Development of the GABAergic and glutamatergic neurons of the lateral hypothalamus.

In the last few years we assist to an unexpected deluge of genomic data on hypothalamic development and structure. Perhaps most surprisingly, the Lateral Zone has received much attention too. The new information focuses first of all on transcriptional heterogeneity. Many already known and a number of hitherto unknown lateral hypothalamic neurons have been described to an enormous degree of detail. Maybe the most surprising novel discoveries are two: First, some restricted regions of the embryonic forebrain neuroepithelium generate specific LHA neurons, either GABAergic or glutamatergic. Second, evidence is mounting that supports the existence of numerous kinds of "bilingual" lateral hypothalamic neurons, expressing (and releasing) glutamate and GABA both as well as assorted neuropeptides. This is not accepted by all, and it could be that genomic researchers need a common set of rules to interpret their data (sensitivity, significance, age of analysis). In any case, some of the new results appear to confirm hypotheses about the ability of the hypothalamus and in particular its Lateral Zone to achieve physiological flexibility on a fixed connectivity ("biochemical switching"). Furthermore, the results succinctly reviewed here are the basis for future advances, since the transcriptional databases generated can now be mined e.g. for adhesion genes, to figure out the causes of the peculiar histology of the Lateral Zone; or for ion channel genes, to clarify present and future electrophysiological data. And with the specific expression data about small subpopulations of neurons, their connections can now be specifically labeled, revealing novel relations with functional significance.

Mechanisms of N-oleoyldopamine activation of central histaminergic neurons.

Histaminergic (HA) neurons located in the tuberomamillary nucleus (TMN) of the posterior hypothalamus fire exclusively during waking and support many physiological functions. We investigated the role of the endovanilloid N-oleoyldopamine (OLDA) in TMN, where dopamine synthesis and its conjugation with oleic acid likely occur. We show that several known targets of OLDA including TRPV1 and cannabinoid receptors are expressed in HA neurons. In contrast to capsaicin, which failed to increase firing of HA neurons in TRPV1 knockout mice (TRPVI KO), OLDA was still able to induce excitation. This excitation was not sensitive to the blockade of cannabinoid receptors 1 and 2 and could result from OLDA interaction with GPR119, as the ligand of GPR119, oleoylethanolamide (OEA), also increased the firing of HA neurons. However, we ruled out this possibility as OEA- (but not OLDA-) excitation was abolished by the PPAR (peroxisome proliferator activated receptor) alpha antagonist MK886. The dopamine uptake blocker nomifensine blanked OLDA-excitation and dopamine receptor antagonists abolished the OLDA action in TRPV1 KO mice. Therefore OLDA excites HA neurons through multiple targets suggesting a central role of the histaminergic system in the behavioral stimulation seen after systemic OLDA application.

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.

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.

Differential peptidomics assessment of strain and age differences in mice in response to acute cocaine administration.

Neurochemical differences in the hypothalamic-pituitary axis between individuals and between ages may contribute to differential susceptibility to cocaine abuse. This study measured peptide levels in the pituitary gland (Pit) and lateral hypothalamus (LH) in adolescent (age 30 days) and adult (age 65 days) mice from four standard inbred strains, FVB/NJ, DBA/2J, C57BL/6J, and BALB/cByJ, which have previously been characterized for acute locomotor responses to cocaine. Individual peptide profiles were analyzed using mass spectrometric profiling and principal component analysis. Sequences of assigned peptides were verified by tandem mass spectrometry. Principal component analysis classified all strains according to their distinct peptide profiles in Pit samples from adolescent mice, but not adults. Select pro-opiomelanocortin-derived peptides were significantly higher in adolescent BALB/cByJ and DBA/2J mice than in FVB/NJ or C57BL/6J mice. A subset of peptides in the LH, but not in the Pit, was altered by cocaine in adolescents. A 15 mg/kg dose of cocaine induced greater peptide alterations than a 30 mg/kg dose, particularly in FVB/NJ animals, with larger differences in adolescents than adults. Neuropeptides in the LH affected by acute cocaine administration included pro-opiomelanocortin-, myelin basic protein-, and glutamate transporter-derived peptides. The observed peptide differences could contribute to differential behavioral sensitivity to cocaine among strains and ages. Peptides were measured using mass spectrometry (MALDI-TOF) in individual lateral hypothalamus and pituitary samples from four strains and two ages of inbred mice in response to acute cocaine administration. Principal component analyses (PCA) classified the strains according to their peptide profiles from adolescent mice, and a subset of peptides in the lateral hypothalamus was altered by cocaine in adolescents.

