Melanocortin 3 receptor-expressing neurons in the ventromedial hypothalamus promote glucose disposal.

The ventromedial hypothalamus (VMH) is a critical neural node that senses blood glucose and promotes glucose utilization or mobilization during hypoglycemia. The VMH neurons that control these distinct physiologic processes are largely unknown. Here, we show that melanocortin 3 receptor (Mc3R)-expressing VMH neurons (VMHMC3R) sense glucose changes both directly and indirectly via altered excitatory input. We identify presynaptic nodes that potentially regulate VMHMC3R neuronal activity, including inputs from proopiomelanocortin (POMC)-producing neurons in the arcuate nucleus. We find that VMHMC3R neuron activation blunts, and their silencing enhances glucose excursion following a glucose load. Overall, these findings demonstrate that VMHMC3R neurons are a glucose-responsive hypothalamic subpopulation that promotes glucose disposal upon activation; this highlights a potential site for targeting dysregulated glycemia.

Potassium channel dysfunction underlies Purkinje neuron spiking abnormalities in spinocerebellar ataxia type 2.

Alterations in Purkinje neuron firing often accompany ataxia, but the molecular basis for these changes is poorly understood. In a mouse model of spinocerebellar ataxia type 2 (SCA2), a progressive reduction in Purkinje neuron firing frequency accompanies cell atrophy. We investigated the basis for altered Purkinje neuron firing in SCA2. A reduction in the expression of large-conductance calcium-activated potassium (BK) channels and Kv3.3 voltage-gated potassium channels accompanies the inability of Purkinje neurons early in disease to maintain repetitive spiking. In association with prominent Purkinje neuron atrophy, repetitive spiking is restored, although at a greatly reduced firing frequency. In spite of a continued impairment in spike repolarization and a persistently reduced BK channel mediated afterhyperpolarization (AHP), repetitive spiking is maintained, through the increased activity of barium-sensitive potassium channels, most consistent with inwardly rectifying potassium (Kir) channels. Increased activity of Kir channels results in the generation of a novel AHP not seen in wild-type Purkinje neurons that also accounts for the reduced firing frequency late in disease. Homeostatic changes in Purkinje neuron morphology that help to preserve repetitive spiking can also therefore have deleterious consequences for spike frequency. These results suggest that the basis for spiking abnormalities in SCA2 differ depending on disease stage, and interventions targeted towards correcting potassium channel dysfunction in ataxia need to be tailored to the specific stage in the degenerative process.

Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease.

Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.

Neuronal Atrophy Early in Degenerative Ataxia Is a Compensatory Mechanism to Regulate Membrane Excitability.

Neuronal atrophy in neurodegenerative diseases is commonly viewed as an early event in a continuum that ultimately results in neuronal loss. In a mouse model of the polyglutamine disorder spinocerebellar ataxia type 1 (SCA1), we tested the hypothesis that cerebellar Purkinje neuron atrophy serves an adaptive role rather than being simply a nonspecific response to injury. In acute cerebellar slices from SCA1 mice, we find that Purkinje neuron pacemaker firing is initially normal but, with the onset of motor dysfunction, becomes disrupted, accompanied by abnormal depolarization. Remarkably, subsequent Purkinje cell atrophy is associated with a restoration of pacemaker firing. The early inability of Purkinje neurons to support repetitive spiking is due to unopposed calcium currents resulting from a reduction in large-conductance calcium-activated potassium (BK) and subthreshold-activated potassium channels. The subsequent restoration of SCA1 Purkinje neuron firing correlates with the recovery of the density of these potassium channels that accompanies cell atrophy. Supporting a critical role for BK channels, viral-mediated increases in BK channel expression in SCA1 Purkinje neurons improves motor dysfunction and partially restores Purkinje neuron morphology. Cerebellar perfusion of flufenamic acid, an agent that restores the depolarized membrane potential of SCA1 Purkinje neurons by activating potassium channels, prevents Purkinje neuron dendritic atrophy. These results suggest that Purkinje neuron dendritic remodeling in ataxia is an adaptive response to increases in intrinsic membrane excitability. Similar adaptive remodeling could apply to other vulnerable neuronal populations in neurodegenerative disease. SIGNIFICANCE STATEMENT: In neurodegenerative disease, neuronal atrophy has long been assumed to be an early nonspecific event preceding neuronal loss. However, in a mouse model of spinocerebellar ataxia type 1 (SCA1), we identify a previously unappreciated compensatory role for neuronal shrinkage. Purkinje neuron firing in these mice is initially normal, but is followed by abnormal membrane depolarization resulting from a reduction in potassium channels. Subsequently, these electrophysiological effects are counteracted by cell atrophy, which by restoring normal potassium channel membrane density, re-establishes pacemaker firing. Reversing the initial membrane depolarization improved motor function and Purkinje neuron morphology in the SCA1 mice. These results suggest that Purkinje neuron remodeling in ataxia is an active compensatory response that serves to normalize intrinsic membrane excitability.

