Marina Cholanian

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• Levine, R. B., Wealing, J. C., Levine, R. B., Fregosi, R. F., Flanigan, E. G., & Cholanian, M. (2019). Diverse physiological properties of hypoglossal motoneurons innervating intrinsic and extrinsic tongue muscles.. Journal of neurophysiology, 122(5), 2054-2060. doi:10.1152/jn.00478.2019
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  The mammalian tongue contains eight muscles that collaborate to ensure that suckling, swallowing, and other critical functions are robust and reliable. Seven of the eight tongue muscles are innervated by hypoglossal motoneurons (XIIMNs). A somatotopic organization of the XII motor nucleus, defined in part by the mechanical action of a neuron's target muscle, has been described, but whether or not XIIMNs within a compartment are functionally specialized is unsettled. We hypothesize that developing XIIMNs are assigned unique functional properties that reflect the challenges that their target muscle faces upon the transition from in utero to terrestrial life. To address this, we studied XIIMNs that innervate intrinsic and extrinsic tongue muscles, because intrinsic muscles play a more prominent role in suckling than the extrinsic muscles. We injected dextran-rhodamine into the intrinsic longitudinal muscles (IL) and the extrinsic genioglossus, and physiologically characterized the labeled XIIMNs. Consistent with earlier work, IL XIIMNs (n = 150) were located more dorsally within the nucleus, and GG XIIMNs (n = 55) more ventrally. Whole cell recordings showed that resting membrane potential was, on average, 9 mV more depolarized in IL than in GG XIIMNs (P = 0.0019), and the firing threshold in response to current injection was lower in IL (-31 ± 23 pA) than in GG XIIMNs (225 ± 39 pA; P < 0.0001). We also found that the appearance of net outward currents in GG XIIMNs occurred at more hyperpolarized membrane potentials than IL XIIMNs, consistent with lower excitability in GG XIIMNs. These observations document muscle-specific functional specializations among XIIMNs.NEW & NOTEWORTHY The hypoglossal motor nucleus contains motoneurons responsible for innervating one of seven different muscles with notably different biomechanics and patterns of use. Whether or not motoneurons innervating the different muscles also have unique functional properties (e.g., spiking behavior, synaptic physiology) is poorly understood. In this work we show that neonatal hypoglossal motoneurons innervating muscles that shape the tongue (intrinsic longitudinal muscles) have different electrical properties than those innervating the genioglossus, which controls tongue position.
• Levine, R. B., Powell, G. L., Levine, R. B., Fregosi, R. F., & Cholanian, M. (2017). Influence of developmental nicotine exposure on glutamatergic neurotransmission in rhythmically active hypoglossal motoneurons.. Experimental neurology, 287(Pt 2), 254-260. doi:10.1016/j.expneurol.2016.07.023
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  Developmental nicotine exposure (DNE) is associated with increased risk of cardiorespiratory, intellectual, and behavioral abnormalities in neonates, and is a risk factor for apnea of prematurity, altered arousal responses and Sudden Infant Death Syndrome. Alterations in nicotinic acetylcholine receptor signaling (nAChRs) after DNE lead to changes in excitatory neurotransmission in neural networks that control breathing, including a heightened excitatory response to AMPA microinjection into the hypoglossal motor nucleus. Here, we report on experiments designed to probe possible postsynaptic and presynaptic mechanisms that may underlie this plasticity. Pregnant dams were exposed to nicotine or saline via an osmotic mini-pump implanted on the 5th day of gestation. We used whole-cell patch clamp electrophysiology to record from hypoglossal motoneurons (XIIMNs) in thick medullary slices from neonatal rat pups (N=26 control and 24 DNE cells). To enable the translation of our findings to breathing-related consequences of DNE, we only studied XIIMNs that were receiving rhythmic excitatory drive from the respiratory central pattern generator. Tetrodotoxin was used to isolate XIIMNs from presynaptic input, and their postsynaptic responses to bath application of l-glutamic acid (glutamate) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were studied under voltage clamp. DNE had no influence on inward current magnitude evoked by either glutamate or AMPA. However, in cells from DNE animals, bath application of AMPA was associated with a right shift in the amplitude distribution (P=0.0004), but no change in the inter-event interval distribution of miniature excitatory postsynaptic currents (mEPSCs). DNE had no influence on mEPSC amplitude or frequency evoked by glutamate application, or under (unstimulated) baseline conditions. Thus, in the presence of AMPA, DNE is associated with a small but significant increase in quantal size, but no change in the probability of glutamate release.
