Joke Wortel

Anatomical and functional demonstration of a multisynaptic suprachiasmatic nucleus adrenal (cortex) pathway

In view of mounting evidence that the suprachiasmatic nucleus (SCN) is directly involved in the setting of sensitivity of the adrenal cortex to ACTH, the present study investigated possible anatomical and functional connections between SCN and adrenal. Transneuronal virus tracing from the adrenal revealed first order labelling in neurons in the intermedio‐lateral column of the spinal cord that were shown to receive an input from oxytocin fibres and subsequently second‐order labelling in neurons of the autonomic division of the paraventricular nucleus. The latter neurons were shown to receive an input from vasopressin or vasoactive intestinal peptide (VIP) containing SCN efferents. The true character of this SCN input to second‐order neurons was also demonstrated by the fact that third‐order labelling was present within the SCN, vasopressin or VIP neurons. The functional presence of the SCN–adrenal connection was demonstrated by a light‐induced fast decrease in plasma corticosterone that could not be attributed to a decrease in ACTH. Using intact and SCN‐lesioned animals, the immediate decrease in plasma corticosterone was only observed in intact animals and only at the beginning of the dark period. This fast decrease of corticosterone was accompanied by constant basal levels of blood adrenaline and noradrenaline, and is proposed to be due to a direct inhibition of the neuronal output to the adrenal cortex by light‐mediated activation of SCN neurons. As a consequence, it is proposed that the SCN utilizes neuronal pathways to spread its time of the day message, not only to the pineal, but also to other organs, including the adrenal, utilizing the autonomic nervous system.

The suprachiasmatic nucleus balances sympathetic and parasympathetic output to peripheral organs through separate preautonomic neurons

Opposing parasympathetic and sympathetic signals determine the autonomic output of the brain to the body and the change in balance over the sleep‐wake cycle. The suprachiasmatic nucleus (SCN) organizes the activity/inactivity cycle and the behaviors that go along with it, but it is unclear how the hypothalamus, in particular the SCN, with its high daytime electrical activity, influences this differentiated autonomic balance. In a first series of experiments, we visualized hypothalamic pre‐sympathetic neurons by injecting the retrograde tracer Fluoro‐Gold into the thoracic sympathetic nuclei of the spinal cord. Pre‐parasympathetic neurons were revealed by injection of the retrograde trans‐synaptic tracer pseudorabies virus (PRV) into the liver and by sympathetic liver denervation, forcing the virus to infect via the vagus nerve only. This approach revealed separate pre‐sympathetic and pre‐parasympathetic neurons in the brainstem and hypothalamus. Next, selective retrograde tracing with two unique reporter PRV strains, one injected into the adrenal and the other into the sympathetic denervated liver, demonstrated that there are two separate populations of pre‐sympathetic and pre‐parasympathetic neurons within the paraventricular nucleus of the hypothalamus. Interestingly, this segregation persists into the SCN, where, as a result, the day‐night balance in autonomic function of the organs is affected by specialized pre‐sympathetic or pre‐parasympathetic SCN neurons. These separate preautonomic SCN neurons provide the anatomical basis for the circadian‐driven regulation of the parasympathetic and sympathetic autonomic output. J. Comp. Neurol. 464:36–48, 2003.

The suprachiasmatic nucleus controls the daily variation of plasma glucose via the autonomic output to the liver: are the clock genes involved?

In order to drive tissue‐specific rhythmic outputs, the master clock, located in the suprachiasmatic nucleus (SCN), is thought to reset peripheral oscillators via either chemical and hormonal cues or neural connections. Recently, the daily rhythm of plasma glucose (characterized by a peak before the onset of the activity period) has been shown to be directly driven by the SCN, independently of the SCN control of rhythmic feeding behaviour. Indeed, the daily variation in glucose was not impaired unless the scheduled feeding regimen (six‐meal schedule) was associated with an SCN lesion. Here we show that the rhythmicity of both clock‐gene mRNA expression in the liver and plasma glucose is not abolished under such a regular feeding schedule. Because the onset of the activity period and hyperglycemia are correlated with an increased sympathetic tonus, we investigated whether this autonomic branch is involved in the SCN control of plasma glucose rhythm and liver rhythmicity. Interestingly, hepatic sympathectomy combined with a six‐meal feeding schedule resulted in a disruption of the plasma glucose rhythmicity without affecting the daily variation in clock‐gene mRNA expression in the liver. Taking all these data together, we conclude that (i) the SCN needs the sympathetic pathway to the liver to generate the 24‐h rhythm in plasma glucose concentrations, (ii) rhythmic clock‐gene expression in the liver is not dependent on the sympathetic liver innervation and (iii) clock‐gene rhythmicity in liver cells is not sufficient for sustaining a circadian rhythm in plasma glucose concentrations.

