Marian H. Lewandowski

Ultradian rhythmic neuronal oscillation in the intergeniculate leaflet

Our paper is the first to describe ultradian rhythmic neuronal oscillation in the intergeniculate leaflet (IGL) of the rat. We recorded a multiple-unit neuronal activity (MUA) from dorsal to ventral parts of the lateral geniculate nucleus (LGN) in anaesthetized rats. In all the subdivisions of the lateral geniculate complex we observed spontaneous irregular firing rates of cells. However only at the anatomical localisation of the IGL, after the light was on, those responses exhibited burst firing with a constant interburst interval, which lasted several hours until the light was off. The duration of that rhythmic oscillation obtained by means of Fouriers analysis was ∼124 s. To date we have not had sufficient data to discuss possible mechanisms of this neuronal rhythmicity. We can only conclude that light is the most important stimulus not only for suprachiasmatic nuclei (SCN), but also for the IGL. On the other hand, we can neither exclude nor confirm that in order to evoke ultradian rhythmical oscillation in the IGL, in addition to light also non-photic information is necessary.

Endogenous rhythm of absence epilepsy: relationship with general motor activity and sleep-wake states.

The rhythms of spontaneously occurring seizures (spike-wave discharges, SWD) and motor activity, as well as the relationship between SWD and sleep-wake states were investigated in the WAG/Rij rat model of absence epilepsy. In order to establish whether SWD are controlled by external (Zeitgebers) or by endogenous factors such as circadian influences or the state of vigilance, the study was performed in entrained and constant dim light conditions. EEG and motor activity were recorded in the 12:12 light-dark cycle and in constant dim light conditions. Circadian rhythmicity was found both for motor activity and the occurrence of SWD in conditions of entrainment. In constant dim light conditions also circadian rhythms emerged, however, the change in circadian parameters was opposite for the rhythm of SWD and motor activity. SWD were preceded mostly by passive wakefulness and by slow-wave sleep in both experimental conditions. It can be concluded that the rhythm of SWD seems to be generated and controlled by an endogenous mechanism distinct from that which controls the rhythm of motor activity. The relationship between SWD and sleep-wake states preceding their occurrences appeared to be unchanged, suggesting that the mechanism of generation of SWD is independent of the circadian timing system.

The influence of orexins on the firing rate and pattern of rat intergeniculate leaflet neurons--electrophysiological and immunohistological studies.

Orexins influence various physiological processes associated with feeding behaviour, endocrine functions and wakefulness. One component of mammalian circadian timing systems, intergeniculate leaflet (IGL) of the lateral geniculate nucleus, is thought to contribute to circadian entrainment by processing photic and non‐photic/arousal‐related signals. Because the IGL is possibly innervated by the orexinergic system, using in vitro extracellular recording techniques we evaluated the influence of orexin A (OXA) and orexin B (OXB) on the rate and pattern of neuronal firing in this structure. Significant increases in the activity of 33 and 28% of IGL cells were observed after locally applied OXA (1 μm) and OXB (1 μm), respectively. In the great majority of neurons responses to OXA were maintained in the presence of orexin‐1 receptor OX1R antagonist, SB 334867 (10 μm). Additionally, 75% of the OXB‐responsive neurons were also sensitive to an orexin‐2 receptor (OX2R)‐selective agonist, [Ala11, D‐Leu15]‐OXB (1 μm). Immunohistochemical stainings showed putative synaptic contacts between OXA‐ and OXB‐immunoreactive fibres and neuropeptide Y, and enkephalin‐positive neurons in the investigated area. The outcome of our experiments reinforces previous reports indicating the possible linkage between the orexinergic and circadian systems. To our knowledge the presented findings are the first showing the direct influence of orexins on the IGL activity, mostly through activation of OX2R.

Differential firing pattern and response to lighting conditions of rat intergeniculate leaflet neurons projecting to suprachiasmatic nucleus or contralateral intergeniculate leaflet

