AMPK facilitates the hypoxic ventilatory response through non-adrenergic mechanisms at the brainstem.

We recently demonstrated that the hypoxic ventilatory response (HVR) is facilitated by the AMP-activated protein kinase (AMPK) in catecholaminergic neural networks that likely lie downstream of the carotid bodies within the caudal brainstem. Here, we further subcategorise the neurons involved, by cross-comparison of mice in which the genes encoding the AMPK-α1 (Prkaa1) and AMPK-α2 (Prkaa2) catalytic subunits were deleted in catecholaminergic (TH-Cre) or adrenergic (PNMT-Cre) neurons. As expected, the HVR was markedly attenuated in mice with AMPK-α1/α2 deletion in catecholaminergic neurons, but surprisingly was modestly augmented in mice with AMPK-α1/α2 deletion in adrenergic neurons when compared against a variety of controls (TH-Cre, PNMT-Cre, AMPK-α1/α2 floxed). Moreover, AMPK-α1/α2 deletion in catecholaminergic neurons precipitated marked hypoventilation and apnoea during poikilocapnic hypoxia, relative to controls, while mice with AMPK-α1/α2 deletion in adrenergic neurons entered relative hyperventilation with reduced apnoea frequency and duration. We conclude, therefore, that AMPK-dependent modulation of non-adrenergic networks may facilitate increases in ventilatory drive that shape the classical HVR, whereas AMPK-dependent modulation of adrenergic networks may provide some form of negative feedback or inhibitory input to moderate HVR, which could, for example, protect against hyperventilation-induced hypocapnia and respiratory alkalosis.

The catecholaminergic innervation of the claustrum of the pig.

Over the past decades, the number of studies employing the pig brain as a model for neurochemical studies has dramatically increased. The key translational features of the pig brain are the similarities with the cortical and subcortical structures of the human brain. In addition, the caudalmost part of the pig claustrum (CL) is characterized by a wide enlargement called posterior puddle, an ideal structure for physiological recordings. Several hypotheses have been proposed for CL function, the key factor being its reciprocal connectivity with most areas of the cerebral cortex and selected subcortical structures. However, afferents from the brainstem could also be involved. The brainstem is the main source of catecholaminergic axons that play an important neuromodulatory action in different brain functions. To study a possible role of the CL in catecholaminergic pathways, we analyzed the presence and the distribution of afferents immunostained with antibodies against tyrosine hydroxylase (TH) and dopamine betahydroxylase (DBH) in the pig CL. Here we show that the CL contains significant TH immunoreactive axons contacting perikarya, whereas projections staining for DBH are very scarce. Our findings hint at the possibility that brainstem catecholaminergic afferents project to the CL, suggesting (i) a possible role of this nucleus in functions controlled by brainstem structures; and, consequently, (ii) its potential involvement in the pathophysiology of neurodegenerative pathologies, including Parkinson's disease (PD).

Selective inhibition of dopamine-beta-hydroxylase enhances dopamine release from noradrenergic terminals in the medial prefrontal cortex.

INTRODUCTION: Disulfiram has been claimed to be useful in cocaine addiction therapy, its efficacy being attributed to dopamine-beta-hydroxylase (DBH) inhibition. Our previous results indicate that disulfiram and the selective DBH inhibitor nepicastat increase extracellular dopamine (DA) in the rat medial prefrontal cortex (mPFC), and markedly potentiated cocaine-induced increase. Concomitantly, in rats with cocaine self-administration history, cocaine-seeking behavior induced by drug priming was prevented, probably through overstimulation of D1 receptors due to the DA increase. The present research was aimed at studying the neurochemical mechanisms originating the enhanced DA release. METHODS: Noradrenergic system ablation was attained by intracerebroventricular (i.c.v.) administration of the neurotoxin anti-DBH-saporin (aDBH-sap). DA, noradrenaline (NA), and DOPAC were assessed by HPLC after ex vivo tissue extraction or in vivo microdialysis. Control and denervated rats were subjected to microdialysis in the mPFC and caudate nucleus to evaluate the effect of nepicastat-cocaine combination on extracellular DA levels and their regulation by α2-adrenoceptors. RESULTS: Fifteen days after neurotoxin or its vehicle administration, tissue and extracellular NA were reduced to less than 2% the control value, while extracellular DA was increased by approximately 100%. In control rats, nepicastat given alone and in combination with cocaine increased extracellular DA by about 250% and 1100%, respectively. In denervated rats, nepicastat slightly affected extracellular DA, while in combination with cocaine increased extracellular DA by 250%. No differences were found in the caudate nucleus. Clonidine almost totally reversed the extracellular DA elevation produced by nepicastat-cocaine combination, while it was ineffective in denervated rats. CONCLUSIONS: This research shows that the increase of extracellular DA produced by nepicastat alone or in combination with cocaine was prevented by noradrenergic denervation. The results indicate that nepicastat enhances DA release from noradrenergic terminals supposedly by removing NA from α2-autoreceptors. In addition to the inhibition of DA uptake, the latter mechanism may explain the synergistic effect of cocaine on nepicastat-induced DA release.

Functional impact of glycogen degradation on astrocytic signalling.

Astrocytic glycogen degradation is an important factor in metabolic support of brain function, particularly during increased neuronal firing. In this context, glycogen is commonly thought of as a source for the provision of energy substrates, such as lactate, to neurons. However, the signalling pathways eliciting glycogen degradation inside astrocytes are themselves energy-demanding processes, a fact that has been emphasized in recent studies, demonstrating dependence of these signalling mechanisms on glycogenolytic ATP.

