattori, K., Circadian rhythmic changes of neuronal activity in the suprachiasmatic nucleus of the rat hypothalamic slice, Brain Res., 247 (1982) 154-158. 25 Shibata, S., Liou, S.Y., Ueki, S. and Oomura, Y., Influence of environmental light dark cycle and enucleation on activity of suprachiasmatic neurones in slice preparations, Brain Res., 302 (1984) 75 8 I. 26 Shibata, S., Shiratsuchk A., Liou, S,Y. and Ueki, S., The role of calcium ions in circadian rhythm of suprachiasmatic nucleus neuron activity in rat hypothalamic slices, Neurosci. Lett., 52 (1984) 181 184. 27 Shibata, S., Oomura, Y., Liou, S.Y. and Ueki, S., Electrophysiological studies of the development of suprachiasmatic neuronal activity in hypothalamic slice preparations, Dev. Brain Res., 13 (1984) 29 35. 28 Thompson, A.M., Slow, regular discharge in suprachiasmatic neurones is calcium dependent, in slices of rat brain, Neuroscience, 13 (1984) 761 767. 29 Uchizono, K., Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat, Nature (London) 207 (1965) 642~43. 30 Van den Pol, A.N. and Tsujimoto, K.L., Neurotransmitters of the hypothalamic suprachiasmatic nucleus: immunocytochemical analysis of 25 neuronal antigens, Neuroscience, 15 (1985) 1049 1086.