Trophic action of leptin on hypothalamic neurons that regulate feeding.

In adult mammals, the adipocyte-derived hormone leptin acts on the brain to reduce food intake by regulating the activity of neurons in the arcuate nucleus of the hypothalamus (ARH). Here, we report that neural projection pathways from the ARH are permanently disrupted in leptin-deficient (Lepob/Lepob) mice and leptin treatment in adulthood does not reverse these neuroanatomical defects. However, treatment of Lepob/Lepob neonates with exogenous leptin rescues the development of ARH projections, and leptin promotes neurite outgrowth from ARH neurons in vitro. These results suggest that leptin plays a neurotrophic role during the development of the hypothalamus and that this activity is restricted to a neonatal critical period that precedes leptin's acute regulation of food intake in adults.

Formation of projection pathways from the arcuate nucleus of the hypothalamus to hypothalamic regions implicated in the neural control of feeding behavior in mice.

The arcuate nucleus of the hypothalamus (ARH) is a critical component of forebrain pathways that regulate a variety of neuroendocrine functions, including an important role in relaying leptin signals to other parts of the hypothalamus. However, neonatal rodents do not lose weight in response to leptin treatment in the same way as do adults, suggesting that certain aspects of leptin signaling pathways in the hypothalamus may not be mature. We tested this possibility by using DiI axonal labeling to examine the development of projections from the ARH to other parts of the hypothalamus in neonatal mice, paying particular attention to the innervation of the paraventricular nucleus (PVH), the dorsomedial nucleus (DMH), and the lateral hypothalamic area (LHA), each of which have been implicated in the regulation of feeding. The results indicate that ARH projections are quite immature at birth and appear to innervate the DMH, PVH, and LHA in succession, within distinct temporal domains. The projections from the ARH to the DMH develop rapidly and are established by the sixth postnatal day (P6), whereas those to the PVH develop significantly later, with the mature pattern of innervation first apparent between postnatal day 8 (P8)-P10. Furthermore, the ability of leptin to activate Fos in the PVH, DMH, and LHA appears to be age-dependent and correlates with the arrival of ARH projections to each nucleus. Taken together, these findings provide new insight into development of hypothalamic circuits and suggest an anatomical basis for the delayed postnatal regulation of food intake and body weight by leptin.

Lateral hypothalamus: early developmental expression and response to hypocretin (orexin).

Hypocretin is a recently discovered peptide that is synthesized by neurons in the lateral hypothalamic area (LH) and is believed to play a role in sleep regulation, arousal, endocrine control, and food intake. These functions are critical for the development of independent survival. We investigated the developmental profile of the hypocretin system in rats. Northern blot analysis showed that the expression of hypocretin mRNA increased from postnatal day 1 to adulthood. Both of the identified hypocretin receptor mRNAs were strongly expressed very early in hypothalamic development, and expression subsequently decreased in the mature brain. Immunocytochemistry revealed hypocretin-2 peptide expression in the cell bodies of LH neurons and in axons in the brain and spinal cord as early as embryonic day 19. Whole-cell patch clamp recordings from postnatal P1-P14 LH slices demonstrated a robust increase in synaptic activity in all LH neurons tested (n = 20) with a 383% increase in the frequency of spontaneous activity upon hypocretin-2 (1.5 microM) application. A similar increase in activity was found with hypocretin-1 application to LH slices. Hypocretin-2 evoked a robust increase in synaptic activity even on the earliest day tested, the day of birth. Furthermore, voltage-clamp recordings and calcium digital imaging experiments using cultured LH cells revealed that both hypocretin-1 and -2 induced enhancement of neuronal activity occurred as early as synaptic activity was detected. Thus, as in the adult central nervous system, hypocretin exerts a profound excitatory influence on neuronal activity early in development, which might contribute to the development of arousal, sleep regulation, feeding, and endocrine control.

Dietary composition during fetal and neonatal life affects neuropeptide Y functioning in adult offspring.