The intergeniculate leaflet (IGL) of the lateral geniculate body in the rat is a population of GABAergic neurons that can be divided into two, anatomically and neurochemically distinct populations. One population comprises neuropeptide-Y (NPY)-positive neurons that form the geniculohypothalamic tract innervating the suprachiasmatic nuclei (SCN) and the other population comprises enkephalin-positive (ENK) neurons giving rise to the geniculo-geniculate tract innervating the contralateral IGL (cIGL). Previous electrophysiological studies have observed various patterns of firing and different responses to changes in lighting conditions of IGL neurons in vitro and in vivo. The aim of the present study was to determine if these distinct properties could be ascribed to differentially projecting IGL neurons. Neuron activity was recorded extracellularly in the IGL of anaesthetised rats under different lighting conditions (i.e. light/dark). Antidromic activation was used to identify recorded cells as projecting to the SCN or the contralateral IGL. All IGL neurons identified as projecting to the contralateral IGL displayed infra-slow oscillatory activity (ISO; i.e. slow rhythmic bursts of action potentials). ISO of these neurons was sustained in the light and was diminished in the darkness. In contrast, all IGL neurons identified as projecting to the SCN displayed a low level of firing in the light and a majority of these cells increased firing in the darkness. All IGL neurons projecting to the SCN were characterised by an irregular pattern of firing in the light and dark. These data are the first to demonstrate that differentially projecting rat intergeniculate leaflet neurons are characterised by distinct firing patterns and opposite responses to light and dark conditions.

Relaxin-3 innervation of the intergeniculate leaflet of the rat thalamus - neuronal tract-tracing and in vitro electrophysiological studies

Behavioural state is controlled by a range of neural systems that are sensitive to internal and external stimuli. The relaxin‐3 and relaxin family peptide receptor 3 (RXFP3) system has emerged as a putative ascending arousal network with putative involvement in regulation of stress responses, neuroendocrine control, feeding and metabolism, circadian activity and cognition. Relaxin‐3/γ‐aminobutyric acid neuron populations have been identified in the nucleus incertus, pontine raphe nucleus, periaqueductal grey (PAG) and an area dorsal to the substantia nigra. Relaxin‐3‐positive fibres/terminals densely innervate arousal‐related structures in the brainstem, hypothalamus and limbic forebrain, but the functional significance of the heterogeneous relaxin‐3 neuron distribution and its inputs to specific brain areas are unclear. Therefore, in this study, we used neuronal tract‐tracing and immunofluorescence staining to explore the source of the dense relaxin‐3 innervation of the intergeniculate leaflet (IGL) of the thalamus, a component of the neural circadian timing system. Confocal microscopy analysis revealed that relaxin‐3‐positive neurons retrogradely labelled from the IGL were predominantly present in the PAG and these neurons expressed corticotropin‐releasing factor receptor‐like immunoreactivity. Subsequently, whole‐cell patch‐clamp recordings revealed heterogeneous effects of RXFP3 activation in the IGL by the RXFP3 agonist, relaxin‐3 B‐chain/insulin‐like peptide‐5 A‐chain (R3/I5). Identified, neuropeptide Y‐positive IGL neurons, known to influence suprachiasmatic nucleus activity, were excited by R3/I5, whereas neurons of unidentified neurotransmitter content were either depolarized or displayed a decrease in action potential firing and/or membrane potential hyperpolarization. Our data identify a PAG to IGL relaxin‐3/RXFP3 pathway that might convey stress‐related information to key elements of the circadian system and influence behavioural state rhythmicity.

Effects of intergeniculate leaflet lesions on circadian rhythms in the mouse

We investigated effects of bilateral electrolytic intergeniculate leaflet (IGL) lesions (IGLX) on circadian rhythms of the wheel-running activity in mice kept under constant darkness conditions (DD). As a result of complete bilateral IGL lesions, the period of a free-running rhythm was lengthened by ca. 45 min in comparison with a pre-operational one. The present findings confirm results obtained previously with other animals. However, for the first time locomotor activity levels were precisely assessed in mice (the number of wheel revolutions/day) before and after IGL lesions. A computer analysis of data showed a considerable decrease in that activity (by 68% on an average) in mice with complete bilateral IGL lesions in comparison with IGL-non-lesioned individuals. The obtained results show that also in mice IGL constitutes an anatomically important element of the mechanism of a circadian time-keeping system, which mediates the transfer of non-photic information to the suprachiasmatic nuclei (SCN) by modulating their activity.

Dorsal raphe nucleus modulates neuronal activity in rat intergeniculate leaflet

Serotonergic input from midbrain raphe nuclei is believed to have a significant effect on mammalian circadian timing system. The suprachiasmatic nucleus (SCN) receives its serotonergic input from the median raphe nucleus, while the intergeniculate leaflet (IGL) receives serotonergic innervation from the dorsal raphe nucleus (DRN). The present paper was aimed at determining whether projection from the DRN affected rhythmic neuronal oscillations in the IGL of rats. We investigated the impact of electrolytic lesions and electric stimulation of the DRN on spontaneous isoperiodic (i.e. burst firing with a constant interburst interval) neuronal activity recorded in the IGL. In all our experiments a complete lesion of the DRN always caused a significant increase (ca. 100%) of spontaneous activity of IGL neurons, their oscillatory character having been maintained, though. On the other hand, electric stimulation of the DRN produced a transient decrease in firing rate oscillations of the IGL neurons. The obtained results indicate that the neuronal projection from the DRN has a substantial modulating effect on IGL activity-an important element of the mechanism of the circadian time-keeping system that mediates the transfer of non-photic information to the SCN by modulating its activity. The observed increase of isoperiodic activity in the IGL after DRN lesion and a transient decrease in this activity after electric stimulation indicate an inhibitory character of this effect. The present findings corroborate the hypothesis that the DRN is a one of the major and extremely important source of the modulatory inputs to the mammalian circadian time-keeping system.

