Meenakshi Alreja

Regulation of NKB pathways and their roles in the control of Kiss1 neurons in the arcuate nucleus of the male mouse.

Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17β-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.

Molecular control of locus coeruleus neurotransmission

The locus coeruleus (LC) is the major noradrenergic nucleus in the brain and innervates large segments of the neuraxis. LC neurons are thought to regulate states of attention and vigilance as well as activity of the sympathetic nervous system. These neurons also have been implicated in the actions of stress, antidepressants, and opiates on the brain. Aided in part by the fact that the LC is relatively homogeneous, it has been possible to understand some of the cellular and molecular mechanisms that control their functional state. This review focuses on the role played by the cAMP pathway in regulation of LC neurons, particularly after chronic perturbations. Thus, several components of this intracellular signaling pathway are upregulated in the LC after chronic stress or chronic opiate treatment, but downregulated after chronic antidepressant treatment. LC neurons exhibit a pacemaker activity, which appears to be mediated, at least in part, by a nonspecific cation current that is activated by protein kinase A. As a result, stimuli that upregulate the cAMP pathway after chronic administration (e.g., stress or opiates) increase the excitability of LC neurons, whereas stimuli that downregulate the cAMP pathway (e.g., antidepressants) exert the opposite effect. Such molecular adaptations could contribute to the behavioral plasticity that is associated with these various conditions.

Hypocretin/orexin innervation and excitation of identified septohippocampal cholinergic neurons.

Hypothalamic fibers containing the wake-promoting peptides, hypocretins (Hcrts) or orexins, provide a dense innervation to the medial septum–diagonal band of Broca (MSDB), a sleep-associated brain region that has been suggested to show intense axonal degeneration in canine narcoleptics. The MSDB, via its cholinergic and GABAergic projections to the hippocampus, controls the hippocampal theta rhythm and associated learning and memory functions. Neurons of the MSDB express very high levels of the Hcrt receptor 2, which is mutated in canine narcoleptics. In the present study, we investigated the electrophysiological effects of Hcrt peptides on septohippocampal cholinergic neurons that were identified in living brain slices of the MSDB using a selective fluorescent marker. Hcrt activation of septohippocampal cholinergic neurons was reversible, reproducible, and concentration dependent and mediated via a direct postsynaptic mechanism. Both Hcrt1 and Hcrt2 activated septohippocampal cholinergic neurons with similar EC50 values. The Hcrt effect was dependent on external Na+, reduced by external Ba2+, and also reduced in recordings with CsCl-containing electrodes, suggesting a dual underlying ionic mechanism that involved inhibition of a K+ current, presumably an inward rectifier, and a Na+-dependent component. The Na+ component was dependent on internal Ca2+, blocked by replacing external Na+ with Li+, and also blocked by bath-applied Ni2+ and KB-R7943, suggesting involvement of the Na+–Ca2+ exchanger. Using double-immunolabeling studies at light and ultrastructural levels, we also provide definitive evidence for a hypocretin innervation of cholinergic neurons. Thus Hcrt effects within the septum should increase hippocampal acetylcholine release and thereby promote hippocampal arousal.

Molecular and cellular mechanisms of opiate action: Studies in the rat locus coeruleus

We have studied the molecular and cellular mechanisms underlying the acute and chronic effects of opiate on neurons of the rat locus coeruleus (LC). Acutely, opiates inhibit LC neurons by activating K+ channels and inhibiting a novel sodium-dependent inward current. Both of these actions are mediated via pertussis toxin-sensitive G-proteins, and regulation of the sodium current occurs through inhibition of the cyclic AMP pathway. In contrast to the acute effects of opiates, chronic treatment of rats with opiates increases levels of specific G-protein subunits, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the possibility that this upregulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these noradrenergic LC neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms by which opiates regulate the expression of these intracellular messenger proteins in the LC. These studies will lead to an improved understanding of the molecular and cellular basis of opiate addiction.