• Levine, R. B., Wealing, J., Levine, R. B., Fregosi, R. F., & Cholanian, M. (2017). Developmental nicotine exposure alters potassium currents in hypoglossal motoneurons of neonatal rat.. Journal of neurophysiology, 117(4), 1544-1552. doi:10.1152/jn.00774.2016
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  We previously showed that nicotine exposure in utero and after birth via breast milk [developmental nicotine exposure (DNE)] is associated with many changes in the structure and function of hypoglossal motoneurons (XIIMNs), including a reduction in the size of the dendritic arbor and an increase in cell excitability. Interestingly, the elevated excitability was associated with a reduction in the expression of glutamate receptors on the cell body. Together, these observations are consistent with a homeostatic compensation aimed at restoring cell excitability. Compensation for increased cell excitability could also occur by changing potassium conductance, which plays a critical role in regulating resting potential, spike threshold, and repetitive spiking behavior. Here we test the hypothesis that the previously observed increase in the excitability of XIIMNs from DNE animals is associated with an increase in whole cell potassium currents. Potassium currents were measured in XIIMNs in brain stem slices derived from DNE and control rat pups ranging in age from 0 to 4 days by whole cell patch-clamp electrophysiology. All currents were measured after blockade of action potential-dependent synaptic transmission with tetrodotoxin. Compared with control cells, XIIMNs from DNE animals showed significantly larger transient and sustained potassium currents, but this was observed only under conditions of increased cell and network excitability, which we evoked by raising extracellular potassium from 3 to 9 mM. These observations suggest that the larger potassium currents in nicotine-exposed neurons are an important homeostatic compensation that prevents "runaway" excitability under stressful conditions, when neurons are receiving elevated excitatory synaptic input.NEW & NOTEWORTHY Developmental nicotine exposure is associated with increased cell excitability, which is often accompanied by compensatory changes aimed at normalizing excitability. Here we show that whole cell potassium currents are also increased in hypoglossal motoneurons from nicotine-exposed neonatal rats under conditions of increased cell and network excitability. This is consistent with a compensatory response aimed at preventing instability under conditions in which excitatory synaptic input is high and is compatible with the concept of homeostatic plasticity.
• Cholanian, M., Fregosi, R. F., & Levine, R. B. (2015). Developmental Nicotine Exposure Results in Exaggerated Response to AMPA Receptor Activation in Hypoglossal Motoneurons in Neonatal Rats. The FASEB Journal. doi:10.1096/fasebj.29.1_supplement.1032.14
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  Nicotine is the primary neurotoxin in tobacco products, as it has been strongly associated with altered development of neurons throughout the brain, including brainstem neurons involved in the control of breathing. Chronic nicotine exposure causes widespread desensitization of nicotinic acetylcholine receptors (nAChRs), leading to increased receptor expression, but a reduction in synaptic efficacy. Our previous work in rhythmically active hypoglossal motoneurons (XIIMNs) showed that developmental nicotine exposure (DNE) desensitizes their nAChRs and reduces excitatory synaptic input to these cells. We also showed that expression of somatic glutamate receptors was reduced, suggesting a reduction in post-synaptic efficacy. Here we hypothesize that DNE results in increased postsynaptic responses to AMPA receptor activation in XIIMNs from neonatal rats. Whole cell voltage clamp recordings of rhythmically active XIIMNs in brainstem slices from control and DNE animals were made. AMPA (2.5 mM) was bath-applied in the presence of TTX to isolate fast postsynaptic glutamatergic responses. Preliminary data suggest a larger AMPA-mediated inward current and a higher charge transfer in DNE XIIMNs (DNE, n = 10; control, n = 12). All responses were blocked by CNQX (20 mM), a selective AMPA and kainate receptor antagonist, indicating that the responses were AMPAergic. These results indicate that DNE: (1) upregulates AMPA receptor expression in XIIMNs, and/or (2) changes the kinetics of AMPA receptors in order to overcome the reduced excitatory synaptic input to XIIMNs.