Knockdown of tyrosine hydroxylase in the nucleus of the solitary tract reduces elevated blood pressure during chronic intermittent hypoxia.

Noradrenergic A2 neurons in nucleus tractus solitarius (NTS) respond to stressors such as hypoxia. We hypothesize that tyrosine hydroxylase (TH) knockdown in NTS reduces cardiovascular responses to chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia observed during sleep apnea in humans. Adult male Sprague-Dawley rats were implanted with radiotelemetry transmitters and adeno-associated viral constructs with green fluorescent protein (GFP) reporter having either short hairpin RNA (shRNA) for TH or scrambled virus (scRNA) were injected into caudal NTS. Virus-injected rats were exposed to 7 days of CIH (alternating periods of 10% O2 and of 21% O2 from 8 AM to 4 PM; from 4 PM to 8 AM rats were exposed to 21% O2). CIH increased mean arterial pressure (MAP) and heart rate (HR) during the day in both the scRNA (n = 14, P < 0.001 MAP and HR) and shRNA (n = 13, P < 0.001 MAP and HR) groups. During the night, MAP and HR remained elevated in the scRNA rats (P < 0.001 MAP and HR) but not in the shRNA group. TH immunoreactivity and protein were reduced in the shRNA group. FosB/ΔFosB immunoreactivity was decreased in paraventricular nucleus (PVN) of shRNA group (P < 0.001). However, the shRNA group did not show any change in the FosB/ΔFosB immunoreactivity in the rostral ventrolateral medulla. Exposure to CIH increased MAP which persisted beyond the period of exposure to CIH. Knockdown of TH in the NTS reduced this CIH-induced persistent increase in MAP and reduced the transcriptional activation of PVN. This indicates that NTS A2 neurons play a role in the cardiovascular responses to CIH.

Developmental origins of central norepinephrine neuron diversity.

Central norepinephrine-producing neurons comprise a diverse population of cells differing in anatomical location, connectivity, function and response to disease and environmental insult. The mechanisms that generate this diversity are unknown. Here we elucidate the lineal relationship between molecularly distinct progenitor populations in the developing mouse hindbrain and mature norepinephrine neuron subtype identity. We have identified four genetically separable subpopulations of mature norepinephrine neurons differing in their anatomical location, axon morphology and efferent projection pattern. One of the subpopulations showed an unexpected projection to the prefrontal cortex, challenging the long-held belief that the locus coeruleus is the sole source of norepinephrine projections to the cortex. These findings reveal the embryonic origins of central norepinephrine neurons and provide multiple molecular points of entry for future study of individual norepinephrine circuits in complex behavioral and physiological processes including arousal, attention, mood, memory, appetite and homeostasis.

Regional and cell-type specific distribution of HDAC2 in the adult mouse brain.

The effects of epigenetics on brain functions are not completely understood, but histone deacetylases (HDACs) are known to affect brain function and dysfunction by mediating the acetylation status of target proteins, thereby affecting gene expression. The current study used immunochemistry to illuminate the regional distribution of one member of the HDAC family, HDAC2, in the C57BL/6J mouse brain. Our data show that HDAC2 is ubiquitously expressed throughout the mouse brain and is localized primarily within the cell nucleus. Using double-immunofluorescence, we demonstrated HDAC2 expression in neuronal cells, including cholinergic, serotonergic and catecholaminergic neurons, as well as postsynaptic glutamatergic and GABAergic neurons. HDAC2 was also observed in oligodendrocytes, but not in astrocytes or microglia. These detailed immunological studies illuminate the distribution of HDAC2 throughout the mouse brain and will facilitate investigation of the roles of HDAC2 in brain function and neurological disorders.

Differential effects of systemic interleukin-1ß on gene expression in brainstem noradrenergic nuclei.

AIMS: The cytokine, interleukin-1ß (IL-1ß), is known to produce specific effects on the neuroendocrine system such as suppression of the reproductive axis and stimulation of the stress axis. The mechanism by which IL-1ß produces these differential effects is not clear. Since norepinephrine (NE) is involved in these effects, we hypothesized that IL-1ß acts on brainstem noradrenergic nuclei to affect gene transcription of NE synthesizing enzymes, cytokines and associated transcription factors. MAIN METHODS: Adult female Sprague Dawley rats in proestrus were divided into two groups. Control animals received PBS-BSA and the treatment group received 5 µg of rat recombinant IL-1ß i.p. at noon. They were sacrificed in groups at 1, 3 and 5 pm (n=6/group) for measurement of tyrosine hydroxylase (TH) mRNA by qPCR or at 3 pm for mRNA analysis by qPCR array. KEY FINDINGS: TH mRNA levels decreased gradually with time in both control and IL-1ß-treated rats in the ventrolateral medulla. In the nucleus of solitary tract, TH mRNA levels were significantly reduced by IL-1ß treatment at 5 pm. In the locus coeruleus, TH mRNA levels increased significantly at 5 pm with IL-1ß treatment compared to controls. In the second set of animals analyzed by qPCR array, there were several fold increases in the expression of certain cytokines, chemokines, and transcription factors in specific noradrenergic nuclei. SIGNIFICANCE: Systemic administration of IL-1ß causes significant changes in the expression of tyrosine hydroxylase and several chemokines in brain stem noradrenergic nuclei, thereby mediating its neuroendocrine effects.