The aim of this study was to examine the impact of maternal diet during the gestation and lactation periods on the neuropeptide Y (NPY) system in adult offspring. Male Long-Evans rats were obtained from dams fed either on a well-balanced diet (C), a high carbohydrate diet (HC) or a high-fat diet (HF) and fed themselves on the well-balanced diet for their whole life. At 6 months of age, their feeding response to various doses of NPY injected in the lateral brain ventricle was measured in one group and NPY concentrations in microdissected nuclei of the hypothalamic were measured in a second group. The HF rats were lighter than the two other groups (P<0.001). The control rats showed a typical dose-dependent feeding response to NPY. The HC rats showed a continuous increase in the response, starting at the intermediate dose (1.0 microg) only while the HF rats had a maximal response at the lowest dose (0.5 microg). The HF rats ate twice as much as the HC rats at the lowest dose tested 1 h after injection (4.4+/-0.6 vs. 2.7+/-0.4 g; P<0.05), showing therefore the greatest sensitivity to NPY. This change in the sensitivity was not related to hypothalamic NPY concentration as it was not modified in the arcuate and paraventricular nuclei. The diet imposed on the mother could have long-lasting effects on body weight regulation of the offsprings and alter the NPY system likely through modifications at the receptor level.

Retinohypothalamic tract development in the hamster and rat.

The development of the retinohypothalamic tract (RHT) in the albino rat and golden hamster was studied using anterograde transport of cholera toxin conjugated to horseradish peroxidase (CT-HRP). The RHT has three components in the adult: (1) a dense projection to the ventrolateral subdivision of the suprachiasmatic nucleus (SCN) with some fibers extending into the dorsomedial SCN; (2) a projection to adjacent areas, the anterior hypothalamic area (AHA) and retrochiasmatic area (RCA) and in the hamsters, into the preoptic area (POA); (3) a projection to the lateral hypothalamic area (LHA). In the rat, the projection to the SCN and adjacent areas first appears as scattered varicosities at the ventral border of the SCN at postnatal day 1 (P1) and gradually increases until the adult pattern is achieved at approximately P10. The projections to the AHA and RCA are seen first at P2-P3 and gradually increase to the adult appearance by P15. Both the projection to the SCN and adjacent areas and to the LHA, initially are more extensive than in the adult. Many of the axons extend well beyond the zone of the adult pattern but these anomalous fibers are eliminated by P6-P10. The LHA projection first appears at embryonic day 21-22 (E 21-22) and gradually increases in density from P1-P6. In the hamster the projections to the SCN, AHA and LHA appear first on P4 and gradually increase in density to reach the adult pattern by P15. The projections to the RCA and POA are present by P6 and reach the adult pattern by P15. None of the RHT projections in the hamster has the initial extended growth followed by pruning back that characterizes RHT development in the rat. Thus, the development of the RHT in both the rat and the hamster is complex with components of the projection appearing at different times with differing patterns of development that indicate specialized interactions of the developing axons with their target neurons. Synaptogenesis in the hamster hypothalamus was analyzed using an antiserum to synapsin I. Few synapses are present at E16, the last day of gestation, in the LHA, SCN and AHA. From P1-P3, synaptogenesis proceeds rapidly and the adult pattern is achieved in all three areas by P4.

The pharmaco-ontogeny of the perifornical lateral hypothalamic beta 2-adrenergic and dopaminergic receptor systems mediating epinephrine- and dopamine-induced suppression of feeding in the rat.

The functional ontogeny of beta 2-adrenergic and dopaminergic receptors in the perifornical lateral hypothalamus (PLH) that mediate adrenergic and dopaminergic suppression of feeding in rats was investigated. Rat pups, ranging in age from 2 to 15 days, were removed from their mothers and implanted with a brain cannula directed unilaterally at the PLH or a more rostral site lateral to the anterior nucleus of the hypothalamus. On the next day, following a 22-h period of food and water deprivation, each pup was implanted with an intra-oral cannula for oral infusion of milk that could be swallowed or rejected. Subsequently, each pup received an intracerebral injection of saline, or a single dose of epinephrine (EPI, 0.1-30.0 nmol), the beta 2-adrenergic receptor agonist salbutamol (1.0-30.0 nmol) or the dopaminergic receptor agonist apomorphine (1.0-30.0 nmol). Milk intake was then assessed following a 1-h period of infusion. The results showed significant dose-dependent suppression of milk intake in pups as young as 2 days of age in response to PLH injection of EPI, salbutamol and apomorphine. In contrast to its effectiveness in the PLH at 2 days of age, EPI failed to suppress milk intake at this age following injection into a more rostral site lateral to the anterior nucleus of the hypothalamus. Together, these findings suggest that both beta 2-adrenergic and dopaminergic receptors, mediating adrenergic and dopaminergic suppression of feeding, are functionally mature very early in the postnatal development of the rat. Moreover, consistent with evidence in adult rats, these catecholaminergic receptors in young pups appear to be located in the region of the PLH.