Single amino acid deletion in transmembrane segment D4S6 of sodium channel Scn8a (Nav1.6) in a mouse mutant with a chronic movement disorder.

Mutations of the neuronal sodium channel gene SCN8A are associated with lethal movement disorders in the mouse and with human epileptic encephalopathy. We describe a spontaneous mouse mutation, Scn8a(9J), that is associated with a chronic movement disorder with early onset tremor and adult onset dystonia. Scn8a(9J) homozygotes have a shortened lifespan, with only 50% of mutants surviving beyond 6 months of age. The 3 bp in-frame deletion removes 1 of the 3 adjacent isoleucine residues in transmembrane segment DIVS6 of Nav1.6 (p.Ile1750del). The altered helical orientation of the transmembrane segment displaces pore-lining amino acids with important roles in channel activation and inactivation. The predicted impact on channel activity was confirmed by analysis of cerebellar Purkinje neurons from mutant mice, which lack spontaneous and induced repetitive firing. In a heterologous expression system, the activity of the mutant channel was below the threshold for detection. Observations of decreased nerve conduction velocity and impaired behavior in an open field are also consistent with reduced activity of Nav1.6. The Nav1.6Δ1750 protein is only partially glycosylated. The abundance of mutant Nav1.6 is reduced at nodes of Ranvier and is not detectable at the axon initial segment. Despite a severe reduction in channel activity, the lifespan and motor function of Scn8a(9J/9J) mice are significantly better than null mutants lacking channel protein. The clinical phenotype of this severe hypomorphic mutant expands the spectrum of Scn8a disease to include a recessively inherited, chronic and progressive movement disorder.

Early changes in cerebellar physiology accompany motor dysfunction in the polyglutamine disease spinocerebellar ataxia type 3.

The relationship between cerebellar dysfunction, motor symptoms, and neuronal loss in the inherited ataxias, including the polyglutamine disease spinocerebellar ataxia type 3 (SCA3), remains poorly understood. We demonstrate that before neurodegeneration, Purkinje neurons in a mouse model of SCA3 exhibit increased intrinsic excitability resulting in depolarization block and the loss of the ability to sustain spontaneous repetitive firing. These alterations in intrinsic firing are associated with increased inactivation of voltage-activated potassium currents. Administration of an activator of calcium-activated potassium channels, SKA-31, partially corrects abnormal Purkinje cell firing and improves motor function in SCA3 mice. Finally, expression of the disease protein, ataxin-3, in transfected cells increases the inactivation of Kv3.1 channels and shifts the activation of Kv1.2 channels to more depolarized potentials. Our results suggest that in SCA3, early Purkinje neuron dysfunction is associated with altered physiology of voltage-activated potassium channels. We further suggest that the observed changes in Purkinje neuron physiology contribute to disease pathogenesis, underlie at least some motor symptoms, and represent a promising therapeutic target in SCA3.

Ventromedial hypothalamic nucleus neuronal subset regulates blood glucose independently of insulin.