Gonadotropin inhibitory hormone inhibits basal forebrain vGluT2-gonadotropin-releasing hormone neurons via a direct postsynaptic mechanism

The novel hypothalamic peptides avian gonadotropin inhibitory hormone (GnIH) and its mammalian analogue RFRP‐3, are emerging as key negative regulators of reproductive functions across species. GnIH/RFRP‐3 reduces gonadotropin release and may play an inhibitory role in ovulation and seasonal reproduction, actions opposite to that of the puberty‐promoting kisspeptins. GnIH directly inhibits gonadotropin release from the anterior pituitary in birds. GnIH/RFRP‐3‐immunoreactive fibres also abut the preoptic‐septal gonadotropin‐releasing hormone (GnRH) neurons, suggesting an additional site of action that has not been studied at the cellular level. Using anatomical labelling and electrophysiological recordings in septal brain slices from GnRH‐GFP, vGluT2‐GFP and GAD67‐GFP mice, we report inhibitory actions of GnIH/RFRP‐3 on kisspeptin‐activated vGluT2 (vesicular glutamate transporter 2)‐GnRH neurons as well as on kisspeptin‐insensitive GnRH neurons, but not on cholinergic or GABAergic neurons (n= 531). GnIH and RFRP‐3 produced a strikingly similar non‐desensitizing hyperpolarization following brief 15 s applications (GnIH: 9.3 ± 1.9 mV; RFRP‐3: 9.0 ± 0.9 mV) with IC50 values of 34 and 37 nm, respectively. The inhibitory effect was mediated via a direct postsynaptic Ba2+‐sensitive K+ current mechanism and could prevent or interrupt kisspeptin‐induced activation of vGluT2‐GnRH neurons. GnIH‐immunoreactive fibres were in apparent contact with vGluT2‐GFP neurons. Thus, GnIH/RFRP‐3 could reduce GnRH and glutamate release in target brain regions and in the median eminence via a direct inhibition of vGluT2‐GnRH neurons. This in turn could suppress gonadotropin release, influence reproductive development and alter sex behaviour.

Excitatory Effects of the Puberty-Initiating Peptide Kisspeptin and Group I Metabotropic Glutamate Receptor Agonists Differentiate Two Distinct Subpopulations of Gonadotropin-Releasing Hormone Neurons

Activation of the G-protein-coupled receptor GPR54 by kisspeptins during normal puberty promotes the central release of gonadotropin-releasing hormone (GnRH) that, in turn, leads to reproductive maturation. In humans and mice, a loss of function mutations of GPR54 prevents the onset of puberty and leads to hypogonadotropic hypogonadism and infertility. Using electrophysiological, morphological, molecular, and retrograde-labeling techniques in brain slices prepared from vGluT2-GFP and GnRH-GFP mice, we demonstrate the existence of two physiologically distinct subpopulations of GnRH neurons. The first subpopulation is comprised of septal GnRH neurons that colocalize vesicular glutamate transporter 2 and green fluorescent protein and is insensitive to metabotropic glutamate receptor agonists, but is exquisitely sensitive to kisspeptin which closes potassium channels to dramatically initiate a long-lasting activation in neurons from prepubertal and postpubertal mice of both sexes. A second subpopulation is insensitive to kisspeptin but is uniquely activated by group I metabotropic glutamate receptor agonists. These two physiologically distinct classes of GnRH cells may subserve different functions in the central control of reproduction and fertility.

Melanin-concentrating hormone directly inhibits GnRH neurons and blocks kisspeptin activation, linking energy balance to reproduction

A link between energy balance and reproduction is critical for the survival of all species. Energy-consuming reproductive processes need to be aborted in the face of a negative energy balance, yet knowledge of the pathways mediating this link remains limited. Fasting and food restriction that inhibit fertility also upregulate the hypothalamic melanin-concentrating hormone (MCH) system that promotes feeding and decreases energy expenditure; MCH knockout mice are lean and have a higher metabolism but remain fertile. MCH also modulates sleep, drug abuse behavior, and mood, and MCH receptor antagonists are currently being developed as antiobesity and antidepressant drugs. Despite the clinical implications of MCH, the direct postsynaptic effects of MCH have never been reported in CNS neurons. Using patch-clamp recordings in brain slices from multiple lines of transgenic GFP mice, we demonstrate a strong inhibitory effect of MCH on an exclusive population of septal vGluT2-GnRH neurons that is activated by the puberty-triggering and preovulatory luteinizing hormone surge-mediating peptide, kisspeptin. MCH has no effect on kisspeptin-insensitive GnRH, vGluT2, cholinergic, or GABAergic neurons located within the same nucleus. The inhibitory effects of MCH are reproducible and nondesensitizing and are mediated via a direct postsynaptic Ba2+-sensitive K+ channel mechanism involving the MCHR1 receptor. MCH immunoreactive fibers are in close proximity to vGluT2-GFP and GnRH-GFP neurons. Importantly, MCH blocks the excitatory effect of kisspeptin on vGluT2-GnRH neurons. Considering the role of MCH in regulating energy balance and of GnRH and kisspeptin in triggering puberty and maintaining fertility, MCH may provide a critical link between energy balance and reproduction directly at the level of the kisspeptin-activated vGluT2-GnRH neuron.