• Mcmullen, N. T., Rance, N. E., Krajewski-hall, S. J., & Cholanian, M. (2015). Chronic oestradiol reduces the dendritic spine density of KNDy (kisspeptin/neurokinin B/dynorphin) neurones in the arcuate nucleus of ovariectomised Tac2-enhanced green fluorescent protein transgenic mice.. Journal of neuroendocrinology, 27(4), 253-63. doi:10.1111/jne.12263
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  Neurones in the arcuate nucleus that express neurokinin B (NKB), kisspeptin and dynorphin (KNDy) play an important role in the reproductive axis. Oestradiol modulates the gene expression and somatic size of these neurones, although there is limited information available about whether their dendritic structure, a correlate of cellular plasticity, is altered by oestrogens. In the present study, we investigated the morphology of KNDy neurones by filling fluorescent neurones in the arcuate nucleus of Tac2-enhanced green fluorescent protein (EGFP) transgenic mice with biocytin. Filled neurones from ovariectomised (OVX) or OVX plus 17β-oestradiol (E2)-treated mice were visualised with anti-biotin immunohistochemistry and reconstructed in three dimensions with computer-assisted microscopy. KNDy neurones exhibited two primary dendrites, each with a few branches confined to the arcuate nucleus. Quantitative analysis revealed that E2 treatment of OVX mice decreased the cell size and dendritic spine density of KNDy neurones. The axons of KNDy neurones originated from the cell body or proximal dendrite and gave rise to local branches that appeared to terminate within the arcuate nucleus. Numerous terminal boutons were also visualised within the ependymal layer of the third ventricle adjacent to the arcuate nucleus. Axonal branches also projected to the adjacent median eminence and exited the arcuate nucleus. Confocal microscopy revealed close apposition of EGFP and gonadotrophin-releasing hormone-immunoreactive fibres within the median eminence and confirmed the presence of KNDy axon terminals in the ependymal layer of the third ventricle. The axonal branching pattern of KNDy neurones suggests that a single KNDy neurone could influence multiple arcuate neurones, tanycytes in the wall of the third ventricle, axon terminals in the median eminence and numerous areas outside of the arcuate nucleus. In parallel with its inhibitory effects on electrical excitability, E2 treatment of OVX Tac2-EGFP mice induces structural changes in the somata and dendrites of KNDy neurones.
• Levine, R. B., Mcmullen, N. T., Rance, N. E., Krajewski-hall, S. J., & Cholanian, M. (2014). Electrophysiology of arcuate neurokinin B neurons in female Tac2-EGFP transgenic mice.. Endocrinology, 155(7), 2555-65. doi:10.1210/en.2014-1065
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  Neurons in the arcuate nucleus that coexpress kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) play an important role in the modulation of reproduction by estrogens. Here, we study the anatomical and electrophysiological properties of arcuate NKB neurons in heterozygous female transgenic mice with enhanced green fluorescent protein (EGFP) under the control of the Tac2 (NKB) promoter (Tac2-EGFP mice). The onset of puberty, estrous cyclicity, and serum LH were comparable between Tac2-EGFP and wild-type mice. The location of EGFP-immunoreactive neurons was consistent with previous descriptions of Tac2 mRNA-expressing neurons in the rodent. In the arcuate nucleus, nearly 80% of EGFP neurons expressed pro-NKB-immunoreactivity. Moreover, EGFP fluorescent intensity in arcuate neurons was increased by ovariectomy and reduced by 17β-estradiol (E2) treatment. Electrophysiology of single cells in tissue slices was used to examine the effects of chronic E2 treatment on Tac2-EGFP neurons in the arcuate nucleus of ovariectomized mice. Whole-cell recordings revealed arcuate NKB neurons to be either spontaneously active or silent in both groups. E2 had no significant effect on the basic electrophysiological properties or spontaneous firing frequencies. Arcuate NKB neurons exhibited either tonic or phasic firing patterns in response to a series of square-pulse current injections. Notably, E2 reduced the number of action potentials evoked by depolarizing current injections. This study demonstrates the utility of the Tac2-EGFP mouse for electrophysiological and morphological studies of KNDy neurons in tissue slices. In parallel to E2 negative feedback on LH secretion, E2 decreased the intensity of the EGFP signal and reduced the excitability of NKB neurons in the arcuate nucleus of ovariectomized Tac2-EGFP mice.