Suprachiasmatic control of melatonin synthesis in rats: inhibitory and stimulatory mechanisms

The suprachiasmatic nucleus (SCN) controls the circadian rhythm of melatonin synthesis in the mammalian pineal gland by a multisynaptic pathway including, successively, preautonomic neurons of the paraventricular nucleus (PVN), sympathetic preganglionic neurons in the spinal cord and noradrenergic neurons of the superior cervical ganglion (SCG). In order to clarify the role of each of these structures in the generation of the melatonin synthesis rhythm, we first investigated the day‐ and night‐time capacity of the rat pineal gland to produce melatonin after bilateral SCN lesions, PVN lesions or SCG removal, by measurements of arylalkylamine N‐acetyltransferase (AA‐NAT) gene expression and pineal melatonin content. In addition, we followed the endogenous 48 h‐pattern of melatonin secretion in SCN‐lesioned vs. intact rats, by microdialysis in the pineal gland. Corticosterone content was measured in the same dialysates to assess the SCN lesions effectiveness. All treatments completely eliminated the day/night difference in melatonin synthesis. In PVN‐lesioned and ganglionectomised rats, AA‐NAT levels and pineal melatonin content were low (i.e. 12% of night‐time control levels) for both day‐ and night‐time periods. In SCN‐lesioned rats, AA‐NAT levels were intermediate (i.e. 30% of night‐time control levels) and the 48‐h secretion of melatonin presented constant levels not exceeding 20% of night‐time control levels. The present results show that ablation of the SCN not only removes an inhibitory input but also a stimulatory input to the melatonin rhythm generating system. Combination of inhibitory and stimulatory SCN outputs could be of a great interest for the mechanism of adaptation to day‐length (i.e. adaptation to seasons).

Polysynaptic neural pathways between the hypothalamus, including the suprachiasmatic nucleus, and the liver.

The suprachiasmatic nucleus of the hypothalamus is responsible for a 24-h rhythm in basal glucose levels in the rat. The neural pathways used by the suprachiasmatic nucleus to mediate this rhythm in plasma glucose have not yet been identified. In the present study we examined whether there are any connections between hypothalamic centers, including the suprachiasmatic nucleus, and the liver, which is the main site for glucose production and storage. Transneuronal virus tracing from the liver showed that after injection of pseudorabies virus, specific neuronal cell populations in the central nervous system were labeled retrogradely, suggesting that specific sites in the central nervous system may control liver metabolism. First-order neurons belonged to the sympathetic and parasympathetic system, while second-order and third-order neurons were present in both the brainstem and hypothalamus. The presence of third-order neurons in the suprachiasmatic nucleus suggests an involvement of the biological clock in the neural control of the liver.

Evidence from Confocal Fluorescence Microscopy for a Dense, Reciprocal Innervation Between AVP-,somatostatin-, VIP/PHI-, GRP- and VIP/PHI/GRP-immunoreactive Neurons in the Rat Suprachiasmatic Nucleus

The rat suprachiasmatic nucleus (SCN) consists of several classes of neurons which can be identified by their transmitter content. Knowledge of putative interaction between these different cell types is essential in order to understand the possibilities of information processing within the SCN. The aim of the present study was therefore to obtain more information about the mutual innervation between the main cell classes in the rat SCN, viz. those containing the neuropeptides arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), gastrin‐releasing peptide (GRP) and somatostatin respectively. For this purpose, vibratome sections were double‐immunolabelled for seven different peptide combinations and subsequently analysed by high‐resolution confocal laser scanning fluorescence microscopy. Attention was focused on axosomatic appositions, the occurrence and frequency of which were quantitatively estimated. Our analysis of double‐immunolabelled sections demonstrated that some of the VIP‐ and some of the GRP‐immunoreactive nerve cells and endings showed colocalization. Assuming, on the basis of literature data, that VIP and PHI are always colocalized at the cellular level, the five main cell classes in the SCN appeared to be interconnected, at least axosomatically, in the following reciprocal way: AVP ↔ VIP/PHI, AVP ↔ GRP, AVP ↔ somatostatin, somatostatin ↔ VIP/PHI, somatostatin ↔ GRP, VIP/PHI ↔ GRP, VIP/PHI/GRP ↔ GRP, VIP/PHI/GRP ↔ VIP/ PHI. In addition to this heterologous axosomatic innervation, these cell groups also showed substantial homologous innervation. Supported by electron microscope data from the literature showing the existence of axodendritic synapses for some of these peptide combinations, our findings strongly suggest that the rat SCN comprises a complex synaptic network with strong interactive capabilities, which is probably a requisite for its biological clock function.