Excitatory actions of serotonin on GABAergic neurons of the medial septum and diagonal band of broca

The physiological and pharmacological actions of serotonin (5‐HT) on neurons in the medial septum and diagonal band of Broca (MSDB) were examined using extracellular and intracellular recording techniques in an in vitro rat brain‐slice preparation. In addition to previously described inhibitory effects, novel excitatory actions of 5‐HT on GABA‐type cells were observed. In intracellular recordings with KCl‐containing electrodes, bath‐applied 5‐HT induced a bicuculline and tetrodotoxin‐sensitive increase in the number of reverse IPSPs in both cholinergic‐ and noncholinergic‐type neurons (presumably GABAergic). In brain slices where all structures neighboring the MSDB, including the lateral septum, had been excised, a similar increase in 5‐HT‐induced IPSPs occurred, indicating that 5‐HT‐induced IPSPs in both cholinergic‐ and noncholinergic‐type neurons originate from GABAergic neurons within the MSDB itself. Accordingly, GABA‐type neurons in the MSDB were found to be directly excited by 5‐HT. MDL 100,907, a selective 5‐HT2A antagonist, blocked 5‐HT‐induced excitations in a majority of neurons (58%). ICS 205‐930, a 5‐HT3/5‐HT4 antagonist, or mianserin, a nonselective 5‐HT antagonist, blocked most MDL‐resistant responses, indicating a role for multiple 5‐HT receptor subtypes.

Pacemaker activity of locus coeruleus neurons: whole-cell recordings in brain slices show dependence on cAMP and protein kinase A

Noradrenergic neurons of the rat locus coeruleus (LC) are endogenous pacemakers that exhibit slow, tonic firing even in the complete absence of synaptic inputs. In the present study a time-dependent decline in LC spontaneous firing activity was found on intracellular dialysis during whole-cell recording with low-resistance patch electrodes; this decline was accentuated by a specific inhibitor of cAMP-dependent protein kinase (PKI5-24). Conversely, the inclusion of cAMP, 8-Br-cAMP, or the catalytic subunit of cAMP-dependent protein kinase (PKAcat) in the patch pipettes dose-dependently increased firing rate; intracellular PKI5-24 blocked both 8-Br-cAMP and PKAcat-induced firing in LC neurons. These results indicate that endogenous cAMP, via a phosphorylation-dependent route, drives tonic pacemaker activity in LC neurons.

Nicotine recruits a local glutamatergic circuit to excite septohippocampal GABAergic neurons

Tonic impulse flow in the septohippocampal GABAergic pathway is essential for normal cognitive functioning and is sustained, in part, by acetylcholine (ACh) that is released locally via axon collaterals of septohippocampal cholinergic neurons. Septohippocampal cholinergic neurons degenerate in Alzheimers disease and other neurodegenerative disorders. While the importance of the muscarinic effects of ACh on septohippocampal GABAergic neurons is well recognized, the nicotinic effects of ACh remain unstudied despite the reported benefits of nicotine on cognitive functioning. In the present study, using electrophysiological recordings in a rat brain slice preparation, rapid applications of nicotine excited 90% of retrogradely labelled septohippocampal GABA‐type neurons with an EC50 of 17 µm and increased the frequency of spontaneously occurring, impulse‐dependent fast GABAergic and glutamatergic synaptic currents via the α4β2‐nicotinic receptor. Interestingly, tetrodotoxin blocked all effects of nicotine on septohippocampal GABAergic type neurons, suggesting involvement of indirect mechanisms. We demonstrate that the effects of nicotine on septohippocampal GABA‐type neurons involve recruitment of a novel, local glutamatergic circuitry as (i) Group I metabotropic glutamatergic receptor antagonists reduced the effects of nicotine; (ii) the number of nicotine responsive neurons was significantly reduced in recordings from slices that had been trimmed so as to reduce the number of glutamate‐containing neurons within the slice preparation; (iii) in light and ultrastructural double immunocytochemical labelling studies vesicular glutamate 2 transporter immunoreactive terminals made synaptic contacts with parvalbumin‐immunoreactive septohippocampal GABAergic neurons. The discovery of a local glutamatergic circuit within the septum may provide another avenue for restoring septohippocampal GABAergic functions in neurodegenerative disorders associated with a loss of septohippocampal cholinergic neurons.