To identify neurons that specifically increase blood glucose from among the diversely functioning cell types in the ventromedial hypothalamic nucleus (VMN), we studied the cholecystokinin receptor B-expressing (CCKBR-expressing) VMN targets of glucose-elevating parabrachial nucleus neurons. Activation of these VMNCCKBR neurons increased blood glucose. Furthermore, although silencing the broader VMN decreased energy expenditure and promoted weight gain without altering blood glucose levels, silencing VMNCCKBR neurons decreased hIepatic glucose production, insulin-independently decreasing blood glucose without altering energy balance. Silencing VMNCCKBR neurons also impaired the counterregulatory response to insulin-induced hypoglycemia and glucoprivation and replicated hypoglycemia-associated autonomic failure. Hence, VMNCCKBR cells represent a specialized subset of VMN cells that function to elevate glucose. These cells not only mediate the allostatic response to hypoglycemia but also modulate the homeostatic setpoint for blood glucose in an insulin-independent manner, consistent with a role for the brain in the insulin-independent control of glucose homeostasis.

Fetal programming: excess prenatal testosterone reduces postnatal luteinizing hormone, but not follicle-stimulating hormone responsiveness, to estradiol negative feedback in the female.

Exposure of female sheep fetuses to excess testosterone (T) during early to midgestation produces postnatal hypergonadotropism manifest as a selective increase in LH. This hypergonadotropism may result from reduced sensitivity to estradiol (E2) negative feedback and/or increased pituitary sensitivity to GnRH. We tested the hypothesis that excess T before birth reduces responsiveness of LH and FSH to E2 negative feedback after birth. Pregnant ewes were treated with T propionate (100 mg/kg in cotton seed oil) or vehicle twice weekly from d 30-90 gestation. Responsiveness to E2 negative feedback was assessed at 12 and 24 wk of age in the ovary-intact female offspring. Our experimental strategy was first to arrest follicular growth and reduce endogenous E2 by administering the GnRH antagonist (GnRH-A), Nal-Glu (50 microg/kg sc every 12 h for 72 h), and then provide a fixed amount of exogenous E2 via an implant. Blood samples were obtained every 20 min at 12 wk and every 10 min at 24 wk before treatment, during and after GnRH-A treatment both before and after E2 implant. GnRH-A ablated LH pulsatility, reduced FSH by approximately 25%, and E2 production diminished to near detection limit of assay at both ages in both groups. Prenatal T treatment produced a precocious and selective reduction in responsiveness of LH but not FSH to E2 negative feedback, which was manifest mainly at the level of LH/GnRH pulse frequency. Collectively, these findings support the hypothesis that prenatal exposure to excess T decreases postnatal responsiveness to E2 inhibitory feedback of LH/GnRH secretion to contribute to the development of hypergonadotropism.

The influence of subthalamic nucleus lesions on sign-tracking to stimuli paired with food and drug rewards: facilitation of incentive salience attribution?

It is well known that the subthalamic nucleus (STN) plays an important role in regulating motor function, but recent studies suggest the STN is also involved in regulating motivated behavior. For example, bilateral lesions of the STN increase motivation for both food and cocaine as assessed by 'breakpoint' on a progressive ratio schedule. However, the psychological mechanism(s) by which STN lesions increase motivation for rewards is unknown. We hypothesized that STN lesions might influence one specific component of motivation, the attribution of incentive value (incentive salience) to reward-related cues. We tested this hypothesis by quantifying the ability of a discrete cue that had been paired with the non-contingent delivery of either food or cocaine to elicit approach towards it (ie, to produce a 'sign-tracking' conditioned response, CR). STN lesions made prior to training increased asymptotic levels of sign-tracking behavior directed towards a cue paired with either food or cocaine. In addition, when STN lesions were made after animals had already undergone Pavlovian training, and animals were tested under extinction conditions, the STN lesion still facilitated a sign-tracking CR. Finally, reintroduction of the US (food) following extinction immediately restored robust sign-tracking behavior in animals with STN lesions. We speculate, therefore, that the STN is part of a neural system that moderates the amount of incentive salience attributed to reward-related stimuli. Activity in this neural system may help mitigate the development of compulsive behavioral disorders, such as addiction, which are characterized by pathological control over behavior by reward-associated cues.