Elsevier

Brain Research

Volume 1748, 1 December 2020, 147125
Brain Research

Research report
LIM homeobox 6 (Lhx6)+ neurons in the ventral zona incerta project to the core portion of the lateral supramammillary nucleus in the rat

https://doi.org/10.1016/j.brainres.2020.147125Get rights and content

Highlights

  • Lhx6+ neurons projecting to the SUMl occupied the ventral border (or one-third) of the ventral ZI.

  • The proportion of SUMl-projecting, Lhx6+ neurons over the total Lhx6+ ZI cells was 7.8%.

  • The proportion of SUMl-projecting, Lhx6+ and MCH+ cells over SUMl-projecting ZI neurons was 43.7%.

Abstract

There was a recent report suggesting that LIM homeobox 6 (Lhx6)+ GABA-releasing neurons of the ventral zona incerta (ZI) promote sleep. We demonstrated in the previous study that Lhx6+ ZI neurons are activated during paradoxical sleep (PS) hypersomnia which was induced by 48-hour PS deprivation, implying their roles in the control of PS like melanin-concentrating hormone (MCH) cells. Since the core portion of the lateral supramammillary nucleus (SUMl) is the major hypothalamic area activating the dentate gyrus as well as other limbic cortices during PS, we examined in the present study whether Lhx6+ ZI cells provide efferent projections to the SUMl, using the retrograde-tracing method. The majority of Lhx6+ neurons projecting to the SUMl occupied the ventral border (or ventral one-third) of the ventral ZI. Based on the quantitative analysis, the mean number of retrogradely-labeled Lhx6+ neurons was comparable to that of retrogradely-labeled MCH cells in the ZI. However, the total (i.e., single- plus double-labeled) number of Lhx6+ cells was approximately three times larger than that of MCH cells in the ZI. Thus, the proportion (about 7.8%) of retrogradely-labeled Lhx6+ neurons over the total Lhx6+ cells was approximately one-third of the percentage (about 20.9%) of retrogradely-labeled MCH neurons over the total MCH cells. On the other hand, a combination of retrogradely-labeled, Lhx6 and MCH cells occupied approximately 43.7% of the total retrogradely-labeled neurons in the ventral ZI. The present observations suggested that Lhx6+ neurons in the ventral ZI might play an important role in the regulation of PS, partly via the neural network involving the SUMl.

Introduction

It is well known that hypocretin and γ-aminobutyric acid (GABA)-releasing neurons of the lateral hypothalamic area (LHA) promote the transition from slow wave sleep (SWS) and paradoxical sleep (PS) to wakefulness and that optogenetic stimulation of GABAergic neurons in the LHA as well as the zona incerta (ZI) induces waking rather than PS (Lee et al., 2005, Saper et al., 2010, Herrera et al., 2016). However, the vast majority of cFos+ neurons located in the LHA and ZI following PS hypersomnia also express glutamic acid decarboxylase (GAD) 67 and are thus GABAergic (Sapin et al., 2009, Sapin et al., 2010, Brown et al., 2017). For example, melanin-concentrating hormone (MCH) neurons specifically active during PS are mostly GABAergic and opto- and chemogenetic activation of these neurons induces an increase in PS quantity (Hassani et al., 2009, Jego et al., 2013, Peever and Fuller, 2017, Varin et al., 2018). Considering the observation that about 80% of the Fos+/GAD+ neurons in the tuberal hypothalamus after PS hypersomnia do not contain MCH (Sapin et al., 2010), further studies are needed to characterize PS-on GABAergic neurons in the region.