Orexins/hypocretins modulate the activity of NPY-positive and -negative neurons in the rat intergeniculate leaflet via OX1 and OX2 receptors

Orexins/hypocretins (OXA and OXB) are two hypothalamic peptides involved in the regulation of many physiological processes including the sleep-wake cycle, food intake and arousal. The orexinergic system of the lateral hypothalamus is considered a non-specific peptidergic system, and its nerve fibers innervate numerous brain areas. Among many targets of orexinergic neurons is the intergeniculate leaflet (IGL) of the thalamus - a small but important structure of the mammalian biological clock. In rats, the IGL consists of GABAergic cells which also synthesize different neuropeptides. One group of neurons produces neuropeptide Y (NPY) and sends its axons to the master biological clock known as the suprachiasmatic nuclei. Another neuronal group produces enkephalin and is known to connect contralateral IGLs. This study evaluated the effects of orexins on identified IGL neurons revealing that 58% of the recorded neurons were sensitive to OXA (200nM) and OXB (200nM) administration. Both NPY-positive and -negative neurons were depolarized by these neuropeptides. Experiments using selective orexin receptor antagonists (SB-334867, 10μM and TCS-OX2-29, 10μM) suggested that both orexin receptors participate in the recorded OXA effects. In addition, IGL neurons were either directly depolarized by OXA or their activity was altered by changes in presynaptic inputs. We observed an increase of GABA release onto the investigated IGL neuron after OXA application, consistent with a presynaptic localization of the orexin receptors. An increase in miniature excitatory postsynaptic current frequency was not observed within the IGL. Our findings reinforce the connection between circadian clock physiology and the orexinergic system.

The serotonergic inhibition of slowly bursting cells in the intergeniculate leaflet of the rat.

Electrophysiological studies combined with local neurotoxic lesions were conducted on anaesthetized rats in order to determine whether the dorsal raphe nucleus (DRN) inhibits the intergeniculate leaflet (IGL) of the lateral geniculate nucleus by means of innervation by serotonin‐containing fibres. In the control animals, electrical stimulation of the DRN induced the long‐latency and long‐lasting inhibition of the neuronal firing of the IGL cells that are characterized by rhythmic, slow‐bursting activity in light conditions. The electrical destruction of the DRN resulted in an increase in the firing rate of the recorded IGL cells, whilst at the same time not affecting the rhythmic, bursting pattern of the activity. In the second group of animals, local neurotoxic lesion of serotonergic fibres was performed by injection of the toxin 5,7‐dihydroxytryptamine into the IGL. After 10 days of postoperative recovery, electrophysiological experiments were performed on the toxin‐treated rats. In these animals, electrical stimulation as well as electrical lesion of the DRN did not induce any change in the firing of the slowly bursting cells in the 5,7‐dihydroxytryptamine‐injected IGL. The results obtained provide evidence that inhibition of the IGL slowly bursting cells, by innervation from the dorsal raphe, is mediated by the release of serotonin. Furthermore, the observed serotonergic inhibition of the light‐dependent activity of slowly bursting cells can contribute to the neuronal mechanism gating the information that flows through this nucleus to the vestibular, visuomotor, circadian and sleep/arousal systems, with which the IGL is strongly interconnected.

Are ultra-slow isoperiodic oscillations in rat intergeniculate leaflet neurons dependent on reciprocal connection with its contralaterally located counterpart?

We investigated effects of the electrical lesion and/or chemical inactivation of intergeniculate leaflet (IGL) neurons on the ultra-slow isoperiodic neuronal oscillation of the contralaterally located IGL. The spontaneous extracellular activity of neurons, recorded simultaneously in both leaflets of the lateral geniculate nucleus, showed an ultradian oscillatory pattern. In all our experiments, both the electrical lesion and the inactivation of neurons via the blockade of action potential generation did not cause any changes in the neuronal activity pattern in the contralaterally located geniculate leaflet. The obtained results show that a bilateral IGL connection is not necessary for the pattern of neuronal oscillation in the IGL. Hence the functional significance of a reciprocal connection between both lateral geniculate nucleus leaflets is still an open question.