Developmental Responses of the Lateral Hypothalamus to Leptin in Neonatal Rats, and its Implications for the Development of Functional Connections with the Ventral Tegmental Area

Abstract

Food intake is regulated by a close communication between the hypothalamus and the mesocorticolimbic pathways, which are still developing during the perinatal period in the rat, and are known targets for peripheral metabolic hormones such as leptin. A key region for this communication is the lateral hypothalamus (LH), although the onset of leptin responsiveness in the LH is unknown. We examined the activation of cellular signalling molecules in identified LH neurones on postnatal day (PND)10 and 16 and determined whether leptin directly targets orexin A (ORX-A) or neurotensin (NT) LH neurones through the detection of leptin receptors (ObRb) mRNA on these neurones. Next, using retrograde labelling in PND6 pups, we tested whether phenotypically identified neurones of the LH that respond to leptin project to ventral tegmental area (VTA) neurones. Leptin significantly induced phosphorylated extracellular signal-regulated kinase (pERK)1/2 and phosphorylated signal transducer activator of transcription (pSTAT)3 in the LH on PND16, whereas, on PND10, modest pERK1/2- and sparse pSTAT3-positive cells were identified. On PND16, most pERK1/2-activated neurones contain ORX-A and leptin-induced pSTAT3 was observed in other unidentified neurones. Afferents to the VTA were observed on PND6, including a large input from the LH, which contained both ORX-A-positive and non-ORX-A neurones, with some of these ORX-A neurones being activated by leptin treatment. Leptin receptor (ObRb) mRNA in the LH did not colocalise with ORX-A neurones on PND10, and only a few NT-positive neurones displayed ObRb mRNA expression. Thus, functional responsiveness to leptin in LH neurones is only partially achieved prior to the onset of independent feeding on PND16, and ORX-A neurones are indirectly activated by leptin. The presence of anatomical connections between the LH and the VTA in the first week of life, prior to the development of leptin responsiveness in both structures, suggests that tissue responsiveness to leptin, rather than the maturation of neuronal connections, critically regulates the onset of independent feeding.

Increased Hypothalamic Projections to the Lateral Hypothalamus and Responses to Leptin in Rat Neonates From High Fat Fed Mothers

“…Figure 5 illustrates the changes in pSTAT3 immunostaining in the LHA of PND16 pups following either vehicle or leptin administration in CD and HFD pups. On PND10, there was no significant treatment or diet effect and no treatment X diet interaction on the expression of pSTAT3 immunostaining in the LHA ORX-A field as shown in an earlier study (Gjerde et al, 2016). In contrast, on PND16, HFD pups exhibited significantly higher pSTAT3 activation in response to leptin compared to CD pups.…”

“…One limitation to our study is that in our experiments, retrograde fluorescent microbeads were injected into the region of the LHA containing ORX-A cells, but they may have been taken up by synapses formed with other cell types as well. For instance, we have previously shown that ORX-A neurons are intermingled with Neurotensin neurons in neonates (Gjerde et al, 2016) and thus, microbeads may have been transported by afferent fibers innervating these or other cells in this area.…”

“…The production of leptininduced intracellular signaling molecules such as phospho-STAT3 (pSTAT3) and phospho-ERK (pERK) (Kwon et al, 2016) might not only indicate the presence of functional LeptR b receptors, but also provide an index of tissue responsiveness and/or resistance to increased circulating leptin levels. We previously documented a gradual and site-specific emergence of tissue responsiveness to leptin in naïve neonates (Naef et al, 2014;Gjerde et al, 2016) with significant pSTAT3 production in the ARC already by PND10 and by PND16 in the VTA. In the LHA, increases in pERK were documented on PND16.…”

“…Injections were made to target the medial and lateral LHA ORX-A field between the adult coordinates of −2.28 and −3.36 mm anterior/posterior (A/P) from the bregma (Paxinos and Watson, 2005). These adult coordinates were adapted by a factor of 0.663 (and further adjusted based on trial and error in preliminary surgeries) to make injections to neonatal pups (Gjerde et al, 2016). The resulting coordinates used for targeting the medial and lateral portions of the LH ORX cell field were the following: A/P −1.99 to −2.6 mm, lateral: 0.8-1.2 mm, and ventral: −5.95 to −6.08 mm.…”

“…While the development of hypothalamic homeostatic connections has been well studied, much less is known on the development of efferent projections from the LHA to the VTA. Using retrograde tracing, we previously showed that functional connections already exist between these two nuclei by PND6 and that some of these originated from ORX-A neurons (Gjerde et al, 2016). Thus, by PND10, the LHA is already connected to both homeostatic and hedonic centers even though independent food intake does not start until PND17 in rat pups from control mothers or PND16 in pups from high fat diet (HFD)-fed mothers (Doerflinger and Swithers, 2004).…”