The development of the diencephalon of the rainbow trout (Salmo gairdneri Richardson). Thalamus and hypothalamus.

This article describes a study of the development of the thalamus and hypothalamus of the rainbow trout from the embryo to the juvenile fish. The areas from which the various nuclei derived are reported together with the order of their appearance, the earliest being the nucleus geniculatus lateralis, the nucleus pretectalis, the nucleus of the torus lateralis hypothalami, the nucleus preglomerulosus and the nucleus diffusus of the lateral lobes, all of were beginning to be distinguishable in 9 mm embryos. The lateral lobes of the hypothalamus developed mainly after hatching, and medial regions generally developed later than lateral areas, though certain periventricular structures appeared very soon. Cell proliferation persisted in 35 mm juveniles, especially in the walls of the lateral and posterior recesses.

Postnatal development of sensory influences on neurons in the ventromedial hypothalamic nucleus of the rat.

Extracellular unitary records were obtained from neurons in the ventromedial hypothalamic nucleus (VMH) of very young (1-25 days of postnatal age) and adult rats. Spontaneous unitary activity and evoked responses to both external (somatic, gustatory, and olfactory) and internal sensory (systemic administration of hypertonic saline and glucose solutions) stimulation were determined in order to assess the functional development of VMH neurons and their afferents. The basic electrophysiological characteristics of VMH neurons were established prenatally. From the date of birth, many VMH neurons had: spontaneous action potential generation; evoked responses to external or internal sensory stimulation; and convergent sensory inputs. In contrast, the major developmental change in the neurophysiological properties of VMH neurons was the diminution with increasing age of the convergence of external and internal sensory influences. This developmental 'fine-tuning' of a complex functional feature of VMH neurons is important because the maturation of convergence coincides with a 'critical period' of VMH ontogenesis demonstrated in behavioral and experimental brain damage reports.

Postnatal development of sensory influences on lateral hypothalamic neurons of the rat.

Extracellular unitary records were obtained from neurons in the lateral hypothalamic area (LHA) of very young (1-25 days of postnatal age) and adult rats. Spontaneous unitary activity and evoked responses to external sensory (somatosensory, gustatory and olfactory) and internal sensory (administration of hypertonic saline and glucose solutions) stimulation were determined in these neurons in order to assess their functional development. The basic electrophysiological characteristics of LHA neurons were established prenatally. From the day of birth, many LHA neurons had: (1) spontaneous spike generation; (2) evoked responses to external and internal sensory stimulation; and (3) convergent sensory inputs. However, each of these neurophysiological properties was modified considerably during postnatal ontogenesis: (4) spontaneous activity of LHA neurons with evoked responses to sensory stimulation increased with age; (5) internal sensory influences tended to diminish while evoked response magnitude and complexity were augmented developmentally; and (6) convergence of sensory influences decreased in older rats. This developmental 'fine tuning' of the functional features of LHA neurons is important because their maturation coincides with certain 'critical periods' of LHA ontogenesis demonstrated in behavioral, morphological, pharmacological and experimental brain damage reports.

Organized behavioral responses to lateral hypothalamic electrical stimulation in infant rats.

Brief trains of electrical stimulation (500 msec) were administered to 3-, 6-, 10-, and 15-day-old rat pups through electrodes directed at the medical forebrain bundle (MFB) at the level of the lateral hypothalamus. Stimulation was given at three different frequencies: once per 30 sec, once per 20 sec, and once per 10 sec. Pups 10 days of age and younger became behaviorally activated by stimulation and reliably emitted a series of behavioral responses including mouthing, licking, pawing, gaping, probing, and stretch and lordosis responses. Behavior increased with increasing frequency of stimulation and became more organized with age. Sequences of organized behavior patterns are elicited by MFB stimulation which in their culmination take on the characteristics of motivational responses. Fifteen-day-old pups did not respond to these stimulation parameters. These findings indicate that the components of motivational systems are present and can be elicited at an early age. As development progresses, responses become more organized until stimulation parameters effective in rats 3 to 10 days of age become ineffective in activating behavior in day 15 pups.