• Yelleswarapu, C. S., Lobzova, A., Donaldson, S. T., Das, B., & Cholanian, M. (2014). Digital holographic microscopy discriminates sex differences in medial prefrontal cortex GABA neurons following amphetamine sensitization.. Pharmacology, biochemistry, and behavior, 124, 326-32. doi:10.1016/j.pbb.2014.06.026
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  Sex differences have been noted in patterns of drug use and relapse, and in particular with amphetamine abuse, implicating estradiol in mediating female neurobehavioral responses. To investigate the interaction of estradiol with amphetamine-induced hyperactivity, we compared male, intact female (INTACT), ovariectomized (OVX) and ovariectomized estradiol-treated (OVX+EB) female rats receiving repeated amphetamine (AMPH) treatment. All rats received intermittent AMPH injections for three days, and baseline and post-injection locomotor activity as well as fine-motor movements were recorded. Upon completion of behavioral experiments, immunohistochemistry was performed to assess parvalbumin-immunoreactive (PV-IR) GABAergic neurons in the medial prefrontal cortex (mPFC). Results indicate that AMPH induced greater behavioral response during habituation among the INTACT animals, and post-injection hyperactivity was apparent on days 2 and 3, among INTACT and OVX+EB females. For INTACT animals, the hyperactivity was most pronounced when estrogen levels were high. Immunohistochemical analysis using digital holographic microscopy revealed INTACT and OVX+EB females had less expression and smaller somatic area of PV-IR neurons in the mPFC. These data provide evidence for rapid development of sex differences in response to AMPH that correlates with sexually dimorphic alterations in a subset of mPFC GABAergic neurons implicated in modulating forebrain dopamine projections.
• Smith, M. A., Rance, N. E., Krajewski, S. J., Dacks, P. A., & Cholanian, M. (2010). Neurokinin B and the hypothalamic regulation of reproduction.. Brain research, 1364, 116-28. doi:10.1016/j.brainres.2010.08.059
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  Loss-of-function mutations in the genes encoding either neurokinin B (NKB) or its receptor, NK3 (NK3R), result in hypogonadotropic hypogonadism, characterized by an absence of pubertal development and low circulating levels of LH and gonadal steroids. These studies implicate NKB and NK3R as essential elements of the human reproductive axis. Studies over the last two decades provide evidence that a group of neurons in the hypothalamic infundibular/arcuate nucleus form an important component of this regulatory circuit. These neurons are steroid-responsive and coexpress NKB, kisspeptin, dynorphin, NK3R, and estrogen receptor α (ERα) in a variety of mammalian species. Compelling evidence in the human indicates these neurons function in the hypothalamic circuitry regulating estrogen negative feedback on gonadotropin-releasing hormone (GnRH) secretion. Moreover, in the rat, they form a bilateral, interconnected network that projects to NK3R-expressing GnRH terminals in the median eminence. This network provides an anatomical framework to explain how coordination among NKB/kisspeptin/dynorphin/NK3R/ERα neurons could mediate feedback information from the gonads to modulate pulsatile GnRH secretion. There is substantial (but indirect) evidence that this network may be part of the neural circuitry known as the "GnRH pulse generator," with NK3R signaling as an important component. This theory provides a compelling explanation for the occurrence of hypogonadotropic hypogonadism in patients with inactivating mutations in the TAC3 or TACR3 genes. Future studies will be needed to determine whether NKB signaling plays a permissive role in the onset of puberty or is part of the driving force initiating the maturation of reproductive function.

Other Teaching Materials

• Cholanian, M. (2022. AZ Online Principles of Neuroanatomy: Cells to Systems Course. Arizona Online.