A recent report suggested that LIM homeobox 6 (Lhx6)+ GABA-releasing neurons of the ventral ZI express cFos at the end of the dark period or during total sleep deprivation and that selective loss of Lhx6 expression in the diencephalon reduces both SWS and PS (Liu et al., 2017). They demonstrated that Lhx6+ neurons represent 45% of all Slc32A1+ and 33% of Gad1+ cells in the ventral ZI, receiving synaptic inputs from various sleep-regulating regions and providing axon terminals to the amygdala as well as several, sleep-related brainstem regions. In contrast to the Lhx6+ cellular aggregate in the ventral ZI, Lhx6+ cells in the LHA were few in number with diffuse distribution and their involvement in sleep regulation has not been elucidated (Shimogori et al., 2010, Liu et al., 2017). Using a new mouse genetic model employing the targeted recombination in active populations (TRAP), we further demonstrated that Lhx6+ neurons in the ventral ZI are activated during PS hypersomnia, suggesting their roles in the control of PS similar to MCH cells (Lee et al., 2020).

Several limbic cortices contained neurons with an increased expression of immediate early genes during vivid dreaming of PS state, among which the dentate gyrus (DG) is the only cortical structure containing more cFos+ cells during PS than during waking (Renouard et al., 2015). Furthermore, cFos overexpression occurring in the cortex during PS hypersomnia is due to projections from the supramammillary nucleus (SUM) as well as the claustrum. In fact, the SUM heavily projects to the claustrum (Barbier and Risold, 2019), implying the importance of its position as their final common origin. The medial portion of the SUM (i.e., the parvicellular portion or SUMm) contains small dopaminergic (or substance P-containing) cells which provide a major input to the lateral septum, while the lateral SUM (SUMl) surrounding the principal mammillary tract (pm) contains large cells (i.e., the grandicellular portion or the core of the SUMl) that project directly to the hippocampus and medial septum (MS); in addition, the third, lateral-most region called the shell portion projects mainly to the entorhinal cortex (for review, see Pan and McNaughton, 2004, Vertes, 2015). Only the core of the SUMl contains the largest number (or sizes) of cells which project to the hippocampus, especially into dorsal hippocampus (Maglóczky et al., 1994, Ohara et al., 2013, Vertes, 2015). Thus, we examined in the present study the Lhx6+ ZI neuronal projection to the core portion of the SUMl using retrograde tracing studies, comparing it quantitatively with MCH+ ZI cellular projection to the important PS-on region.

Section snippets

Results

In order to examine the projection pattern of Lhx6+ (or MCH+) ZI neurons to the SUMl, red RetrobeadsTM was injected into the SUMl, particularly the core region around the pm (Fig. 1A). Cocaine- and amphetamine-regulated transcript (CART) immunostaining was employed in this study to determine the cytoarchitectonic borders of the SUMl (Fig. 1B). Due to extensive fibers as well as somata, CART method could serve as a superb background staining compared with NeuN (Pan and McNaughton, 2004, Renouard

Discussion

Based on the reports that Lhx6+ neurons in the ventral ZI might play a role in the control of PS (Liu et al., 2017, Lee et al., 2020), we examined in the present study whether Lhx6+ cells provided efferent projections to the SUMl, which is the major subcortical structure activating limbic cortices during PS (Renouard et al., 2015). The present study demonstrated that the majority of Lhx6+ neurons projecting to the SUMl occupied the ventral border (or ventral one-third) of the ventral ZI. The

Experimental procedures

Every effort was made to minimize the number of animals utilized. A total of 18 male Sprague-Dawley rats weighing 300–320 g were used to examine Lhx6+ (or MCH+) ZI neuronal projection to the SUMl. The animals were housed in cages that were kept at constant room temperature (23 ± 2 °C) and relative humidity (55 ± 5%) under a 12 h light/dark cycle, with free access to rat-chow and water. Rats were treated according to guidelines approved by local institutional animal care and use committee

CRediT authorship contribution statement

Sung-Gyoon Oh: Methodology, Software, Writing - original draft. Young-Gi Hwang: Data curation, Visualization. Hyun-Sook Lee: Conceptualization, Supervision, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

Both S.G. Oh and Y.G. Hwang were supported by Brain Korea 21+ Project from the Ministry of Education and received KU Excellent Research Scholarship (Type B) from Konkuk University.

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