Proteomics, Ultrastructure, and Physiology of Hippocampal Synapses in a Fragile X Syndrome Mouse Model Reveal Presynaptic Phenotype

Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development.

Changes in molecular composition of rat medial prefrontal cortex synapses during adolescent development

Postnatal brain development continues throughout adolescence into young adulthood. In particular, synapse strengthening and elimination are prominent processes during adolescence. However, molecular data of this relatively late stage of synaptic development are sparse. In this study, we used iTRAQ (isobaric tag for relative and absolute quantification)‐based proteomics and electron microscopy to investigate the molecular composition of a synaptic membrane fraction from adolescent postnatal day (P)34 and P44 and adult (P78) rat medial prefrontal cortex. Differential expression of proteins was most prominent between early adolescence and young adulthood (35%, P34–P78), with an over‐representation of cell‐membrane proteins during adolescent development (between P34 and P44), and synaptic vesicle proteins between late adolescence and young adulthood (P44–P78). Indicative of the critical period of development, we found that, between P34 and P44, a substantial number of proteins was differentially expressed (14%), much more than during the period after adolescence, i.e. between P44 and P78 (5%). A striking observation was the developmental non‐stoichiometric regulation of distinct classes of proteins from the synaptic vesicle and the presynaptic release machinery. Electron microscopy demonstrated a small change in the number of docked vesicles between P34 and P44, but not in the total number of synaptic vesicles and in the size of the vesicle cluster. We conclude that the molecular composition of synapses, and more specifically the synaptic release machinery, of the medial prefrontal cortex changes drastically during adolescent development.

Development of dendritic tonic GABAergic inhibition regulates excitability and plasticity in CA1 pyramidal neurons

Synaptic plasticity rules change during development: while hippocampal synapses can be potentiated by a single action potential pairing protocol in young neurons, mature neurons require burst firing to induce synaptic potentiation. An essential component for spike timing-dependent plasticity is the backpropagating action potential (BAP). BAP along the dendrites can be modulated by morphology and ion channel composition, both of which change during late postnatal development. However, it is unclear whether these dendritic changes can explain the developmental changes in synaptic plasticity induction rules. Here, we show that tonic GABAergic inhibition regulates dendritic action potential backpropagation in adolescent, but not preadolescent, CA1 pyramidal neurons. These developmental changes in tonic inhibition also altered the induction threshold for spike timing-dependent plasticity in adolescent neurons. This GABAergic regulatory effect on backpropagation is restricted to distal regions of apical dendrites (>200 μm) and mediated by α5-containing GABA(A) receptors. Direct dendritic recordings demonstrate α5-mediated tonic GABA(A) currents in adolescent neurons which can modulate BAPs. These developmental modulations in dendritic excitability could not be explained by concurrent changes in dendritic morphology. To explain our data, model simulations propose a distally increasing or localized distal expression of dendritic α5 tonic inhibition in mature neurons. Overall, our results demonstrate that dendritic integration and plasticity in more mature dendrites are significantly altered by tonic α5 inhibition in a dendritic region-specific and developmentally regulated manner.

Adult mouse eIF2Bϵ Arg191His astrocytes display a normal integrated stress response in vitro

Vanishing white matter (VWM) is a genetic childhood white matter disorder, characterized by chronic as well as episodic, stress provoked, neurological deterioration. Treatment is unavailable and patients often die within a few years after onset. VWM is caused by recessive mutations in the eukaryotic initiation factor 2B (eIF2B). eIF2B regulates protein synthesis rates in every cell of the body. In normal cells, various types of cellular stress inhibit eIF2B activity and induce the integrated stress response (ISR). We have developed a VWM mouse model homozygous for the pathogenic Arg191His mutation in eIF2Bε (2b5ho), representative of the human disease. Neuropathological examination of VWM patient and mouse brain tissue suggests that astrocytes are primarily affected. We hypothesized that VWM astrocytes are selectively hypersensitive to ISR induction, resulting in a heightened response. We cultured astrocytes from wildtype and VWM mice and investigated the ISR in assays that measure transcriptional induction of stress genes, protein synthesis rates and cell viability. We investigated the effects of short- and long-term stress as well as stress recovery. We detected congruent results amongst the various assays and did not detect a hyperactive ISR in VWM mouse astrocytes.